T-Cell Receptors Binding Orientation over Peptide/MHC Class I Is Driven by Long-Range Interactions.

Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

Molecular modeling study of the binding and signaling properties of the TCRpMHC complex

The activation of the specific immune response against tumor cells is based on the recognition by the CD8+ Cytotoxic Τ Lymphocytes (CTL), of antigenic peptides (p) presented at the surface of the cell by the class I major histocompatibility complex (MHC). The ability of the so-called T-Cell Receptors (TCR) to discriminate between self and non-self peptides constitutes the most important specific control mechanism against infected cells. The TCR/pMHC interaction has been the subject of much attention in cancer therapy since the design of the adoptive transfer approach, in which Τ lymphocytes presenting an interesting response against tumor cells are extracted from the patient, expanded in vitro, and reinfