Research in Drug Development (RD3)

Microbiology, Bioorganic and Macromolecular Chemistry

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The goals of my research are to provide new insight into protein structure and function, and to decipher how proteins interact together and with other molecules, particularly with pharmacologically active molecules.

Top ten recent articles

Articles dans des revues avec comité de lecture

2024

Intriguing link between fetal intracranial hemorrhage and X-linked recessive chondrodysplasia punctata.

Létard, P., Wintjens, R., Van-Gils, J., Martinovic, J., Laffargue, F., Dufernez, F., & Egloff, M. (2024). Intriguing link between fetal intracranial hemorrhage and X-linked recessive chondrodysplasia punctata. Ultrasound in obstetrics & gynecology. doi:10.1002/uog.27573  
https://dipot.ulb.ac.be/dspace/bitstream/2013/368258/3/Letard-2024.pdf

 

2023

Evaluation of in vitro antiplasmodial, antiproliferative activities, and in vivo oral acute toxicity of Spathodea campanulata flowers

Mbosso Teinkela, J. E., Oumarou, H., Siwe Noundou, X., Meyer, F., Megalizzi, V., Hoppe, H. H., Krause, R. W. M., & Wintjens, R. (2023). Evaluation of in vitro antiplasmodial, antiproliferative activities, and in vivo oral acute toxicity of Spathodea campanulata flowers. Scientific African, 21, e01871. doi:10.1016/j.sciaf.2023.e01871  
https://dipot.ulb.ac.be/dspace/bitstream/2013/368319/5/Mbosso_2023.pdf

 

Two new siderophores produced by Pseudomonas sp. NCIMB 10586: The anti-oomycete non-ribosomal peptide synthetase-dependent mupirochelin and the NRPS-independent triabactin

Grosse, C., Brandt, N., Van Antwerpen, P., Wintjens, R., & Matthijs, S. (2023). Two new siderophores produced by Pseudomonas sp. NCIMB 10586: The anti-oomycete non-ribosomal peptide synthetase-dependent mupirochelin and the NRPS-independent triabactin. Frontiers in microbiology, 14. doi:10.3389/fmicb.2023.1143861  

Introduction Globisporangium ultimum is an oomycetal pathogen causing damping-off on over 300 different plant hosts. Currently, as for many phytopathogens, its control relies in the use of chemicals with negative impact on health and ecosystems. Therefore, many biocontrol strategies are under investigation to reduce the use of fungicides. Results In this study, the soil bacterium Pseudomonas sp. NCIMB 10586 demonstrates a strong iron-repressed in vitro antagonism against G. ultimum MUCL 38045. This antagonism does not depend on the secretion of the broad-range antibiotic mupirocin or of the siderophore pyoverdine by the bacterial strain. The inhibitor molecule was identified as a novel non-ribosomal peptide synthetase (NRPS) siderophore named mupirochelin. Its putative structure bears similarities to other siderophores and bioactive compounds. The transcription of its gene cluster is affected by the biosynthesis of pyoverdine, the major known siderophore of the strain. Besides mupirochelin, we observed the production of a third and novel NRPS-independent siderophore (NIS), here termed triabactin. The iron-responsive transcriptional repression of the two newly identified siderophore gene clusters corroborates their role as iron scavengers. However, their respective contributions to the strain fitness are dissimilar. Bacterial growth in iron-deprived conditions is greatly supported by pyoverdine production and, to a lesser extent, by triabactin. On the contrary, mupirochelin does not contribute to the strain fitness under the studied conditions. Conclusion Altogether, we have demonstrated here that besides pyoverdine, Pseudomonas sp. NCIMB 10586 produces two newly identified siderophores, namely mupirochelin, a weak siderophore with strong antagonism activity against G. ultimum, and the potent siderophore triabactin.

https://dipot.ulb.ac.be/dspace/bitstream/2013/357326/1/doi_340970.pdf

 

2022

Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection.

Sawant, M., Benamrouz-Vanneste, S., Meloni, D., Gantois, N., Even, G., Guyot, K., Creusy, C., Duval, E., Wintjens, R., Weitzman, J. J., Chabe, M., Viscogliosi, E., & Certad, G. (2022). Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection. Virulence, 13(1), 1632-1650. doi:10.1080/21505594.2022.2123363  

Cryptosporidium parvum is a leading cause of diarrhoeal illness worldwide being a significant threat to young children and immunocompromised patients, but the pathogenesis caused by this parasite remains poorly understood. C. parvum was recently linked with oncogenesis. Notably, the mechanisms of gene expression regulation are unexplored in Cryptosporidium and little is known about how the parasite impact host genome regulation. Here, we investigated potential histone lysine methylation, a dynamic epigenetic modification, during the life cycle of the parasite. We identified SET-domain containing proteins, putative lysine methyltransferases (KMTs), in the C. parvum genome and classified them phylogenetically into distinct subfamilies (namely CpSET1, CpSET2, CpSET8, CpKMTox and CpAKMT). Our structural analysis further characterized CpSET1, CpSET2 and CpSET8 as histone lysine methyltransferases (HKMTs). The expression of the CpSET genes varies considerably during the parasite life cycle and specific methyl-lysine antibodies showed dynamic changes in parasite histone methylation during development (CpSET1:H3K4; CpSET2:H3K36; CpSET8:H4K20). We investigated the impact of C. parvum infection on the host histone lysine methylation. Remarkably, parasite infection led to a considerable decrease in host H3K36me3 and H3K27me3 levels, highlighting the potential of the parasite to exploit the host epigenetic regulation to its advantage. This is the first study to describe epigenetic mechanisms occurring throughout the parasite life cycle and during the host-parasite interaction. A better understanding of histone methylation in both parasite and host genomes may highlight novel infection control strategies.

https://dipot.ulb.ac.be/dspace/bitstream/2013/355346/3/Sawant-2022.pdf

 

The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis.

Flipo, M., Frita, R., Bourotte, M., Martínez-Martínez, M. S., Boesche, M., Boyle, G. W., Derimanov, G., Drewes, G., Gamallo, P., Ghidelli-Disse, S., Gresham, S., Jiménez, E., de Mercado, J., Pérez-Herrán, E., Porras-De Francisco, E., Rullas, J., Casado, P., Leroux, F., Piveteau, C., Kiass, M., Mathys, V., Soetaert, K., Megalizzi, V., Tanina, A., Wintjens, R., Antoine, R., Brodin, P., Delorme, V., Moune, M., Djaout, K., Slupek, S., Kemmer, C., Gitzinger, M., Ballell, L., Mendoza-Losana, A., Lociuro, S., Déprez, B., Barros-Aguirre, D., Remuiñán, M. M., Willand, N., & Baulard, A. R. (2022). The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis. Science Translational Medicine, 14(643), eaaz6280. doi:10.1126/scitranslmed.aaz6280  

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

https://dipot.ulb.ac.be/dspace/bitstream/2013/349752/3/aaz6280_STM_Main_EditionV7.pdf

 

Case Report: Inactivating PTH/PTHrP Signaling Disorder Type 1 Presenting With PTH Resistance.

Demaret, T., Wintjens, R., Sana, G., Docquir, J., Bertin, F., Ide, C., Monestier, O., Karadurmus, D., Benoit, V., & Maystadt, I. (2022). Case Report: Inactivating PTH/PTHrP Signaling Disorder Type 1 Presenting With PTH Resistance. Frontiers in endocrinology, 13, 928284. doi:10.3389/fendo.2022.928284  

PTH resistance is characterized by elevated parathyroid hormone (PTH) levels, hypocalcemia, hyperphosphatemia and it is classically associated with GNAS locus genetic or epigenetic defects. Inactivating PTH/PTHrP signaling disorders (iPPSD) define overlapping phenotypes based on their molecular etiology. iPPSD1 is associated with PTH1R variants and variable phenotypes including ossification anomalies and primary failure of tooth eruption but no endocrine disorder. Here we report on a 10-month-old child born from consanguineous parents, who presented with mild neurodevelopmental delay, seizures, enlarged fontanelles, round face, and bilateral clinodactyly. Hand x-rays showed diffuse delayed bone age, osteopenia, short metacarpal bones and cone-shaped distal phalanges. A diagnosis of PTH resistance was made on the basis of severe hypocalcemia, hyperphosphatemia, elevated PTH and normal vitamin D levels on blood sample. The patient was treated with calcium carbonate and alfacalcidol leading to rapid bio-clinical improvement. Follow-up revealed multiple agenesis of primary teeth and delayed teeth eruption, as well as Arnold-Chiari type 1 malformation requiring a ventriculoperitoneal shunt placement. GNAS gene analysis showed no pathogenic variation, but a likely pathogenic homozygous substitution c.723C>G p.(Asp241Glu) in PTH1R gene was found by trio-based whole exome sequencing. We studied the deleterious impact of the variant on the protein conformation with bioinformatics tools. In conclusion, our study reports for the first time PTH resistance in a child with a biallelic PTH1R mutation, extending thereby the clinical spectrum of iPPSD1 phenotypes.

https://dipot.ulb.ac.be/dspace/bitstream/2013/349751/5/Demaret-2022.pdf

 

2021

Child to adulthood clinical description of MDPL syndrome due to a novel variant in POLD1

Battisti, G., Wintjens, R., Decottignies, A., Merhi, A., Fervaille, C., Sokal, E. M., Karadurmus, D., Benoit, V., Claessens, A., Martinet, J.-P., Martiat, B., Kinzinger, P., & Maystadt, I. (2021). Child to adulthood clinical description of MDPL syndrome due to a novel variant in POLD1. European journal of medical genetics, 64(12), 104333. doi:10.1016/j.ejmg.2021.104333  

Mandibular hypoplasia, Deafness, Progeroid features, and Lipodystrophy (MDPL) syndrome is a rare autosomal dominant disorder caused by mutations in POLD1 gene and characterized by mandibular hypoplasia, deafness, progeroid features and lipodystrophy. One recurrent mutation p.(Ser605del) was reported in almost all affected patients. We report a novel de novo c.3214A>C p.(Thr1072Pro) variant in POLD1 in a 28-year-old male with MDPL syndrome. We provide a clinical description, molecular/immunohistological results, and literature review.

https://dipot.ulb.ac.be/dspace/bitstream/2013/336663/3/Gladys-2021.pdf

 

2020

Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein

Wintjens, R., Bifani, A. M., & Bifani, P. (2020). Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein. NPJ vaccines, 5(1), 81. doi:10.1038/s41541-020-00237-9  

The SARS-CoV-2 outbreak originated in China in late 2019 and has since spread to pandemic proportions. Diagnostics, therapeutics and vaccines are urgently needed. We model the trimeric Spike protein, including flexible loops and all N-glycosylation sites, in order to elucidate accessible epitopes for antibody-based diagnostics, therapeutics and vaccine development. Based on published experimental data, six homogeneous glycosylation patterns and two heterogeneous ones were used for the analysis. The glycan chains alter the accessible surface areas on the S-protein, impeding antibody-antigen recognition. In presence of glycan, epitopes on the S1 subunit, that notably contains the receptor binding domain, remain mostly accessible to antibodies while those present on the S2 subunit are predominantly inaccessible. We identify 28 B-cell epitopes in the Spike structure and group them as non-affected by the glycan cloud versus those which are strongly masked by the glycan cloud, resulting in a list of favourable epitopes as targets for vaccine development, antibody-based therapy and diagnostics.

https://dipot.ulb.ac.be/dspace/bitstream/2013/313085/1/doi_296729.pdf

 

Compound isolation and biological activities of Piptadeniastrum africanum (hook.f.) Brennan roots

Mbosso Teinkela, J. E., Siwe Noundou, X., Zeh Mimba, J. E., Meyer, F., Tabouguia, O. M., Assob Nguedia, J. C., Hoppe, H. H., Krause, R. W. M., Wintjens, R., & Azebaze, G. A. B. (2020). Compound isolation and biological activities of Piptadeniastrum africanum (hook.f.) Brennan roots. Journal of ethnopharmacology, 255, 112716. doi:10.1016/j.jep.2020.112716  

Ethnopharmacological relevance: The dicotyledonous plant Piptadeniastrum africanum (hook.f.) Brennan (Fabaceae) is used in traditional medicine to treat various human complaints including bronchitis, coughing, urino-genital ailments, meningitis, abdominal pain, treatment of wounds, malaria and gastrointestinal ailments, and is used as a purgative and worm expeller. Aim of the study: The present study describes the phytochemical investigation and the determination of the antimicrobial, antiplasmodial and antitrypanosomal activities of crude extract, fractions and compounds extracted from Piptadeniastrum africanum roots. Materials and methods: Isolated compounds were obtained using several chromatographic techniques. The structures of all compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR) and by comparing their NMR data with those found in literature. In vitro antimicrobial activity of samples was evaluated using the microdilution method on bacterial (Escherichia coli, Proteus mirabilis, Staphylococcus aureus) and fungal (Candida krusei) strains, while in vitro cell-growth inhibition activities were assessed against two parasites (Trypanosoma brucei brucei and Plasmodium falciparum strain 3D7). The cytotoxicity properties of samples were assayed against HeLa human cervical carcinoma. Results: Five compounds were isolated and identified as: tricosanol 1, 5α-stigmasta-7,22-dien-3-β-ol 2, betulinic acid 3, oleanolic acid 4 and piptadenamide 5. This is the first report of the isolation of these five compounds from the roots of P. africanum. The (Hex:EtOAc 50:50) fraction exhibited moderate antibacterial activity against P. mirabilis (MIC 250 μg/mL), while the other fractions and isolated compounds had weak antimicrobial activities. Only the EtOAc fraction presented a moderate antimalarial activity with an IC50 of 16.5 μg/mL. The MeOH crude extract and three fractions (Hexane, Hexane-EtOAc 25% and EtOAc-MeOH 25%) exhibited significant trypanocidal activity with IC50 values of 3.0, 37.5, 3.8 and 9.5 μg/mL, respectively. Conclusion: These results demonstrated a scientific rational of the traditional uses of P. africanum and indicate that this plant should be further investigated to identify some of the chemical components that exhibited the activities reported in this study and therefore may constitute new lead candidates in parasiticidal drug discovery.

https://dipot.ulb.ac.be/dspace/bitstream/2013/307387/3/Mbosso-2020.pdfhttps://dipot.ulb.ac.be/dspace/bitstream/2013/307387/4/Mbosso-2020-Manuscript-final.pdf

 

Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a fragment-based screening.

Faïon, L., Djaout, K., Frita, R., Pintiala, C., Cantrelle, F.-X., Moune, M., Vandeputte, A., Bourbiaux, K., Piveteau, C., Herledan, A., Biela, A., Leroux, F., Kremer, L., Blaise, M., Tanina, A., Wintjens, R., Hanoulle, X., Déprez, B., Willand, N., Baulard, A. R., & Flipo, M. (2020). Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a fragment-based screening. European journal of medicinal chemistry, 200, 112440. doi:10.1016/j.ejmech.2020.112440  

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments.

https://dipot.ulb.ac.be/dspace/bitstream/2013/308695/1/elsevier_292339.pdf

 

Fragment-Based Optimized EthR Inhibitors with in Vivo Ethionamide Boosting Activity.

Villemagne, B., Machelart, A., Tran, N. C., Flipo, M., Moune, M., Leroux, F., Piveteau, C., Wohlkönig, A., Wintjens, R., Li, X., Gref, R., Brodin, P., Déprez, B., Baulard, A. R., & Willand, N. (2020). Fragment-Based Optimized EthR Inhibitors with in Vivo Ethionamide Boosting Activity. ACS infectious diseases, 6(3), 366-378. doi:10.1021/acsinfecdis.9b00277  

Killing more than one million people each year, tuberculosis remains the leading cause of death from a single infectious agent. The growing threat of multidrug-resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. EthR, a mycobacterial transcriptional regulator, is involved in the control of the bioactivation of the second-line drug ethionamide. We have previously reported the discovery of in vitro nanomolar boosters of ethionamide through fragment-based approaches. In this study, we have further explored the structure-activity and structure-property relationships in this chemical family. By combining structure-based drug design and in vitro evaluation of the compounds, we identified a new oxadiazole compound as the first fragment-based ethionamide booster which proved to be active in vivo, in an acute model of tuberculosis infection.

https://dipot.ulb.ac.be/dspace/bitstream/2013/308699/3/Villemagne_et_al_submission.pdf

 

Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor.

Brito, A. S., Neuhäuser, B., Wintjens, R., Marini, A. M., & Boeckstaens, M. (2020). Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor. PLOS genetics, 16(2), e1008634. doi:10.1371/journal.pgen.1008634  

The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of a putative partner to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. Finally, we show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans.

https://dipot.ulb.ac.be/dspace/bitstream/2013/303445/1/doi_287089.pdf

 

2019

In vitro antimalarial, antitrypanosomal and HIV-1 integrase inhibitory activities of two Cameroonian medicinal plants: Antrocaryon klaineanum (Anacardiaceae)and Diospyros conocarpa (Ebenaceae)

Fouokeng, Y., Feumo Feusso, H., Mbosso Teinkela, J. E., Siwe Noundou, X., Wintjens, R., Isaacs, M., Hoppe, H. H., Krause, R. W. M., Azebaze, A. G. B., & Vardamides, J. C. (2019). In vitro antimalarial, antitrypanosomal and HIV-1 integrase inhibitory activities of two Cameroonian medicinal plants: Antrocaryon klaineanum (Anacardiaceae)and Diospyros conocarpa (Ebenaceae). South African journal of botany, 122, 510-517. doi:10.1016/j.sajb.2018.10.008  

Antiplasmodial, antitrypanosomal and anti-HIV-1 activities of crude extracts, fractions and some isolated compounds from two Cameroonian medicinal plants: Antrocaryon klaineanum Pierre (Anacardiaceae)and Diospyros conocarpa Gürke ex K. Schum. (Ebenaceae)were assessed. The phytochemical studies led to the isolation of eight compounds (1-8)from Diospyros conocarpa and six compounds (6, 9-13)from Antrocaryon klaineanum. These compounds were identified as mangiferolic acid (1), 3β, 22(S)-dihydroxycycloart-24E-en-26-oic acid (2), lupeol (3), aridanin (4), betulin (5), betulinic acid (6), bergenin (7), D-quercitol(8), entilin C(9), entilin A(10), antrocarine A(11), 7R,20(S)-dihydroxy-4,24(28)-ergostadien-3-one(12)and stigmasterol glucoside (13). The criteria for activity were set as follows: an IC50 value < 10 μg/mL for crude extracts and < 1 μg/mL for pure compounds. The hexane/ethyl acetate (1:1)fraction of A.klaineanum root bark (AKERF1)and the hexane/ethyl acetate (1:1)fraction of A.klaineanum trunk bark (AKETF1)presented the strongest antiplasmodial activities with IC50 values of 0.4 and 4.4 μg/mL, respectively. Aridanin (4)and antrocarine A(11), as well as the crude extract of D.conocarpa roots (EDCR), AKERF1 and AKETF1 showed moderate trypanocidal effects. The crude extract of A.klaineanum root bark (AKER)and AKETF1 exhibited attractive activities on HIV-1 integrase with IC50 values of 1.96 and 24.04 μg/mL, respectively. The results provide baseline information on the use of A.klaineanum and D.conocarpa extracts, as well as certain components, as sources of new antiplasmodial, antitrypanosomal and anti-HIV drugs.

https://dipot.ulb.ac.be/dspace/bitstream/2013/297090/3/Fouokeng-et-al-AuthorManuscript.pdf

 

A fragment-based approach towards the discovery of N-substituted tropinones as inhibitors of Mycobacterium tuberculosis transcriptional regulator EthR2

Prevet, H., Moune, M., Tanina, A., Kemmer, C., Herledan, A., Frita, R., Wohlkonig, A., Bourotte, M., Villemagne, B., Leroux, F., Gitzinger, M., Baulard, A., Déprez, B., Wintjens, R., Willand, N., & Flipo, M. (2019). A fragment-based approach towards the discovery of N-substituted tropinones as inhibitors of Mycobacterium tuberculosis transcriptional regulator EthR2. European journal of medicinal chemistry, 167, 426-438. doi:10.1016/j.ejmech.2019.02.023  

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis, represents one of the most challenging threat to public health worldwide, and with the increasing resistance to approved TB drugs, it is needed to develop new strategies to address this issue. Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant TB. It is a prodrug that requires activation by mycobacterial monooxygenases to inhibit the enoyl-ACP reductase InhA, which is involved in mycolic acid biosynthesis. Very recently, we identified that inhibition of a transcriptional repressor, termed EthR2, derepresses a new bioactivation pathway that results in the boosting of ethionamide activation. Herein, we describe the identification of potent EthR2 inhibitors using fragment-based screening and structure-based optimization. A target-based screening of a fragment library using thermal shift assay followed by X-ray crystallography identified 5 hits. Rapid optimization of the tropinone chemical series led to compounds with improved in vitro potency.

https://dipot.ulb.ac.be/dspace/bitstream/2013/284895/1/Elsevier_268522.pdf

 

A comprehensive analysis of the protein-ligand interactions in crystal structures of Mycobacterium tuberculosis EthR

Tanina, A., Wohlkonig, A., Soror, S. H., Flipo, M., Villemagne, B., Prevet, H., Déprez, B., Moune, M., Perée, H., Meyer, F., Baulard, A., Willand, N., & Wintjens, R. (2019). A comprehensive analysis of the protein-ligand interactions in crystal structures of Mycobacterium tuberculosis EthR. Biochimica et biophysica acta. Proteins and proteomics, 1867(3), 248-258. doi:10.1016/j.bbapap.2018.12.003  

The Mycobacterium tuberculosis EthR is a member of the TetR family of repressors, controlling the expression of EthA, a mono-oxygenase responsible for the bioactivation of the prodrug ethionamide. This protein was established as a promising therapeutic target against tuberculosis, allowing, when inhibited by a drug-like molecule, to boost the action of ethionamide. Dozens of EthR crystal structures have been solved in complex with ligands. Herein, we disclose EthR structures in complex with 18 different small molecules and then performed in-depth analysis on the complete set of EthR structures that provides insights on EthR-ligand interactions. The 81 molecules solved in complex with EthR show a large diversity of chemical structures that were split up into several chemical clusters. Two of the most striking common points of EthR-ligand interactions are the quasi-omnipresence of a hydrogen bond bridging compounds with Asn179 and the high occurrence of π-π interactions involving Phe110. A systematic analysis of the protein-ligand contacts identified eight hot spot residues that defined the basic structural features governing the binding mode of small molecules to EthR. Implications for the design of new potent inhibitors are discussed.

https://dipot.ulb.ac.be/dspace/bitstream/2013/284532/1/Elsevier_268159.pdf

 

2018

Crystal packing and theoretical analysis of halogen- and hydrogen-bonded hydrazones from pharmaceuticals. Evidence of type I and II halogen bonds in extended chains of dichloromethane

Berger, G., Soubhye, J., Wintjens, R., Robeyns, K., & Meyer, F. (2018). Crystal packing and theoretical analysis of halogen- and hydrogen-bonded hydrazones from pharmaceuticals. Evidence of type I and II halogen bonds in extended chains of dichloromethane. Acta Crystallographica Section B: Structural Science, Crystal Engineering and Materials, 74(6), 618-627. doi:10.1107/S2052520618014221  

The supramolecular assembly of halogenated and hydroxyl hydrazones derived from two well known pharmaceuticals, isoniazid (IsX, where X = I, Br, OH) and hydralazine (HyX, where X = I, Br, OH), was studied by X-ray crystallography and theoretical methods. Crystal packing of IsI and HyI shows weak I…N and I…π halogen bonds, whereas the hydrogen bonds are dominant in the brominated scaffolds IsBr and HyBr. Although the calculated I…N interaction strength appears almost three times weaker than the O—H…N contacts in the isoniazid-based hydrazones, the higher directionality of the halogen bonds induces a linear and planar architecture of self-complementary tectons, observed only with the help of a bridging water molecule in the case of IsOH. Finally, the X-ray structure of HyOH is characterized by an unexpected linear arrangement of clathrated dichloromethane molecules bound through type I and II halogen bonds. This rare phenomenon, observed in less than ten structures, was studied by coupled cluster-based energy decomposition.

https://dipot.ulb.ac.be/dspace/bitstream/2013/279895/4/A.pdfhttps://dipot.ulb.ac.be/dspace/bitstream/2013/279895/5/ActaB0710.pdf

 

Emergence of Aspergillus fumigatus azole resistance in azole-naïve patients with chronic obstructive pulmonary disease and their homes.

Dauchy, C., Bautin, N., Nseir, S., Reboux, G., Wintjens, R., Le Rouzic, O., Sendid, B., Viscogliosi, E., Le Pape, P., Arendrup, M. C. A. M., Gosset, P., Fry, S., & Fréalle, E. (2018). Emergence of Aspergillus fumigatus azole resistance in azole-naïve patients with chronic obstructive pulmonary disease and their homes. Indoor air, 28(2), 298-306. doi:10.1111/ina.12436  

Azole-resistant Aspergillus fumigatus (ARAF) has been reported in patients with chronic obstructive pulmonary disease (COPD) but has not been specifically assessed so far. Here, we evaluated ARAF prevalence in azole-naïve COPD patients and their homes, and assessed whether CYP51A mutations were similar in clinical and environmental reservoirs. Sixty respiratory samples from 41 COPD patients with acute exacerbation and environmental samples from 36 of these patient's homes were prospectively collected. A. fumigatus was detected in respiratory samples from 11 of 41 patients (27%) and in 15 of 36 domiciles (42%). Cyp51A sequencing and selection on itraconazole medium of clinical (n = 68) and environmental (n = 48) isolates yielded ARAF detection in 1 of 11 A. fumigatus colonized patients with COPD (9%) and 2 of 15 A. fumigatus-positive patient's homes (13%). The clinical isolate had no CYP51A mutation. Two environmental isolates from two patients harbored TR34/L98H mutation, and one had an H285Y mutation. Coexistence of different cyp51A genotypes and/or azole resistance profiles was detected in 3 of 8 respiratory and 2 of 10 environmental samples with more than one isolate, confirming the need for a systematic screening of all clinically relevant isolates. The high prevalence of ARAF in patients with COPD and their homes supports the need for further studies to assess the prevalence of azole resistance in patients with Aspergillus diseases in Northern France.

 

Biological activities of plant extracts from Ficus elastica and Selaginella vogelli: An antimalarial, antitrypanosomal and cytotoxity evaluation.

Mbosso, E. J. T., Noundou, X. S., Nguemfo, E. L., Meyer, F., Wintjens, R., Isaacs, M., Mpondo, E. A. M. E., Hoppe, H. H., Krause, R. W. M., & Azebaze, G. A. B. (2018). Biological activities of plant extracts from Ficus elastica and Selaginella vogelli: An antimalarial, antitrypanosomal and cytotoxity evaluation. Saudi journal of biological sciences, 25(1), 117-122. doi:10.1016/j.sjbs.2017.07.002  

The cytotoxic, antiplasmodial, and antitrypanosomal activities of two medicinal plants traditionally used in Cameroon were evaluated. Wood of Ficus elastica Roxb. ex Hornem. aerial roots (Moraceae) and Selaginella vogelii Spring (Selaginellaceae) leaves were collected from two different sites in Cameroon. In vitro cell-growth inhibition activities were assessed on methanol extract of plant materials against Plasmodium falciparum strain 3D7 and Trypanosoma brucei brucei, as well as against HeLa human cervical carcinoma cells. Criteria for activity were an IC50 value < 10 μg/mL. The extract of S. vogelii did not significantly reduce the viability of P. falciparum at a concentration of 25 μg/mL but dramatically affected the trypanosome growth with an IC50 of 2.4 μg/mL. In contrast, at the same concentration, the extract of F. elastica exhibited plasmodiacidal activity (IC50 value of 9.5 μg/mL) and trypanocidal (IC50 value of 0.9 μg/mL) activity. Both extracts presented low cytotoxic effects on HeLa cancer cell line. These results indicate that the selected medicinal plants could be further investigated for identifying compounds that may be responsible for the observed activities and that may represent new leads in parasitical drug discovery.

https://dipot.ulb.ac.be/dspace/bitstream/2013/266367/1/doi_249994.pdf

 

2017

Structural analysis of the interaction between spiroisoxazoline SMARt-420 and the Mycobacterium tuberculosis repressor EthR2.

Wohlkonig, A., Remaut, H., Moune, M., Tanina, A., Meyer, F., Desroses, M., Steyaert, J., Willand, N., Baulard, A., & Wintjens, R. (2017). Structural analysis of the interaction between spiroisoxazoline SMARt-420 and the Mycobacterium tuberculosis repressor EthR2. Biochemical and biophysical research communications, 487(2), 403-408. doi:10.1016/j.bbrc.2017.04.074  

Inhibition of transcriptional regulators of bacterial pathogens with the aim of reprogramming their metabolism to modify their antibiotic susceptibility constitutes a promising therapeutic strategy. One example is the bio-activation of the anti-tubercular pro-drug ethionamide, which activity could be enhanced by inhibiting the transcriptional repressor EthR. Recently, we discovered that inhibition of a second transcriptional repressor, EthR2, leads to the awakening of a new ethionamide bio-activation pathway. The x-ray structure of EthR2 was solved at 2.3 Å resolution in complex with a compound called SMARt-420 (Small Molecule Aborting Resistance). Detailed comparison and structural analysis revealed interesting insights for the upcoming structure-based design of EthR2 inhibitors as an alternative to revert ethionamide resistance in Mycobacterium tuberculosis.

https://dipot.ulb.ac.be/dspace/bitstream/2013/252776/1/Elsevier_236403.pdf

 

Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420

Blondiaux, N., Djaout, K., Trebosc, V., Kemmer, C., Wintjens, R., Wohlkonig, A., Antoine, R., Huot, L., Hot, D., Coscolla, M., Feldmann, J., Moune, M., Gagneux, S., Locht, C., Brodin, P., Gitzinger, M., Déprez, B., Willand, N., Baulard, A., Desroses, M., Frita, R., Flipo, M., Mathys, V., Soetaert, K., Kiass, M., & Vincent-Delorme, C. (2017). Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420. Science, 355(6330), 1206-1211. doi:10.1126/science.aag1006  

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

 

2016

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

Lim, M. Y. X., LaMonte, G., Lee, M. C. S., Reimer, C., Tan, B. H., Corey, V., Tjahjadi, B. F., Chua, A., Nachon, M., Wintjens, R., Gedeck, P., Malleret, B., Renia, L., Bonamy, G. M. C., Ho, P. C.-L., Yeung, B. K. S., Chow, E. D., Lim, L., Fidock, D. A., Diagana, T. T., Winzeler, E. A., & Bifani, P. (2016). UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes. Nature microbiology, 16166. doi:10.1038/nmicrobiol.2016.166  

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

 

Identification of compounds with anti-proliferative activity from the wood of Ficus elastica Roxb. ex Hornem. aerial roots.

Mbosso, E. J. T., Noundou, X. S., Nguemfo, E. L., Meyer, F., Djoukoue, A., Van Antwerpen, P., Ngouela, S., Tsamo, E., Mpondo, E. A. M. E., Vardamides, J. C., Azebaze, G. A. B., & Wintjens, R. (2016). Identification of compounds with anti-proliferative activity from the wood of Ficus elastica Roxb. ex Hornem. aerial roots. Fitoterapia, 112, 65. doi:10.1016/j.fitote.2016.05.002  

The natural products fromplants still remain an important source of pharmaceuticals. In the current study, methanolicwood extracts of Ficus elastica Roxb. ex Hornem. (Moraceae) aerial roots were screened for anti-cancer activity. Using bioassay-guided fractionation, three new compounds, elasticamide (1), elastiquinone (2) and ficusoside B (3), together with four known compounds, were isolated. The structures of the new compounds were established by means of extensive spectroscopic analyses (1D and 2D NMR, HR-ESI-MS, as well as IR and UV) and by comparison of their spectroscopic data with those reported for structurally related molecules. The isolated compounds were afterwards evaluated for their anti-proliferative effect on six human cancer cell lines (U373n and Hs683 glioblastoma, MCF7 and A549 NSCLC carcinoma, and B16F10 and SK-MEL-28 melanoma) using colorimetric MTT assay. Most notably, elastiquinone (2) showed cytotoxic activity with IC50 = 14 μM against B16F10 melanoma cells, whereas the peracetylated form of ficusoside B (3a) displayed the lowest IC50 value (11 μM) against U373n gliomacell lines. Elasticamide (1) and ficusoside B (3) exhibited aweak cytotoxicity with IC50 values ≥88 μM. The results of this investigation demonstrate that the wood of F. elastica aerial roots might be a potential source for identifying new compounds with potent anti-proliferative activity.

https://dipot.ulb.ac.be/dspace/bitstream/2013/230622/1/Elsevier_214249.pdf

 

Global molecular analysis and APOE mutations in a cohort of autosomal dominant hypercholesterolemia patients in France

Wintjens, R., Karsenty, A., Dubern, B., Carel, J.-C., Azar-Kolakez, A., Feillet, F., Labarthe, F., Gorsky, A. M. C., Horovitz, A., Tamarindi, C., Kieffer, P., Bozon, D., Lienhardt, A., Lascols, O., Di Filippo, M., Dufernez, F., Belabbas, K., Bou, F. F., Girardet, J. P., Tounian, P., Jolly, M., Boccara, F., & Cohen-Solal, A. (2016). Global molecular analysis and APOE mutations in a cohort of autosomal dominant hypercholesterolemia patients in France. Journal of lipid research, 57(3), 482-491. doi:10.1194/jlr.P055699  

Autosomal dominant hypercholesterolemia (ADH) is a human disorder characterized phenotypically by isolated high-cholesterol levels. Mutations in the low density lipoprotein receptor ( LDLR ), APOB, and proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) genes are well known to be associated with the disease. To characterize the genetic background associated with ADH in France, the three ADHassociated genes were sequenced in a cohort of 120 children and 109 adult patients. Fifty-one percent of the cohort had a possible deleterious variant in LDLR, 3.1% in APOB, and 1.7% in PCSK9. We identifi ed 18 new variants in LDLR and 2 in PCSK9. Three LDLR variants, including two newly identifi ed, were studied by minigene reporter assay confi rming the predicted effects on splicing. Additionally, as recently an in-frame deletion in the APOE gene was found to be linked to ADH, the sequencing of this latter gene was performed in patients without a deleterious variant in the three former genes. An APOE variant was identifi ed in three patients with isolated severe hypercholesterolemia giving a frequency of 1.3% in the cohort. Therefore, even though LDLR mutations are the major cause of ADH with a large mutation spectrum, APOE variants were found to be signifi-cantly associated with the disease. Furthermore, using structural analysis and modeling, the identifi ed APOE sequence changes were predicted to impact protein function.

https://dipot.ulb.ac.be/dspace/bitstream/2013/231147/5/doi_214774.pdf

 

Halogen bonding in multi-connected 1,2,2-triiodo-alkene involving geminal and/or vicinal iodines: A crystallographic and DFT study

Berger, G., Roeben, K., Soubhye, J., Wintjens, R., & Meyer, F. (2016). Halogen bonding in multi-connected 1,2,2-triiodo-alkene involving geminal and/or vicinal iodines: A crystallographic and DFT study. CrystEngComm, 18, 683. doi:10.1039/c5ce02230d  

Four halogen-bonded arrangements of (1,2,2 - triiodovinyl) benzene involving geminai and/or vicinal iodine atoms were studied both by X-ray diffraction and density functional theory (DFT). Crystallographic data show weaker XB-type interactions for the iodine atom geminal to the phenylring, which is corroborated by DFT-optimized structures of 1:1, 1:2 and 1:3 (1,2,2-triiodovinyl)benzene/pyridine complexes. In addition, a theoretical model reflects an interplay existing between these conjugated and multi-connected sites, highlighting the weakening of the halogen bonds when the number of partners is increased.

https://dipot.ulb.ac.be/dspace/bitstream/2013/226451/3/d.pdf

 

In vitro antimicrobial and anti-proliferative activities of plant extracts from Spathodea campanulata, Ficus bubu and Carica papaya

Mbosso, E. J. T., Nguedia, J. C. A., Meyer, F., Donfack, E. V., Ngouela, S., Tsamo, E., Adiogo, D. D., Azebaze, G. A. B., & Wintjens, R. (2016). In vitro antimicrobial and anti-proliferative activities of plant extracts from Spathodea campanulata, Ficus bubu and Carica papaya. Pharmaceutical biology. doi:10.3109/13880209.2015.1103273.  
https://dipot.ulb.ac.be/dspace/bitstream/2013/195432/3/c.pdf

 

2015

In vitro Evaluation of Antimicrobial and Antiproliferative Activities for Compounds Isolated from the Ficus bubu Warb. (Moraceae) Fruits: Chemotaxonomic Significance

Mbosso, E., Kamdem, L., Nguedia, J. C. A., Meyer, F., Ebelle, D., Lenta, B. N., Tchouankeu, J., Tsamo, E., Looze, Y., Adiogo, D. D. D., Azebaze, G. A. B., & Wintjens, R. (2015). In vitro Evaluation of Antimicrobial and Antiproliferative Activities for Compounds Isolated from the Ficus bubu Warb. (Moraceae) Fruits: Chemotaxonomic Significance. Drug delivery letters, 5(2), 122-131.  
https://dipot.ulb.ac.be/dspace/bitstream/2013/222857/3/a.pdf

 

Mutations in genes for the F<inf>420</inf> biosynthetic pathway and a nitroreductase enzyme are the primary resistance determinants in spontaneous in vitro-selected PA-824-resistant mutants of Mycobacterium tuberculosis

Hana L, H., Chua, A., Pramila, G., Lakshminarayana, S. S., Singhal, A., Mathema, B., Wintjens, R., & Bifani, P. (2015). Mutations in genes for the F<inf>420</inf> biosynthetic pathway and a nitroreductase enzyme are the primary resistance determinants in spontaneous in vitro-selected PA-824-resistant mutants of Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy, 59(9), 5316-5323. doi:10.1128/AAC.00308-15  

Alleviating the burden of tuberculosis (TB) requires an understanding of the genetic basis that determines the emergence of drug-resistant mutants. PA-824 (pretomanid) is a bicyclic nitroimidazole class compound presently undergoing the phase III STAND clinical trial, despite lacking identifiable genetic markers for drug-specific resistant Mycobacterium tuberculosis. In the present study, we aimed to characterize the genetic polymorphisms of spontaneously generated PA-824-resistant mutant strains by surveying drug metabolism genes for potential mutations. Of the 183 independently selected PA-824-resistant M. tuberculosis mutants, 83% harbored a single mutation in one of five nonessential genes associated with either PA-824 prodrug activation (ddn, 29%; fgd1, 7%) or the tangential F<inf>420</inf> biosynthetic pathway (fbiA, 19%; fbiB, 2%; fbiC, 26%). Crystal structure analysis indicated that identified mutations were specifically located within the protein catalytic domain that would hinder the activity of the enzymes required for prodrug activation. This systematic analysis conducted of genotypes resistant to PA-824 may contribute to future efforts in monitoring clinical strain susceptibility with this new drug therapy.

https://dipot.ulb.ac.be/dspace/bitstream/2013/218103/3/doi_201730.pdf

 

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32.

Boeckstaens, M., Merhi, A., Llinares, E., Van Vooren, P., Springael, J.-Y., Wintjens, R., & Marini, A. M. (2015). Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32. PLOS genetics, 11(7), e1005382. doi:10.1371/journal.pgen.1005382  

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.

https://dipot.ulb.ac.be/dspace/bitstream/2013/208422/4/doi_192049.pdf

 

Mutations in the F420 biosynthetic pathway and a nitroreductase enzyme are the primary resistance determinants in spontaneous in vitro selected PA-824 mutants of Mycobacterium tuberculosis.

Hana L, H., Chua, A., Pramila, G., Lakshminarayana, S. S., Singhal, A., Mathema, B., Wintjens, R., & Bifani, P. (2015). Mutations in the F420 biosynthetic pathway and a nitroreductase enzyme are the primary resistance determinants in spontaneous in vitro selected PA-824 mutants of Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy. doi:10.1128/AAC.00308-15  

Alleviating the burden of tuberculosis (TB) requires an understanding of the genetic basis that determines the emergence of drug resistant mutants. PA-824 (Pretonamid) is a bicyclic nitroimidazole class compound presently undergoing the phase III STAND clinical trial despite lacking identifiable genetic markers for drug-specific resistant Mycobacterium tuberculosis (Mtb). In the present study, we aimed to characterize the genetic polymorphisms of spontaneously generated PA-824 resistant mutant strains by surveying drug metabolism genes for potential mutations. Of the 183 independently selected PA-824 resistant Mtb mutants, 83% harbored a single mutation in one of five non-essential genes associated in either PA-824 pro-drug activation (ddn - 29%, fgd1 - 7%) or the tangential F420 biosynthetic pathway (fbiA - 19%, fbiB - 2%, fbiC - 26%). Crystal structure analysis indicated that identified mutations were specifically located within the protein catalytic domain that would hinder the activity of the enzymes required for pro-drug activation. This systematic analysis conducted of resistant genotypes to PA-824 may contribute to future efforts in monitoring clinical strain susceptibility with this new drug therapy.

 

Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS.

Dupré, E., Herrou, J., Lensink, M., Wintjens, R., Vagin, A., Lebedev, A., Crosson, S., Villeret, V., Locht, C., Antoine, R., & Jacob-Dubuisson, F. (2015). Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS. P L o S Pathogens, 11(3), e1004700. doi:10.1371/journal.ppat.1004700  

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

 

2014

Ligand Efficiency Driven Design of New Inhibitors of Mycobacterium tuberculosis Transcriptional Repressor EthR Using Fragment Growing, Merging, and Linking Approaches.

Villemagne, B., Flipo, M., Blondiaux, N., Crauste, C., Malaquin, S., Leroux, F., Piveteau, C., Villeret, V., Brodin, P., Villoutreix, B. O., Sperandio, O., Soror, S. H., Wohlkonig, A., Wintjens, R., Déprez, B., Baulard, A., & Willand, N. (2014). Ligand Efficiency Driven Design of New Inhibitors of Mycobacterium tuberculosis Transcriptional Repressor EthR Using Fragment Growing, Merging, and Linking Approaches. Journal of medicinal chemistry, 57(11), 4876-4888. doi:10.1021/jm500422b  

Tuberculosis remains a major cause of mortality and morbidity, killing each year more than one million people. Although the combined use of first line antibiotics (isoniazid, rifampicin, pyrazinamide, and ethambutol) is efficient to treat most patients, the rapid emergence of multidrug resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. Mycobacterial transcriptional repressor EthR is a key player in the control of second-line drugs bioactivation such as ethionamide and has been shown to impair the sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. As a way to identify new potent ligands of this protein, we have developed fragment-based approaches. In the current study, we combined surface plasmon resonance assay, X-ray crystallography, and ligand efficiency driven design for the rapid discovery and optimization of new chemotypes of EthR ligands starting from a fragment. The design, synthesis, and in vitro and ex vivo activities of these compounds will be discussed.

 

Sequence and conformation effects on ionization potential and charge distribution of homo-nucleobase stacks using M06-2X hybrid density functional theory calculations.

Rooman, M., & Wintjens, R. (2014). Sequence and conformation effects on ionization potential and charge distribution of homo-nucleobase stacks using M06-2X hybrid density functional theory calculations. Journal of biomolecular structure & dynamics, 32(4), 532-545. doi:10.1080/07391102.2013.783508  

DNA is subject to oxidative damage due to radiation or by-products of cellular metabolism, thereby creating electron holes that migrate along the DNA stacks. A systematic computational analysis of the dependence of the electronic properties of nucleobase stacks on sequence and conformation was performed here, on the basis of single- and double-stranded homo-nucleobase stacks of 1-10 bases or 1-8 base pairs in standard A-, B-, and Z-conformation. First, several levels of theory were tested for calculating the vertical ionization potentials of individual nucleobases; the M06-2X/6-31G* hybrid density functional theory method was selected by comparison with experimental data. Next, the vertical ionization potential, and the Mulliken charge and spin density distributions were calculated and considered on all nucleobase stacks. We found that (1) the ionization potential decreases with the number of bases, the lowest being reached by Gua≡Cyt tracts; (2) the association of two single strands into a double-stranded tract lowers the ionization potential significantly (3) differences in ionization potential due to sequence variation are roughly three times larger than those due to conformational modifications. The charge and spin density distributions were found (1) to be located toward the 5'-end for single-stranded Gua-stacks and toward the 3'-end for Cyt-stacks and basically delocalized over all bases for Ade- and Thy-stacks; (2) the association into double-stranded tracts empties the Cyt- and Thy-strands of most of the charge and all the spin density and concentrates them on the Gua- and Ade-strands. The possible biological implications of these results for transcription are discussed.

https://dipot.ulb.ac.be/dspace/bitstream/2013/171917/4/PMC3919198.pdf

 

Interplay between halogen bonding and lone pair-Pi interactions: A computational and crystal packing study

Berger, G., Soubhye, J., Van Der Lee, A., Vande Velde, C., Wintjens, R., Dubois, P., Clement, S., & Meyer, F. (2014). Interplay between halogen bonding and lone pair-Pi interactions: A computational and crystal packing study. ChemPlusChem, 79(4), 552-558. doi:10.1002/cplu.201400005  

The supramolecular organization of modified thiophenes resulting from N⋯I halogen bonding (XB) has been studied. X-ray diffraction analyses of two polymorphs of the same supramolecular complex showed an arrangement controlled by halogen-bonded pyridyl and tetrafluoroiodobenzene rings, one case with and the other without the presence of two different lone pair (lp)⋯π interactions, namely O⋯pyridyl and I⋯ tetrafluoroiodobenzene contacts. To shed light on the interplay between these interactions, quantum mechanical calculations were performed at the ωB97X-D/6-31+G(d,p) level on both systems in the gas phase. The longer N⋯I distance observed in the crystal structure without lp⋯π interactions was also corroborated by dispersion-corrected DFT. These results represent the first case in which a synergy between lp⋯π interactions and XB is observed experimentally and confirmed by calculations. Getting organized: X-ray diffraction analyses of two polymorphs of a supramolecular thiophene complex show an arrangement controlled by N⋯I halogen bonding (XB), one case with and the other without the presence of two different lone pair (lp)⋯π interactions, namely O⋯pyridyl and I⋯tetrafluoroiodobenzene contacts (see figure). Quantum mechanical calculations confirm the interplay between concomitant XB and lp⋯π interactions. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

https://dipot.ulb.ac.be/dspace/bitstream/2013/179459/3/BergerChemPlusChem.pdf

 

2013

High-resolution structure of a papaya plant-defence barwin-like protein solved by in-house sulfur-SAD phasing

Huet, J., Mbosso, E. J. T., Soror, S. H., Meyer, F., Looze, Y., Wintjens, R., & Wohlkonig, A. (2013). High-resolution structure of a papaya plant-defence barwin-like protein solved by in-house sulfur-SAD phasing. Acta crystallographica. Section D, Biological crystallography, 69(10), 2017. doi:10.1107/S0907444913018015  

The first crystal structure of a barwin-like protein, named carwin, has been determined at high resolution by single-wavelength anomalous diffraction (SAD) phasing using the six intrinsic S atoms present in the protein. The barwin-like protein was purified from Carica papaya latex and crystallized in the orthorhombic space group P212121. Using in-house Cu Kα X-ray radiation, 16 cumulative diffraction data sets were acquired to increase the signal-to-noise level and thereby the anomalous scattering signal. A sequence-database search on the papaya genome identified two carwin isoforms of 122 residues in length, both containing six S atoms that yield an estimated Bijvoet ratio of 0.93% at 1.54 Å wavelength. A systematic analysis of data quality and redundancy was performed to assess the capacity to locate the S atoms and to phase the data. It was observed that the crystal decay was low during data collection and that successful S-SAD phasing could be obtained with a relatively low data multiplicity of about 7. Using a synchrotron source, high-resolution data (1 Å) were collected from two different crystal forms of the papaya latex carwin. The refined structures showed a central β-barrel of six strands surrounded by several α-helices and loops. The β-barrel of carwin appears to be a common structural module that is shared within several other unrelated proteins. Finally, the possible biological function of the protein is discussed.

https://dipot.ulb.ac.be/dspace/bitstream/2013/149427/3/149427.pdf

 

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis.

Pethe, K., Bifani, P., Jang, J., Kang, S., Park, S., Ahn, S., Jiricek, J., Jung, J., Jeon, H. K., Cechetto, J., Christophe, T., Lee, H., Kempf, M., Jackson, M., Lenaerts, A. J., Pham, H. O., Jones, V., Seo, M. J., Kim, Y. M., Seo, M., Seo, J. J., Park, D., Ko, Y., Choi, I., Kim, R., Kim, S. Y., Lim, S., Yim, S.-A., Nam, J., Kang, H., Kwon, H., Oh, C.-T., Cho, Y., Jang, Y., Kim, J., Chua, A., Tan, B. H., Nanjundappa, M. B., Rao, S. P. S., Barnes, W. S., Wintjens, R., Walker, J. R., Alonso, S., Lee, S., Kim, J., Oh, S., Oh, T., Nehrbass, U., Han, S.-J., No, Z., Lee, J., Brodin, P., Cho, S.-N., Nam, K., & Kim, J. (2013). Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nature medicine, 19(9), 1157-1160. doi:10.1038/nm.3262  

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

 

Chemical constituents from Glyphaea brevis and Monodora myristica: chemotaxonomic significance.

Mbosso, E. J. T., Wintjens, R., Lenta, B. N., Ngouela, S., Rohmer, M., & Tsamo, E. (2013). Chemical constituents from Glyphaea brevis and Monodora myristica: chemotaxonomic significance. Chemistry & biodiversity, 10(2), 224-232. doi:10.1002/cbdv.201100378  

A chemical investigation of the Glyphaea brevis leaves and of the Monodora myristica fruits led to the identification of thirteen compounds, seven linear long-chain aliphatic compounds, 1, 2, 4, 6, and 9-11, three steroids, 3a, 3b, and 7, two triterpenes, 5a and 5b, and one polyol, 8. The compounds 2 and 8, previously mentioned in the literature, are here characterized by their complete (1)H- and (13)C-NMR assignments. This is the first report of a full NMR assignment for linear fatty acid esters of aliphatic primary alcohols and for meso-erythritol. Compound 5b and 8 were isolated for the first time from plant extracts of the Tiliaceae family, and compounds 9-11 from the Annonaceae plant family. Our results constitute the basis for further chemotaxonomic studies on the two species.

https://dipot.ulb.ac.be/dspace/bitstream/2013/145494/3/145494.pdf

 

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay.

Deschuyteneer, A., Boeckstaens, M., De Mees, C., Van Vooren, P., Wintjens, R., & Marini, A. M. (2013). SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay. PloS one, 8(8), e71092. doi:10.1371/journal.pone.0071092  

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCG(R202C) may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.

https://dipot.ulb.ac.be/dspace/bitstream/2013/149413/4/doi_133572.pdf

 

2012

Systematic analysis of pyrazinamide-resistant spontaneous mutants and clinical isolates of Mycobacterium tuberculosis.

Stoffels, K., Mathys, V., Fauville, M., Wintjens, R., & Bifani, P. (2012). Systematic analysis of pyrazinamide-resistant spontaneous mutants and clinical isolates of Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy, 56(10), 5186-5193. doi:10.1128/AAC.05385-11  

Pyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistent Mycobacterium tuberculosis bacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (PZA(r)) spontaneous mutants. The frequency of acquired resistance to PZA was determined to be 10(-5) bacilli in vitro. Selection at a lower concentration of PZA yielded a significantly larger number of spontaneous mutants. The methodical approach employed allowed for determination of the frequency of the PZA(r) phenotype correlated with mutations in the pncA gene, which was 87.5% for the laboratory-selected spontaneous mutants examined in this study. As elucidated by structural analysis, most of the identified mutations were foreseen to affect protein activity through either alteration of an active site residue or destabilization of protein structure, indicating some preferential mutation site rather than random scattering. Twelve percent of the PZA(r) mutants did not have a pncA mutation, strongly indicating the presence of at least one other mechanism(s) of PZA(r).

https://dipot.ulb.ac.be/dspace/bitstream/2013/145495/4/PMC3457413.pdf

 

Ceramide, cerebroside and triterpenoid saponin from the bark of aerial roots of Ficus elastica (Moraceae).

Mbosso, E. J. T., Nguedia, J. C. A., Meyer, F., Lenta, B. N., Ngouela, S., Lallemand, B., Mathieu, V., Van Antwerpen, P., Njunda, A. L., Adiogo, D. D., Tsamo, E., Looze, Y., Kiss, R., & Wintjens, R. (2012). Ceramide, cerebroside and triterpenoid saponin from the bark of aerial roots of Ficus elastica (Moraceae). Phytochemistry, 83, 95-103. doi:10.1016/j.phytochem.2012.07.010  

Three compounds, ficusamide (1), ficusoside (2) and elasticoside (3), were isolated from the bark of aerial roots of Ficus elastica (Moraceae), together with nine known compounds, including four triterpenes, three steroids and two aliphatic linear alcohols. The chemical structures of the three compounds were established by extensive 1D and 2D NMR spectroscopy, mass spectrometry and by comparison with published data. The growth inhibitory effect of the crude extract and isolated compounds was evaluated against several microorganisms and fungi. The cytotoxicity against human cancer cell lines was also assessed. Ficusamide (1) displayed a moderate in vitro growth inhibitory activity against the human A549 lung cancer cell line and a strong activity against Staphylococcus saprophyticus, while elasticoside (3) showed a potent activity on Enterococcus faecalis.

https://dipot.ulb.ac.be/dspace/bitstream/2013/128999/1/Phytochemistry.pdf

 

Discovery of novel N-phenylphenoxyacetamide derivatives as EthR inhibitors and ethionamide boosters by combining high-throughput screening and synthesis.

Flipo, M., Willand, N., Lecat-Guillet, N., Hounsou, C., Desroses, M., Leroux, F., Lens, Z., Villeret, V., Wohlkonig, A., Wintjens, R., Christophe, T., Kyoung Jeon, H., Locht, C., Brodin, P., Baulard, A., & Déprez, B. (2012). Discovery of novel N-phenylphenoxyacetamide derivatives as EthR inhibitors and ethionamide boosters by combining high-throughput screening and synthesis. Journal of medicinal chemistry, 55(14), 6391-6402. doi:10.1021/jm300377g  

In this paper, we describe the screening of a 14640-compound library using a novel whole mycobacteria phenotypic assay to discover inhibitors of EthR, a transcriptional repressor implicated in the innate resistance of Mycobacterium tuberculosis to the second-line antituberculosis drug ethionamide. From this screening a new chemical family of EthR inhibitors bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent ethionamide boosters on M. tuberculosis -infected macrophages. Finally, the cocrystallization of the best optimized analogue with EthR revealed an unexpected reorientation of the ligand in the binding pocket.

 

Evolutionary changes in antimicrobial resistance of invasive Neisseria meningitidis isolates in Belgium from 2000 to 2010: increasing prevalence of penicillin nonsusceptibility.

Bertrand, S., Carion, F., Wintjens, R., Mathys, V., & Vanhoof, R. (2012). Evolutionary changes in antimicrobial resistance of invasive Neisseria meningitidis isolates in Belgium from 2000 to 2010: increasing prevalence of penicillin nonsusceptibility. Antimicrobial agents and chemotherapy, 56(5), 2268-2272. doi:10.1128/AAC.06310-11  

This study was conducted to evaluate the evolution of the antimicrobial susceptibility of Neisseria meningitidis causing invasive diseases in Belgium in the period of January 2000 to December 2010. A total of 1,933 cases of N. meningitidis from invasive infections were analyzed by antimicrobial susceptibility testing at the Belgian Meningococcal Reference Centre. The majority of strains were susceptible to antibiotics that are currently used for the treatment and prophylaxis of meningococcal disease, but the prevalence of clinical isolates with reduced susceptibility to penicillin increased over the years. The phenotyping, genotyping, and determination of MICs of penicillin G were performed. The systematic shift of the curves toward higher penicillin MICs in the susceptible population indicated that this population became less sensitive to penicillin in this period. A 402-bp DNA fragment in the 3' end of penA was sequenced for the 296 nonsusceptible meningococcal strains isolated between 2000 and 2010 to examine the genetic diversity and evolution of their penA gene. In conclusion, the data obtained in our study support the statement that the position of penicillin G as a first choice in the treatment of invasive meningococcal diseases in Belgium should be reexamined. Despite an important number of isolates displaying a reduced susceptibility to penicillin, at present the expanded-spectrum cephalosporins, such as ceftriaxone, are not affected. The follow-up of the evolutionary changes in antimicrobial resistance has also proved to be essential for the recommendation of an appropriate antimicrobial treatment for invasive meningococcal diseases.

https://dipot.ulb.ac.be/dspace/bitstream/2013/145496/4/PMC3346653.pdf

 

Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug design to solve pharmacokinetic issues in a series of potent 1,2,4-oxadiazole EthR inhibitors.

Flipo, M., Desroses, M., Lecat-Guillet, N., Villemagne, B., Blondiaux, N., Leroux, F., Piveteau, C., Mathys, V., Flament, M.-P., Siepmann, J., Villeret, V., Wohlkonig, A., Wintjens, R., Soror, S. H., Christophe, T., Jeon, H. K., Locht, C., Brodin, P., Déprez, B., Baulard, A., & Willand, N. (2012). Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug design to solve pharmacokinetic issues in a series of potent 1,2,4-oxadiazole EthR inhibitors. Journal of medicinal chemistry, 55(1), 68-83. doi:10.1021/jm200825u  

Mycobacterial transcriptional repressor EthR controls the expression of EthA, the bacterial monooxygenase activating ethionamide, and is thus largely responsible for the low sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. We recently reported structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors leading to the discovery of potent ethionamide boosters. Despite high metabolic stability, pharmacokinetic evaluation revealed poor mice exposure; therefore, a second phase of optimization was required. Herein a structure-property relationship study is reported according to the replacement of the two aromatic heterocycles: 2-thienyl and 1,2,4-oxadiazolyl moieties. This work was done using a combination of structure-based drug design and in vitro/ex vivo evaluations of ethionamide boosters on the targeted protein EthR and on the human pathogen Mycobacterium tuberculosis. Thanks to this process, we identified compound 42 (BDM41906), which displays improved efficacy in addition to high exposure to mice after oral administration.

 

2011

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

Carette, X., Blondiaux, N., Willery, E., Hoos, S., Lecat-Guillet, N., Lens, Z., Wohlkonig, A., Wintjens, R., Soror, S. H., Frénois, F., Dirié, B., Villeret, V., England, P., Lippens, G., Déprez, B., Locht, C., Willand, N., & Baulard, A. (2011). Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands. Nucleic acids research. doi:10.1093/nar/gkr1113  

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors.

 

Hypothesis/review: the structural basis of sweetness perception of sweet-tasting plant proteins can be deduced from sequence analysis.

Wintjens, R., Viet, T. M. V. N., Mbosso, E., & Huet, J. (2011). Hypothesis/review: the structural basis of sweetness perception of sweet-tasting plant proteins can be deduced from sequence analysis. Plant science, 181(4), 347-354. doi:10.1016/j.plantsci.2011.06.009  

Human perception of sweetness, behind the felt pleasure, is thought to play a role as an indicator of energy density of foods. For humans, only a small number of plant proteins taste sweet. As non-caloric sweeteners, these plant proteins have attracted attention as candidates for the control of obesity, oral health and diabetic management. Significant advances have been made in the characterization of the sweet-tasting plant proteins, as well as their binding interactions with the appropriate receptors. The elucidation of sweet-taste receptor gene sequences represents an important step towards the understanding of sweet taste perception. However, many questions on the molecular basis of sweet-taste elicitation by plant proteins remain unanswered. In particular, why homologues of these proteins do not elicit similar responses? This question is discussed in this report, on the basis of available sequences and structures of sweet-tasting proteins, as well as of sweetness-sensing receptors. A simple procedure based on sequence comparisons between sweet-tasting protein and its homologous counterparts was proposed to identify critical residues for sweetness elicitation. The open question on the physiological function of sweet-tasting plant proteins is also considered. In particular, this review leads us to suggest that sweet-tasting proteins may interact with taste receptor in a serendipity manner.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111271/1/Elsevier_91518.pdf

 

Conformations consistent with charge migration observed in DNA and RNA X-ray structures

Rooman, M., Cauet, E., Liévin, J., & Wintjens, R. (2011). Conformations consistent with charge migration observed in DNA and RNA X-ray structures. Journal of biomolecular structure & dynamics, 28, 949-954.  

 

Ester prodrugs of ciprofloxacin as DNA-gyrase inhibitors

Dubar, F., Wintjens, R., Martins-Duarte, E., Vommaro, R., de Souza, W., Dive, D., Pierrot, C., Pradines, B., Wohlkonig, A., Khalife, J., & Biot, C. (2011). Ester prodrugs of ciprofloxacin as DNA-gyrase inhibitors: synthesis, antiparasitic evaluation and docking studies. MedChemComm, 2, 430-435. doi:10.1039/c1md00022e  

Novel ester prodrugs of ciprofloxacin were synthesized, and tested for their antimalarial and antitoxoplasma activity. These new compounds proved to be extremely efficient against these parasites. Molecular modeling and computational calculations were used to understand the mechanisms of action of these drugs. © The Royal Society of Chemistry 2011.

 

2010

Periplasmic domain of the sensor-kinase BvgS reveals a new paradigm for the Venus flytrap mechanism.

Herrou, J., Bompard, C., Wintjens, R., Dupré, E., Willery, E., Villeret, V., Locht, C., Antoine, R., & Jacob-Dubuisson, F. (2010). Periplasmic domain of the sensor-kinase BvgS reveals a new paradigm for the Venus flytrap mechanism. Proceedings of the National Academy of Sciences of the United States of America, 107(40), 17351-17355. doi:10.1073/pnas.1006267107  

Two-component sensory transduction systems control important bacterial programs. In Bordetella pertussis, expression of the virulence regulon is controlled by the unorthodox BvgAS two-component system. BvgS is the prototype of a family of sensor-kinases that harbor periplasmic domains homologous to bacterial solute-binding proteins. Although BvgAS is active under laboratory conditions, no activating signal has been identified, only negative modulators. Here we show that the second periplasmic domain of BvgS interacts with modulators and adopts a Venus flytrap (VFT) fold. X-ray crystallography reveals that the two lobes of VFT2 delimitate a ligand-binding cavity enclosing fortuitous ligands. Most substitutions of putative ligand-binding residues in the VFT2 cavity keep BvgS active, and alteration of the cavity's electrostatic potential affects responsiveness to modulation. The crystal structure of this VFT2 variant conferring constitutive kinase activity to BvgS shows a closed cavity with another nonspecific ligand. Thus, VFT2 is closed and active without a specific agonist ligand, in contrast to typical VFTs. Modulators are antagonists of VFT2 that interrupt signaling. BvgAS is active for most of the B. pertussis infectious cycle, consistent with the proposed mechanism.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111264/4/PMC2951421.pdf

 

Erratum: Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya (Phytochemistry (2009) 70 (970-978))

Looze, Y., Boussard, P., Huet, J., Wintjens, R., Vandenbusche, G., Raussens, V., & Azarkan, M. (2010). Erratum: Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya (Phytochemistry (2009) 70 (970-978)). Phytochemistry, 71(10), 1181. doi:10.1016/j.phytochem.2010.04.010  
https://dipot.ulb.ac.be/dspace/bitstream/2013/203893/1/Elsevier_187520.pdf

 

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs.

Wohlkonig, A., Huet, J., Looze, Y., & Wintjens, R. (2010). Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs. PloS one, 5(11), 15388. doi:10.1371/journal.pone.0015388  

Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111209/4/doi_91457.pdf

 

2009

Molecular genetics of para-aminosalicylic acid resistance in clinical isolates and spontaneous mutants of Mycobacterium tuberculosis.

Mathys, V., Wintjens, R., Lefèvre, P., Bertout, J., Singhal, A., Kiass, M., Kurepina, N., Wang, X.-J., Mathema, B., Baulard, A., Kreiswirth, B. N., & Bifani, J. P. (2009). Molecular genetics of para-aminosalicylic acid resistance in clinical isolates and spontaneous mutants of Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy, 53(5), 2100-2109. doi:10.1128/AAC.01197-08  

The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111261/4/PMC2681553.pdf

 

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya

Looze, Y., Boussard, P., Huet, J., Vandenbussche, G., Raussens, V., & Wintjens, R. (2009). Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya. Phytochemistry, 70(8), 970-978. doi:10.1016/j.phytochem.2009.05.005  

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a Δ Gwater0 of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed. © 2009 Elsevier Ltd. All rights reserved.

https://dipot.ulb.ac.be/dspace/bitstream/2013/171088/1/Elsevier_154718.pdf

 

2008

X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases.

Huet, J., Rucktooa, P., Clantin, B., Azarkan, M., Looze, Y., Villeret, V., & Wintjens, R. (2008). X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases. Biochemistry, 47(32), 8283-8291. doi:10.1021/bi800655u  

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.

 

Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex.

Huet, J., Azarkan, M., Looze, Y., Villeret, V., & Wintjens, R. (2008). Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex. Acta Crystallographica. Section F: Structural Biology and Crystallization Communications Online, 64(Pt 5), 371-374. doi:10.1107/S1744309108007823  

A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 A, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-D-glucosamine, the monomer resulting from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P2(1), with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 A, beta = 95.33 degrees and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 A. Structure refinement is currently in progress.

https://dipot.ulb.ac.be/dspace/bitstream/2013/110375/4/PMC2376395.pdf

 

Molecular characterization of iron-containing superoxide dismutases in the heterotrophic dinoflagellate Crypthecodinium cohnii.

Dufernez, F., Derelle, E., Noël, C., Sanciu, G., Mantini, C., Dive, D., Soyer-Gobillard, M.-O., Capron, M., Pierce, R., Wintjens, R., Guillebault, D., & Viscogliosi, E. (2008). Molecular characterization of iron-containing superoxide dismutases in the heterotrophic dinoflagellate Crypthecodinium cohnii. Protist, 159(2), 223-238. doi:10.1016/j.protis.2007.11.005  

Superoxide dismutases (SODs) are a family of antioxidant enzymes that catalyse the degradation of toxic superoxide radicals in obligate and facultative aerobic organisms. Here, we report the presence of a multi-copy gene family encoding SODs in the heterotrophic dinoflagellate Crypthecodinium cohnii. All the genes identified (sod1 to sod17) have been cloned and sequenced, and shown to encode potentially functional dimeric iron-containing SOD isozymes. Our data revealed a considerable molecular heterogeneity of this enzyme in C. cohnii at both genomic and transcriptional levels. The C. cohnii SOD1, overexpressed in Escherichia coli, was active and its structure obtained by homology modeling using X-ray crystal structures of homologues exhibited the typical fold of dimeric FeSODs. Phylogenetic studies including 110 other dimeric FeSODs and closely related cambialistic dimeric SOD sequences showed that the C. cohnii SODs form a monophyletic group and have all been acquired by the same event of horizontal gene transfer. It also revealed a dichotomy within the C. cohnii SOD sequences that could be explained by an ancestral sod gene duplication followed by subsequent gene duplications within each of the two groups. Enzyme assays of SOD activity indicated the presence of two FeSOD activities in C. cohnii cell lysate whereas MnSOD and Cu/ZnSOD were not detected. These activities contrasted with the SOD repertoire previously characterized in photosynthetic dinoflagellates. To explain these differences, a hypothetical evolutionary scenario is proposed that suggests gains and losses of sod genes in dinoflagellates.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111258/1/Elsevier_91505.pdf

 

Mn/Fe superoxide dismutase interaction fingerprints and prediction of oligomerization and metal cofactor from sequence

Wintjens, R., Gilis, D., & Rooman, M. (2008). Mn/Fe superoxide dismutase interaction fingerprints and prediction of oligomerization and metal cofactor from sequence. Proteins, 70(4), 1564-1577. doi:10.1002/prot.21650  

Fe- and Mn-containing superoxide dismutase (sod) enzymes are closely related and similar in both amino acid sequence and structure, but differ in their mode of oligomerization and in their specificity for the Fe or Mn cofactor. The goal of the present work is to identify and analyze the sequence and structure characteristics that ensure the cofactor specificities and the oligomerization modes. For that purpose, 374 sod sequences and 17 sod crystal structures were collected and aligned. These alignments were searched for residues and interresidue interactions that are conserved within the whole sod family, or alternatively, that are specific to a given sod subfamily sharing common characteristics. This led us to define key residues and interresidue interaction fingerprints in each subfamily. The comparison of these fingerprints allows, on a rational basis, the design of mutants likely to modulate the activity and/or specificity of the target sod, in good agreement with the available experimental results on known mutants. The key residues and interaction fingerprints are furthermore used to predict if a novel sequence corresponds to a sod enzyme, and if so, what type of sod it is. The predictions of this fingerprint method reach much higher scores and present much more discriminative power than the commonly used method that uses pairwise sequence comparisons. © 2007 Wiley-Liss, Inc.

https://dipot.ulb.ac.be/dspace/bitstream/2013/60555/1/2008_Wintjens_Proteins.pdfhttps://dipot.ulb.ac.be/dspace/bitstream/2013/60555/4/60555.pdf

 

SODa: an Mn/Fe superoxide dismutase prediction and design server

Kwasigroch, J.-M., Wintjens, R., Gilis, D., & Rooman, M. (2008). SODa: an Mn/Fe superoxide dismutase prediction and design server. BMC bioinformatics, 9, 257. doi:10.1186/1471-2105-9-257  

Background: Superoxide dismutases (SODs) are ubiquitous metalloenzymes that play an important role in the defense of aerobic organisms against oxidative stress, by converting reactive oxygen species into nontoxic molecules. We focus here on the SOD family that uses Fe or Mn as cofactor. Results: The SODa webtool http://babylone.ulb.ac.be/soda predicts if a target sequence corresponds to an Fe/Mn SOD. If so, it predicts the metal ion specificity (Fe, Mn or cambialistic) and the oligomerization mode (dimer or tetramer) of the target. In addition, SODa proposes a list of residue substitutions likely to improve the predicted preferences for the metal cofactor and oligomerization mode. The method is based on residue fingerprints, consisting of residues conserved in SOD sequences or typical of SOD subgroups, and of interaction fingerprints, containing residue pairs that are in contact in SOD structures. Conclusion: SODa is shown to outperform and to be more discriminative than traditional techniques based on pairwise sequence alignments. Moreover, the fact that it proposes selected mutations makes it a valuable tool for rational protein design. © 2008 Kwasigroch et al; licensee BioMed Central Ltd.

https://dipot.ulb.ac.be/dspace/bitstream/2013/60543/4/doi_36882.pdf

 

2007

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the alpha-chymotrypsin-proflavin interaction

Bruylants, G., Wintjens, R., Looze, Y., Redfield, C., & Bartik, K. (2007). Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the alpha-chymotrypsin-proflavin interaction. European biophysics journal, 37(1), 11-18. doi:10.1007/s00249-007-0148-0  

Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K(obs), DeltaH(obs)(0) ). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between alpha-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between alpha-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria.

https://dipot.ulb.ac.be/dspace/bitstream/2013/54640/3/2007-Bruylants-EurBiophysJ.pdf

 

2006

Structural characterization of two papaya chitinases, a family GH19 of glycosyl hydrolases.

Huet, J., Wyckmans, J., Wintjens, R., Boussard, P., Raussens, V., Vandenbussche, G., Ruysschaert, J. M., Azarkan, M., & Looze, Y. (2006). Structural characterization of two papaya chitinases, a family GH19 of glycosyl hydrolases. Cellular and molecular life sciences, 63(24), 3042-3054. doi:10.1007/s00018-006-6320-3  

Two chitinases, able to use tetra-N-acetylglucosamine, chitin and chitosan as substrates, were characterized after purification from Carica papaya latex. The complete amino acid sequence of the major form and about 40% of the minor one were determined through proteolytic digestions and mass spectroscopy analysis. Sequencing demonstrated that both papaya chitinases are members of the family 19 of glycosyl hydrolases (GH19). Based on the known 3-D structures of other members of family GH19, it was expected that papaya chitinases would adopt all-alpha structures. However, circular dichroism and infrared spectroscopy indicated, for the papaya chitinases, a content of 15-20% of extended structures besides the expected 40% of alpha helices. Since the fully sequenced papaya chitinase contains a large number of proline residues the possibility that papaya chitinase contains polyproline II stretches was examined in the context of their resistance against proteolytic degradation.

 

Exploring the molecular function of PIN1 by nuclear magnetic resonance.

Landrieu, I., Smet, C., Wieruszeski, J.-M., Sambo, A.-V., Wintjens, R., Buée, L., & Lippens, G. (2006). Exploring the molecular function of PIN1 by nuclear magnetic resonance. Current protein and peptide science, 7(3), 179-194.  

PIN1 participates in the regulation of a number of signalling pathways in the cell involving protein phosphorylation/dephosphorylation. Its role seems to be an essential control level in addition to the protein phosphorylation by proline-directed kinases. Its cellular function includes regulation of the cell cycle by interaction with phosphorylated mitotic proteins such as Cdc25 and transcription factors such as p53. PIN1 was shown to be involved in the malignant transformation of cells in breast cancer, by up regulation of cyclinD1 and is thought to be involved in the development of the AD by regulating the function of phosphorylated Tau. We propose here to discuss the molecular function of PIN1 at the atomic level based on data from the recent literature and our own results obtained by the technique of Nuclear Magnetic Resonance. PIN1 specifically interacts with pThr/pSer-Pro motifs and is constituted by two domains: a WW N-terminal domain that binds pThr/pSer-Pro epitopes and a prolyl cis/trans isomerase C-terminal catalytic domain. An exception to this organisation is found in the plant PIN1 homologous enzymes, like PIN1At from Arabidopsis thaliana, that are constituted of the sole catalytic domain. The molecular function of PIN1, binding to and isomerization of pThr/pSer-Pro bonds, are thought to lead to several functional consequences. In a first mode of action, exemplified by its competition with the CKS protein, the interaction with PIN1 prevents interaction with other regulatory proteins, like ubiquitin-ligases that lead to degradation pathways. In a second mode of action, the idea is largely accepted that the local isomerization modifies the global conformation of the protein substrate and hence its intrinsic activity, although this has never been directly demonstrated. Finally, isomerization catalysis is thought to regulate the (de)phosphorylation of specific pThr/pSer-Pro motifs, exemplified by the stimulation of the dephosphorylation of pThr231 of Tau by the PP2A phosphatase.

 

Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases.

Wintjens, R., Belrhali, H., Clantin, B., Azarkan, M., Bompard, C., Volant-Baeyens, D., Looze, Y., & Villeret, V. (2006). Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases. Journal of Molecular Biology, 357(2), 457-470. doi:10.1016/j.jmb.2005.12.029  

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7A resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed beta-propeller, with two additional alpha-helices and one beta hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded beta-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the beta-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase.

 

Structural characterization of the papaya cysteine proteinases at low pH.

Huet, J., Looze, Y., Bartik, K., Raussens, V., Wintjens, R., & Boussard, P. (2006). Structural characterization of the papaya cysteine proteinases at low pH. Biochemical and biophysical research communications, 341(2), 620-626. doi:10.1016/j.bbrc.2005.12.210  

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.

 

The presence of four iron-containing superoxide dismutase isozymes in trypanosomatidae: characterization, subcellular localization, and phylogenetic origin in Trypanosoma brucei.

Dufernez, F., Yernaux, C., Gerbod, D., Noël, C., Chauvenet, M., Wintjens, R., Edgcomb, V. P., Capron, M., Opperdoes, F. R., & Viscogliosi, E. (2006). The presence of four iron-containing superoxide dismutase isozymes in trypanosomatidae: characterization, subcellular localization, and phylogenetic origin in Trypanosoma brucei. Free radical biology & medicine, 40(2), 210-225. doi:10.1016/j.freeradbiomed.2005.06.021  

Metalloenzymes such as the superoxide dismutases (SODs) form part of a defense mechanism that helps protect obligate and facultative aerobic organisms from oxygen toxicity and damage. Here, we report the presence in the trypanosomatid genomes of four SOD genes: soda, sodb1, sodb2, and a newly identified sodc. All four genes of Trypanosoma brucei have been cloned (Tbsods), sequenced, and overexpressed in Escherichia coli and shown to encode active dimeric FeSOD isozymes. Homology modeling of the structures of all four enzymes using available X-ray crystal structures of homologs showed that the four TbSOD structures were nearly identical. Subcellular localization using GFP-fusion proteins in procyclic insect trypomastigotes shows that TbSODB1 is mainly cytosolic, with a minor glycosomal component, TbSODB2 is mainly glycosomal with some activity in the cytosol, and TbSODA and TbSODC are both mitochondrial isozymes. Phylogenetic studies of all available trypanosomatid SODs and 106 dimeric FeSODs and closely related cambialistic dimeric SOD sequences suggest that the trypanosomatid SODs have all been acquired by more than one event of horizontal gene transfer, followed by events of gene duplication.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111250/1/Elsevier_91497.pdf

 

2005

Histidine-aromatic interactions in proteins and protein-ligand complexes

Cauet, E., Rooman, M., Wintjens, R., Liévin, J., & Biot, C. (2005). Histidine-aromatic interactions in proteins and protein-ligand complexes: quantum chemical study of X-ray and model structures. Journal of chemical theory and computation, 1(3), 472-483. doi:10.1021/ct049875k  

His-aromatic complexes, with the His located above the aromatic plane, are stabilized by π-π, δ +-π and/or cation-π interactions according to whether the His is neutral or protonated and the partners are in stacked or T-shape conformations. Here we attempt to probe the relative strength of these interactions as a function of the geometry and protonation state, in gas phase, in water and protein-like environments (acetone, THF and CCl 4), by means of quantum chemistry calculations performed up to second order of the Møller-Plesset pertubation theory. Two sets of conformations are considered for that purpose. The first set contains 89 interactions between His and Phe, Tyr, Trp, or Ade, observed in X-ray structures of proteins and protein-ligand complexes. The second set contains model structures obtained by moving an imidazolium/imida-zole moiety above a benzene ring or an adenine moiety. We found that the protonated complexes are much more stable than the neutral ones in gas phase. This higher stability is due to the electrostatic contributions, the electron correlation contributions being equally important in the two forms. Thus, π-π and δ +-π interactions present essentially favorable electron correlation energy terms, whereas cation-π interactions feature in addition favorable electrostatic energies. The pro-tonated complexes remain more stable than the neutral ones in protein-like environments, but the difference is drastically reduced. Furthermore, the T-shape conformation is undoubtedly more favorable than the stacked one in gas phase. This advantage decreases in the solvents, and the stacked conformation becomes even slightly more favorable in water. The frequent occurrence of His-aromatic interactions in catalytic sites, at protein-DNA or protein-ligand interfaces and in 3D domain swapping proteins emphasize their importance in biological processes. © 2005 American Chemical Society.

 

Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex.

Azarkan, M., Clantin, B., Bompard, C., Belrhali, H., Volant-Baeyens, D., Looze, Y., Villeret, V., & Wintjens, R. (2005). Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex. Acta Crystallographica. Section F: Structural Biology and Crystallization Communications Online, 61(Pt 1), 59-61. doi:10.1107/S1744309104025904  

In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 A and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 A.

https://dipot.ulb.ac.be/dspace/bitstream/2013/110384/4/PMC1952388.pdf

 

Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis.

Noël, C., Dufernez, F., Gerbod, D., Edgcomb, V. P., Delgado-Viscogliosi, P., Ho, L.-C., Singh, M., Wintjens, R., Sogin, M. L., Capron, M., Pierce, R., Zenner, L., & Viscogliosi, E. (2005). Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. Journal of clinical microbiology, 43(1), 348-355. doi:10.1128/JCM.43.1.348-355.2005  

Small-subunit (SSU) rRNA gene sequences were obtained by PCR from 12 Blastocystis isolates from humans, rats, and reptiles for which elongation factor 1alpha (EF-1alpha) gene sequences are already available. These new sequences were analyzed by the Bayesian method in a broad phylogeny including, for the first time, all Blastocystis sequences available in the databases. Phylogenetic trees identified seven well-resolved groups plus several discrete lineages that could represent newly defined clades. Comparative analysis of SSU rRNA- and EF-1alpha-based trees obtained by maximum-likelihood methods from a restricted sampling (13 isolates) revealed overall agreement between the two phylogenies. In spite of their morphological similarity, sequence divergence among Blastocystis isolates reflected considerable genetic diversity that could be correlated with the existence of potentially >/=12 different species within the genus. Based on this analysis and previous PCR-based genotype classification data, six of these major groups might consist of Blastocystis isolates from both humans and other animal hosts, confirming the low host specificity of Blastocystis. Our results also strongly suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111237/4/PMC540115.pdf

 

2004

Identification of a mitochondrial superoxide dismutase with an unusual targeting sequence in Plasmodium falciparum.

Sienkiewicz, N., Daher, W., Dive, D., Wrenger, C., Viscogliosi, E., Wintjens, R., Jouin, H., Capron, M., Müller, S., & Khalife, J. (2004). Identification of a mitochondrial superoxide dismutase with an unusual targeting sequence in Plasmodium falciparum. Molecular and biochemical parasitology, 137(1), 121-132. doi:10.1016/j.molbiopara.2004.05.005  

The intraerythrocytic stages of Plasmodium falciparum are exposed to oxidative stress and require functional anti-oxidant systems to survive. In addition to the parasite's known iron-dependent superoxide dismutase PfSOD1, a second SOD gene (PfSOD2) interrupted by 8 introns was identified on chromosome 6. Molecular modelling shows that the structure of PfSOD2 is similar to other iron-dependent SODs and phylogenetic analysis suggests PfSOD1 and PfSOD2 are the result of an ancestral gene duplication. The deduced amino acid sequence of PfSOD2 is similar to PfSOD1 but has a long N-terminal extension. Immunofluorescence studies show that PfSOD1 is cytosolic, whereas the N-terminal extension of PfSOD2 targets a green fluorescent protein fusion into the parasite's mitochondrion. Both SOD genes are transcribed during the erythrocytic cycle with PfSOD1 mRNA levels up to 35-fold higher than those of PfSOD2. Northern blots demonstrated that the mRNA levels of both SOD genes are up-regulated upon exposure to oxidative stress.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111233/1/Elsevier_91480.pdf

 

Cation-pi/H-bond stair motifs at protein-DNA interfaces

Biot, C., Wintjens, R., & Rooman, M. (2004). Cation-pi/H-bond stair motifs at protein-DNA interfaces: nonadditivity of H-bond, stacking, and cation-π interactions. Journal of the American Chemical Society, 126(20), 6220-6221. doi:10.1021/ja049620g  

At the interface between protein and double-stranded DNA, stair motifs simultaneously involve three different types of pairwise interactions: aromatic base stacking, hydrogen bonding, and cation-π. The relative importance of these interactions is studied in the stair motif occurring in the 1TC3 crystal structure, which involves an arginine and two stacked guanines, by means of Hartree-Fock (HF) and Møller-Plesset energy and free energy calculations, including vibrational, rotational, translational contributions, both in a vacuum and various solvents. The results obtained show an anti-cooperative tendency of the HF energy and vibrational free energy terms, and the cooperativity of the rotational, translational, and solvation free energies. Hence, the cooperativity of the stair motif interactions, in the context of protein-DNA recognition, can be viewed as arising from the environment. Copyright © 2003 American Chemical Society.

https://dipot.ulb.ac.be/dspace/bitstream/2013/71786/4/a73d6ec3-2242-4a29-b1d4-20269e2fc947.txt

 

Specificity and phenetic relationships of iron- and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons.

Wintjens, R., Noël, C., May, A. C. W., Gerbod, D., Dufernez, F., Capron, M., Viscogliosi, E., & Rooman, M. (2004). Specificity and phenetic relationships of iron- and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons. The Journal of biological chemistry, 279(10), 9248-9254. doi:10.1074/jbc.M312329200  

The iron- and manganese-containing superoxide dismutases (Fe/Mn-SOD) share the same chemical function and spatial structure but can be distinguished according to their modes of oligomerization and their metal ion specificity. They appear as homodimers or homotetramers and usually require a specific metal for activity. On the basis of 261 aligned SOD sequences and 12 superimposed x-ray structures, two phenetic trees were constructed, one sequence-based and the other structure-based. Their comparison reveals the imperfect correlation of sequence and structural changes; hyperthermophilicity requires the largest sequence alterations, whereas dimer/tetramer and manganese/iron specificities are induced by the most sizable structural differences within the monomers. A systematic investigation of sequence and structure characteristics conserved in all aligned SOD sequences or in subsets sharing common oligomeric and/or metal specificities was performed. Several residues were identified as guaranteeing the common function and dimeric conformation, others as determining the tetramer formation, and yet others as potentially responsible for metal specificity. Some form cation-pi interactions between an aromatic ring and a fully or partially positively charged group, suggesting that these interactions play a significant role in the structure and function of SOD enzymes. Dimer/tetramer- and iron/manganese-specific fingerprints were derived from the set of conserved residues; they can be used to propose selected residue substitutions in view of the experimental validation of our in silico derived hypotheses.

https://dipot.ulb.ac.be/dspace/bitstream/2013/71787/1/Wintjens-2004.pdf

 

Detection of three wound-induced proteins in papaya latex.

Azarkan, M., Wintjens, R., Looze, Y., & Volant-Baeyens, D. (2004). Detection of three wound-induced proteins in papaya latex. Phytochemistry, 65(5), 525-534. doi:10.1016/j.phytochem.2003.12.006  

The effects of routine mechanical wounding for latex collection from unripe fruits of the tropical Carica papaya tree were investigated. For that purpose, the protein composition of three different latexes was analyzed. The first one, commercially available, was provided in the form of a spray-dried powder, the second one was harvested from fully grown but unripe papaya fruits that are regularly tapped for latex production and the last one, was obtained from similar fruits wounded for the first time. Repeated mechanical wounding was found to profoundly affect the protein content of the latex inducing, among others, activation of papain. Regularly tapped latexes also accumulated several low molecular weight proteins not yet identified, as well as three proteins identified as a trypsin inhibitor, a class-II chitinase and a glutaminyl cyclase on the basis of their enzymatic or inhibitory activities and chromatographic elution profiles. This latter was found here, for the first time, to be a wound-induced protein. The roles of these proteins in the plant defense mechanism are discussed.

 

2003

Pivotal roles of the parasite PGD2 synthase and of the host D prostanoid receptor 1 in schistosome immune evasion.

Hervé, M., Angeli, V., Pinzar, E., Wintjens, R., Faveeuw, C., Narumiya, S., Capron, A., Urade, Y., Capron, M., Riveau, G., & Trottein, F. (2003). Pivotal roles of the parasite PGD2 synthase and of the host D prostanoid receptor 1 in schistosome immune evasion. European Journal of Immunology, 33(10), 2764-2772. doi:10.1002/eji.200324143  

Prostaglandins (PG) are important modulators of immune and inflammatory responses. We recently demonstrated that the production of PGD(2) by the helminthic parasite Schistosoma mansoni inhibits the migration of epidermal Langerhans cells (LC) to the draining lymph nodes (DLN). Here, we identify the responsible parasite enzyme as being a 28-kDa glutathione-S-transferase (termed Sm28GST). Intradermal injection of Sm28GST in wild-type (WT), but not in D prostanoid receptor (DP) 1-deficient mice abrogates the departure of LC from the epidermis after TNF-alpha or FITC treatment. During infection, DP1 deficiency restores LC migration, but does not enhance the rate of T cell proliferation in the skin DLN. However, relative to WT mice, DLN cells from DP1-deficient infected mice produce dramatically less IFN-gamma and IL-10, but equal amount of IL-4. Interestingly, infected DP1-deficient mice develop a more Th2-biased humoral immune response, a significantly reduced parasitemia and a decreased egg-induced inflammatory response in the liver and intestines. Taken together, we propose that DP1 activation by the Sm28GST-derived PGD(2) could represent a strategy for the schistosome to evade host immune defenses. We also suggest that DP1 is important in the Th1/Th2 balance of the immune response and in inflammatory reactions during infection.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111228/3/111228.pdf

 

Basis set and electron correlation effects on ab initio calculations of cation-pi/H-bond stair motifs

Wintjens, R., Biot, C., Rooman, M., & Liévin, J. (2003). Basis set and electron correlation effects on ab initio calculations of cation-pi/H-bond stair motifs. The Journal of Physical Chemistry. A, 107(32), 6249-6258. doi:10.1021/jp034103q  

Cation-π/H-bond stair motifs are recurrently found at the binding interface between protein and DNA. They involve two nucleobases and an amino acid side chain, and encompass three different types of interactions: nucleobase stacking, nucleobase-amino acid H-bond and nucleobase-amino acid cation-π interaction. The interaction energies of the 77 stair motif geometries identified in a data set of 52 high-resolution protein - DNA complexes were investigated by means of ab initio quantum chemistry calculations. Using the standard 6-3IG* basis set, we first establish the value of the Gaussian αd-exponent of d-polarization functions on heavy atoms, which optimizes the MP2 interaction energies. We show that, although the default value of αd = 0.8 is appropriate to minimize the total MP2 energy of a system, the value of αd = 0.2 is optimal for the three types of pairwise interactions studied and yields MP2 interaction energies quite similar to those calculated with more extended basis sets. Indeed, the more diffuse nature of the αd = 0.2 basis functions allows a spatial overlap between the orbitals of the interacting partners. Such functions are also shown to improve the multipole electric moments in the interaction region, which results in a stabilizing polarization effect and a better description of the dispersive energy contributions. Using the MP2 computation level and the 6-31G* basis set with αd = 0.2 instead of αd = 0.8, we computed the interaction energies of the 77 observed stair motif geometries and found that, in a vacuum, the cation-π energy is much less favorable, about 3 times, than the H-bond energy and of the same order of magnitude as the π-π stacking energy. Furthermore, the convergence of the MP perturbation theory expansions was analyzed by computing the MP3 and MP4 corrections on simplified complexes. These expansions exhibited an oscillatory behavior, where MP2 seems to provide a satisfactory approximation, albeit slightly overestimated, to the interaction energy.

https://dipot.ulb.ac.be/dspace/bitstream/2013/72007/4/6c4b27af-f38c-4c30-a20f-96f9c504fe96.txt

 

The different folds adopted by plant cysteine endopeptidases belonging to papain family

Dehon, G., El Moussaoui, A. S., Wintjens, R., Van Wuytswinkel, D., Azarkan, M., & Looze, Y. (2003). The different folds adopted by plant cysteine endopeptidases belonging to papain family. Recent advances in phytochemistry, 97-108.  

 

2002

Evidence that thermodynamic stability of papaya glutamine cyclase is only marginal.

Azarkan, M., Amrani, A., Zerhouni, S., Oberg, K., Ruysschaert, J. M., Wintjens, R., & Looze, Y. (2002). Evidence that thermodynamic stability of papaya glutamine cyclase is only marginal. Biopolymers, 65(5), 325-335. doi:10.1002/bip.10234  

Papaya glutamine cyclase (PQC), a glycoprotein with a molecular mass of 32,980 Da, is a minor constituent of the papaya latex protein fraction. In neutral aqueous solutions, PQC adopts an all-beta conformation and exhibits high resistance to both proteolysis and denaturation. Complete unfolding of PQC requires a combination of an acidic medium and chemical denaturant such as urea or guanidine hydrochloride. The unfolding process takes place through formation of an intermediate A state that accumulates in the absence of chemical denaturants and displays all the features of a molten globule state. The different conformational states-N (native), A (acid-inactivated), and U (unfolded)-have been characterized by means of circular dichroism measurements, fluorescence spectroscopies, Stokes radii determinations, and 8-anilino-1-naphtalenesulfonic acid (ANS) binding characteristics. The unfolding pathways of the enzyme was further studied to estimate thermodynamic parameters characterizing both transitions N if A and A if U. In its A state, PQC is catalytically inefficient and highly susceptible to proteolysis. Also, its thermodynamic stability is decreased by some 3-5 kcal/mol. Conversion of the native to the A state involves digging up of five amino functions together with protonation of four to five acidic groups with pK(a)s, in the native state, around 2.7. It proceeds both cooperatively and reversibly although, in vitro, the refolding process is slow. Unfolding of the A state, on the other hand, occurs with a low degree of cooperativity. The intermediate A state thus seems to be only marginally more stable than the unfolded state. The role of suspected internal ion pairs in the stabilization of the native state of this enzyme is discussed.

https://dipot.ulb.ac.be/dspace/bitstream/2013/77594/4/77594.pdf

 

Pin1: a therapeutic target in Alzheimer neurodegeneration.

Hamdane, M., Smet, C., Sambo, A.-V., Leroy, A., Wieruszeski, J.-M., Delobel, P., Maurage, C.-A., Ghestem, A., Wintjens, R., Bégard, S., Sergeant, N., Delacourte, A., Horvath, D., Landrieu, I., Lippens, G., & Buée, L. (2002). Pin1: a therapeutic target in Alzheimer neurodegeneration. Journal of molecular neuroscience, 19(3), 275-287. doi:10.1385/JMN:19:3:275  

In Alzheimer's disease, the peptidyl prolyl cis/trans isomerase Pin1 binds to phospho-Thr231 on Tau proteins and, hence, is found within degenerating neurons, where it is associated to the large amounts of abnormally phosphorylated Tau proteins. Conversely, Pin1 may restore the tubulin polymerization function of these hyperphosphorylated Tau. In the present work, we investigated, both at the cellular and molecular levels, the role of Pin1 in Alzheimer's disease through the study of its interactions with phosphorylated Tau proteins. We also showed that in neuronal cells, Pin1 upregulates the expression of cyclin D1. This, in turn, could facilitate the transition from quiescence to the G1 phase (re-entry in cell cycle) in a neuron and, subsequently, neuronal dedifferentiation and apoptosis. The involvement of Pin1 in the G0/G1 transition in neurons points to its function as a good target for the development of new therapeutic strategies in neurodegenerative disorders.

 

Probing the energetic and structural role of amino acid/nucleobase cation-pi interactions in protein-ligand complexes.

Biot, C., Buisine, E., Kwasigroch, J.-M., Wintjens, R., & Rooman, M. (2002). Probing the energetic and structural role of amino acid/nucleobase cation-pi interactions in protein-ligand complexes. The Journal of biological chemistry, 277(43), 40816-40822. doi:10.1074/jbc.M205719200  

X-ray structures of proteins bound to ligand molecules containing a nucleic acid base were systematically searched for cation-pi interactions between the base and a positively charged or partially charged side chain group located above it, using geometric criteria. Such interactions were found in 38% of the complexes and are thus even more frequent than pi-pi stacking interactions. They are moreover well conserved in families of related proteins. The overwhelming majority of cation-pi contacts involve Ade bases, as these constitute by far the most frequent ligand building block; Arg-Ade is the most frequent cation-pi pair. Ab initio energy calculations at MP2 level were performed on all recorded pairs. Though cation-pi interactions involving the net positive charge carried by Arg or Lys side chains are the most favorable energetically, those involving the partial positive charge of Asn and Gln side chain amino groups (sometimes referred to as amino-pi interactions) are favorable too, owing to the electron correlation energy contribution. Chains of cation-pi interactions with a nucleobase bound simultaneously to two charged groups or a charged group sandwiched between two aromatic moieties are found in several complexes. The systematic association of these motifs with specific ligand molecules in unrelated protein sequences raises the question of their role in protein-ligand structure, stability, and recognition.

https://dipot.ulb.ac.be/dspace/bitstream/2013/71789/6/bc7ee094-be71-4578-8143-b82558dd98f2.txt

 

Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana.

Landrieu, I., Wieruszeski, J.-M., Wintjens, R., Inzé, D., & Lippens, G. (2002). Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana. Journal of Molecular Biology, 320(2), 321-332. doi:10.1016/S0022-2836(02)00429-1  

The 119-amino acid residue prolyl cis/trans isomerase from Arabidopsis thaliana (PIN1At) is similar to the catalytic domain of the human hPIN1. However, PIN1At lacks the N-terminal WW domain that appears to be essential for the hPIN1 function. Here, the solution structure of PIN1At was determined by three-dimensional nuclear magnetic resonance spectroscopy. The PIN1At fold could be superimposed on that of the catalytic domain of hPIN1 and had a 19 residue flexible loop located between strand beta1 and helix alpha1. The dynamical features of this beta1/alpha1-loop, which are characteristic for a region involved in protein-protein interactions, led to exchange broadening in the NMR spectra. When sodium sulfate salt was added to the protein sample, the beta1/alpha1 loop was stabilized and, hence, a complete backbone resonance assignment was obtained. Previously, with a phospho-Cdc25 peptide as substrate, PIN1At had been shown to catalyze the phosphoserine/phosphothreonine prolyl cis/trans isomerization specifically. To map the catalytic site of PIN1At, the phospho-Cdc25 peptide or sodium sulfate salt was added in excess to the protein and chemical shift changes in the backbone amide protons were monitored in the (1)H(N)-(15)N heteronuclear single quantum coherence spectrum. The peptide caused perturbations in the loops between helix alpha4 and strand beta3, between strands beta3 and beta4, in the alpha3 helix, and in the beta1/alpha1 loop. The amide groups of the residues Arg21 and Arg22 showed large chemical shift perturbations upon phospho-Cdc25 peptide or sulfate addition. We conclude that this basic cluster formed by Arg21 and Arg22, both located in the beta1/alpha1 loop, is homologous to that found in the hPIN1 crystal structure (Arg68 and Arg69), which also is involved in sulfate ion binding. We showed that the sulfate group competed for the interaction between PIN1At and the phospho-Cdc25 peptide. In the absence of the WW domain, three hydrophobic residues (Ile33, Ile34, and Leu35) located in the long flexible loop and specific for the plant PIN-type peptidyl prolyl cis/trans isomerases (PPIases) could be an additional interaction site in PIN1At. However, phospho-peptide addition did not affect the resonances of these residues significantly. Electrostatic potential calculations revealed a negatively charged area not found in hPIN1 on the PIN1At molecular surface, which corresponds to the surface shielded by the WW domain in hPIN1. Based on our experimental results and the molecular specificities of the PIN1At enzyme, functional implications of the lack of WW domains in this plant PIN-type PPIase will be discussed.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111221/1/Elsevier_91467.pdf

 

Biochemical and electron paramagnetic resonance study of the iron superoxide dismutase from Plasmodium falciparum.

Gratepanche, S., Ménage, S., Touati, D., Wintjens, R., Delplace, P., Fontecave, M., Masset, A., Camus, D., & Dive, D. (2002). Biochemical and electron paramagnetic resonance study of the iron superoxide dismutase from Plasmodium falciparum. Molecular and biochemical parasitology, 120(2), 237-246.  

Recombinant iron-containing superoxide dismutase (Fe-SOD) from Plasmodium falciparum was produced in a SOD-deficient strain of Escherichia coli, purified and characterised. The enzyme is a dimer, which contains 1.7 Fe equivalents and is sensitive to hydrogen peroxide (H(2)O(2)). Electron paramagnetic resonance (EPR) analysis showed two different signals, reflecting the presence of two different types of high-spin Fe sites with different symmetries. The role of the W71 residue during inactivation by H(2)O(2) of the P. falciparum Fe-SOD was studied by site-directed mutagenesis. First, the W71V mutation led to a change in the relative proportion of the two Fe-based EPR signals. Second, the mutant protein was almost as active as the wild-type (WT) protein but more sensitive to heat inactivation. Third, resistance to H(2)O(2) was only slightly increased indicating that W71 was marginally responsible for the sensitivity of Fe-SOD to H(2)O(2). A molecular model of the subunit was designed to assist in interpretation of the results. The fact that the parasite SOD does not belong to classes of SOD present in humans may provide a novel approach for the design of antimalarial drugs.

 

Cation-pi/H-bond stair motifs at protein-DNA interfaces

Rooman, M., Liévin, J., Buisine, E., & Wintjens, R. (2002). Cation-pi/H-bond stair motifs at protein-DNA interfaces. Journal of Molecular Biology, 319(1), 67-76. doi:10.1016/S0022-2836(02)00263-2  

H-bonds and cation-π interactions between nucleic acid bases and amino acid side-chains are known to occur often concomitantly at the interface between protein and double-stranded DNA. Here we define and analyze stair-shaped motifs, which simultaneously involve base stacking, H-bond and cation-π interactions. They consist of two successive bases along the DNA stack, one in cation-π interaction with an amino acid side-chain that carries a total or partial positive charge, and the other H-bonded with the same side-chain. A survey of 52 high-resolution structures of protein/DNA complexes reveals the occurrence of such motifs in the majority of the complexes, the most frequent of these motifs involving Arg side-chains and G bases. These stair motifs are sometimes part of larger motifs, called multiple stair motifs, which contain several successive stairs; zinc finger proteins for example exhibit up to quadruple stairs. In another kind of stair motif extension, termed cation-π chain motif, an amino acid side-chain or a nucleic acid base forms simultaneously two cation-π interactions. Such a motif is observed in several homeodomains, where it involves a DNA base in cation-π interactions with an Arg in the minor groove and an Asn in the major groove. A different cation-π chain motif contains an Arg in cation-π with a G and a Tyr, and is found in ets transcription factors. Still another chain motif is encountered in proteins that expulse a base from the DNA stack and replace it by an amino acid side-chain carrying a net or partial positive charge, which forms cation-π interactions with the two neighboring bases along the DNA strand. The striking conservation of typical stair and cation-π chain motifs within families of protein/DNA complexes suggests that they might play a structural and/or functional role and might moreover influence electron migration through the DNA double helix. © 2002 Elsevier Science Ltd. All rights reserved.

https://dipot.ulb.ac.be/dspace/bitstream/2013/145289/1/Elsevier_129112.pdf

 

2001

Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer

Noel, C., Gerbod, D., Fast, N. M., Wintjens, R., Delgado-Viscogliosi, P., Doolittle, W. F., & Viscogliosi, E. (2001). Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer. The Journal of eukaryotic microbiology, 48(6), 647-654. doi:10.1111/j.1550-7408.2001.tb00204.x  

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111212/3/111212.pdfhttps://dipot.ulb.ac.be/dspace/bitstream/2013/111212/4/111212.pdf

 

Phylogenetic relationships of class II fumarase genes from trichomonad species.

Gerbod, D., Edgcomb, V. P., Noel, C., Vanácová, S., Wintjens, R., Tachezy, J., Sogin, M. L., & Viscogliosi, E. (2001). Phylogenetic relationships of class II fumarase genes from trichomonad species. Molecular biology and evolution, 18(8), 1574-1584.  

Class II fumarase sequences were obtained by polymerase chain reaction from five trichomonad species. All residues known to be highly conserved in this enzyme were present. Nuclear run-on assays showed that one of the two genes identified in Tritrichomonas foetus was expressed, whereas no fumarase transcripts were detected in the related species Trichomonas vaginalis. These findings corroborate previous biochemical data. Fumarase genes were also expressed in Monocercomonas sp. and Tetratrichomonas gallinarum but not in Pentatrichomonas hominis, Trichomonas gallinae, Trichomonas tenax, and Trichomitus batrachorum under the culture conditions used. Molecular trees inferred by likelihood methods reveal that trichomonad sequences have no affinity to described class II fumarase genes from other eukaryotes. The absence of functional mitochondria in protists such as trichomonads suggests that they diverged from other eukaryotes prior to the alpha-proteobacterial symbiosis that led to mitochondria. Furthermore, they are basal to other eukaryotes in rRNA analyses. However, support for the early-branching status of trichomonads and other amitochondriate protists based on phylogenetic analyses of multiple data sets has been equivocal. Although the presence of hydrogenosomes suggests that trichomonads once had mitochondria, their class II iron-independent fumarase sequences differ markedly from those of other mitochondriate eukaryotes. All of the class II fumarase genes described from other eukaryotes are of apparent alpha-proteobacterial origin and hence a marker of mitochondrial evolution. In contrast, the class II fumarase from trichomonads emerges among other eubacterial homologs. This is intriguing evidence for an independent acquisition of these genes in trichomonads apart from the mitochondrial endosymbiosis event that gave rise to the form present in other eukaryotes. The ancestral trichomonad class II fumarase may represent a prokaryotic form that was replaced in other eukaryotes after the divergence of trichomonads with the movement of endosymbiont genes into the nucleus. Alternatively, it may have been acquired via a separate endosymbiotic event or lateral gene transfer.

 

1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.

Wintjens, R., Wieruszeski, J. M., Drobecq, H., Rousselot-Pailley, P., Buée, L., Lippens, G., & Landrieu, I. (2001). 1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides. The Journal of biological chemistry, 276(27), 25150-25156. doi:10.1074/jbc.M010327200  

The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its Trp-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro peptide bonds. However, the orientation of the ligand in the aromatic recognition groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A., and Lim, W. A. (2000) Nat. Struct. Biol. 7, 611-613). Because the bound peptide conformation could also differ as observed for peptide ligands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here studied by solution NMR methods. First, the proton resonance perturbations on the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta-hairpin Ser(11)-Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 microm for Cdc25 and tau peptides, respectively, were found. Several intermolecular nuclear Overhauser effects between WW domain and substrates were obtained from a ligand-saturated solution and were used to determine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide ligands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.

 

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence

Gerbod, D., Edgcomb, V. P., Noel, C., Zenner, L., Wintjens, R., Delgado-Viscogliosi, P., Holder, M. E., Sogin, M. L., & Viscogliosi, E. (2001). Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence. The Journal of eukaryotic microbiology, 48(4), 498-504. doi:10.1111/j.1550-7408.2001.tb00185.x  

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111214/3/111214.pdf

 

Identification of amino acid residues in the ETS transcription factor Erg that mediate Erg-Jun/Fos-DNA ternary complex formation.

Verger, A., Buisine, E., Carrère, S., Wintjens, R., Flourens, A., Coll, J. C., Stéhelin, D. J., & Duterque-Coquillaud, M. (2001). Identification of amino acid residues in the ETS transcription factor Erg that mediate Erg-Jun/Fos-DNA ternary complex formation. The Journal of biological chemistry, 276(20), 17181-17189. doi:10.1074/jbc.M010208200  

Jun, Fos, and Ets proteins belong to distinct families of transcription factors that target specific DNA elements often found jointly in gene promoters. Physical and functional interactions between these families play important roles in modulating gene expression. Previous studies have demonstrated a direct interaction between the DNA-binding domains of the two partners. However, the molecular details of the interactions have not been investigated so far. Here we used the known three-dimensional structures of the ETS DNA-binding domain and Jun/Fos heterodimer to model an ETS-Jun/Fos-DNA ternary complex. Docking procedures suggested that certain ETS domain residues in the DNA recognition helix alpha3 interact with the N-terminal basic domain of Jun. To support the model, different Erg ETS domain mutants were obtained by deletion or by single amino acid substitutions and were tested for their ability to mediate DNA binding, Erg-Jun/Fos complex formation, and transcriptional activation. We identified point mutations that affect both the DNA binding properties of Erg and its physical interaction with Jun (R367K), as well as mutations that essentially prevent transcriptional synergy with the Jun/Fos heterodimer (Y371V). These results provide a framework of the ETS/bZIP interaction linked to the manifestation of functional activity in gene regulation.

https://dipot.ulb.ac.be/dspace/bitstream/2013/111218/3/doi_91465.pdf

 

Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism.

El Moussaoui, A. S., Nijs, M., Paul, C., Wintjens, R., Vincentelli, J., Azarkan, M., & Looze, Y. (2001). Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism. Cellular and molecular life sciences, 58(4), 556-570.  

In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree.

 

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

Iurcu-Mustata, G., Van Belle, D., Wintjens, R., Prévost, M., & Rooman, M. (2001). Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations. Biopolymers, 59(3), 145-159. doi:10.1002/1097-0282(200109)59:3<145::AID-BIP1014>3.0.CO;2-Z  
https://dipot.ulb.ac.be/dspace/bitstream/2013/145296/4/145296.pdfhttps://dipot.ulb.ac.be/dspace/bitstream/2013/145296/1/Role-salt-bridges-homeodomains.pdf

 

2000

Contribution of cation-pi interactions to the stability of protein-DNA complexes

Wintjens, R., Liévin, J., Rooman, M., & Buisine, E. (2000). Contribution of cation-pi interactions to the stability of protein-DNA complexes. Journal of Molecular Biology, 302(2), 395-410.  

 

1998

Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all-beta conformation.

Oberg, K., Ruysschaert, J. M., Azarkan, M., Smolders, N., Zerhouni, S., Wintjens, R., Amrani, A., & Looze, Y. (1998). Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all-beta conformation. European journal of biochemistry / FEBS, 258(1), 214-222.  

Glutamine cyclases catalyse the conversion of L-glutaminyl-peptides into 5-oxoprolyl-peptides with the concomitant liberation of ammonia. We report here biophysical characterisation of the glutamine cyclase present in the laticiferous cells of the plant Carica papaya. After purification to near homogeneity, this enzyme was subjected to limited proteolysis and found to exhibit a high resistance to degradation and nicking. The structural reasons for this property were examined using circular dichroism and infrared spectroscopies. By combining the analyses of the infrared and CD spectra of papaya glutamine cyclase, its susceptibility to proteolysis, and its hydrogen-deuterium exchange characteristics, we conclude that this protein contains extensive beta-sheet structure and is likely to have only short immobile loops connecting its beta-strands.

 

Typical interaction patterns in alpha-beta and beta-alpha turn motifs.

Wintjens, R., Wodak, S., & Rooman, M. (1998). Typical interaction patterns in alpha-beta and beta-alpha turn motifs. Protein engineering, 11(7), 505-522.  

A fully automatic classification procedure of short protein fragments is applied to identify connections between alpha-helices and beta-strands in a dataset of 141 protein chains. It yields 15 structural families of alphabeta turns and 15 families of betaalpha turns with at least five members. The sequence and structural features of these turn motifs are analysed with the focus on the local interactions located at alpha-helix and beta-strand ends. This analysis reveals specific interaction patterns that occur frequently among the members of many of the identified turn motifs. For the beta-strands, novel patterns are identified at the strands' entry and exit; they involve side chain/side chain contacts and beta-turns, generally of type I or II. For the alpha-helices, the interaction patterns consist of several backbone/backbone or backbone/side chain hydrogen bonds and of hydrophobic contacts; they generalize the well known N-terminal capping and C-terminal Schellman motifs. The interaction patterns at both ends of alpha-helices and beta-strands are found to constitute favourable structure motifs with low amino acid sequence specificity; their possible stabilizing role is discussed. Finally, the robustness of our classification procedure and of the description of N- and C-cap interaction patterns is validated by repeating our analysis on a larger dataset of 381 protein chains and showing that the results are maintained.

 

1996

Structural classification of HTH DNA-binding domains and protein-DNA interaction modes.

Wintjens, R., & Rooman, M. (1996). Structural classification of HTH DNA-binding domains and protein-DNA interaction modes. Journal of Molecular Biology, 262(2), 294-313. doi:10.1006/jmbi.1996.0514  

This paper constitutes an attempt to rationalize the structural similarities and differences that are observed among the HTH DNA-binding domains, and the various modes of protein-DNA interactions. It consists of classifying all the domains of known structure into families on the basis of the spatial arrangement of their helices, irrespective of the type of loops and the presence of beta-strands, and examining the interaction patterns between amino acids and DNA within each family. It is found that the recognition helix and the preceding helix along the chain have always the same relative orientation. Structural differences arise when considering three helices, corresponding usually to the recognition helix and the two preceding ones, but sometimes to the recognition helix and the two flanking helices. Using an automatic classification procedure, seven main families are obtained, whose members have in common the spatial arrangement of their three key helices, but have sometimes different topology and belong to different species. The structural divergence among these families and the existence of structural intermediates are analyzed. Searching these families systematically for recurrent motifs, leads to identify two specific turns, besides the HTH turn. They both link the two helices preceding the recognition helix and are each characteristic of a given family. Furthermore, the conservation of protein-DNA interaction patterns is examined with respect to the structural alignments. These patterns are found to be relatively well conserved within each family and to be different between the different families. The agreement of the structural classification and the patterns of protein-DNA contacts justify our approach, and suggests its applicability, in particular for modelling protein-DNA interactions.

 

Automatic classification and analysis of alpha alpha-turn motifs in proteins.

Wintjens, R., Rooman, M., & Wodak, S. (1996). Automatic classification and analysis of alpha alpha-turn motifs in proteins. Journal of Molecular Biology, 255(1), 235-253. doi:10.1006/jmbi.1996.0020  

An automatic procedure for the classification of short protein fragments, representing turn motifs between two consecutive secondary structures, is presented. This procedure has two steps. Fragments of given length are first grouped on the basis of their backbone dihedral angle values, and then clustered as a function of the root-mean-square deviation of their superimposed backbone atoms. The classification procedure identifies 63 families of turn motifs with at least five members, in a dataset of 141 proteins. A detailed analysis is presented of the ten identified alpha alpha-turn families, of which four correspond to novel motifs. The sequence and structure features that characterize these families are described. It is found that some features are conserved within the fragments belonging to the same family, but their environment in the parent protein varies considerably. N-capping interactions and helix stop signals are encountered in a number of families, where they seem to stabilize the motif conformation. In two families, one with three residues in the loop, and one with four, an appreciable fraction of the members displays both types of characteristic helix end interactions in the same motif. Interestingly, contrary to most other alpha alpha-turns, the relative frequency of these two motifs is much higher than that of short protein segments with the same loop conformation. Furthermore, the family with three residues in the loop includes the helix-turn-helix motif known to bind DNA. It seems to be the only one among the ten identified families that can be related to biological function.

 

S-pegylthiopapain, a versatile intermediate for the preparation of the fully active form of the cysteine proteinase archetype

Azarkan, M., Wintjens, R., Smolders, N., Nijs, M., & Looze, Y. (1996). S-pegylthiopapain, a versatile intermediate for the preparation of the fully active form of the cysteine proteinase archetype. Journal of chromatography, 724, 185-192.  

 

1995

Synthesis, three-dimensional structure, and specific 15N-labelling of the streptococcal protein G B1-domain.

Boutillon, C., Wintjens, R., Lippens, G., Drobecq, H., & Tartar, A. (1995). Synthesis, three-dimensional structure, and specific 15N-labelling of the streptococcal protein G B1-domain. European journal of biochemistry / FEBS, 231(1), 166-180. doi:10.1111/j.1432-1033.1995.0166f.x  

The 55-amino-acid B1-domain of the streptococcal protein G shows a high binding affinity to IgG isolated from a wide range of mammalian species. Since the B1-domain forms an extremely stable globular folding unit containing the major secondary structure elements and is devoid of proline residues and disulfide bridges, it is also a useful tool for protein folding and stability studies. Its small size makes this protein an ideal candidate for production by chemical synthesis, allowing incorporation of non-natural amino acids with the possibility of assessing the influence of such residues on both the functional and structural characteristics of proteins. In this study, we employed three successive chemical syntheses of the B1-domain in order to define the optimal conditions of coupling and protection. The stepwise solid-phase methodology using the tertbutyloxycarbonyl/benzyl strategy was used for this purpose. First, the sequence assembly difficulties were evaluated. After analyzing of the problems found during assembly, a second optimized synthesis was performed leading to formation of a synthetic B1-domain with a higher yield; the synthetic B1-domain was completely functional in its binding properties to IgG. Three orthogonal purification steps (gel-permeation, reverse-phase and ion-exchange HPLC) were required to obtain a sample suitable for structural analysis by high-resolution NMR. This study led to the conclusion that the synthetic B1-domain adopts a three-dimensional structure identical to that of the molecule obtained by recombinant techniques [Gronenborn, A.M., Filpula, D. R., Essig, N. Z., Achari, A., Whitlow, M., Wingfield, P. T. & Clore, G. M. (1991) Science 253, 657-661]. To demonstrate the usefulness of the chemical approach for the specific introduction of labelled amino acids in the primary structure, fourteen alpha-15N-labelled amino acids were incorporated at selected critical positions during the third synthesis. This analog is the first in a series of molecules planned to study in detail the folding dynamics of the B1-domain.

 

1994

Identification of short turn motifs in proteins using sequence and structure fingerprints

Wintjens, R., Rooman, M., & Wodak, S. (1994). Identification of short turn motifs in proteins using sequence and structure fingerprints. Israel Journal of Chemistry, 34(2), 257-269. doi:10.1002/ijch.199400030  

Families of 20‐residue turn motifs are identified in a dataset of known protein structures using a fully automatic classification procedure that relies on dihedral angle values and atomic root mean square deviations of the polypeptide backbone. Four of the identified motifs, a novel αα connection, and three well‐known αα, βα, and ββ motifs, are used to investigate the possibility of identifying sequences that adopt these motifs in a library of 20‐residue sequence segments from the protein dataset. To this end, several types of fingerprints are derived for individual members of each turn motif family, and for each family as a whole. These fingerprints represent the sequence conservation among family members, or different reduced descriptions of the protein 3D structure that consider the backbone conformation or the tertiary interactions in the context of the parent proteins. All sequence segments in the library are successively mounted onto the fingerprints, without allowing gaps, and the energy of each mount is evaluated using effective potentials derived from known protein structures. The results show that the ability to recognize native sequences associated with a turn motif is improved when different types of fingerprints are combined, and that it fluctuates significantly according to the specific turn family considered. Overall, however, this ability remains rather limited to a level which is much below the native recognition performance generally achieved for full proteins. The fingerprints and their associated potentials are nevertheless found to be quite effective in generating subsets of solutions that are significantly enriched for the correct sequence‐structure combinations. This may have very useful applications in protein folding simulations and in homology modeling. Copyright © 1994 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim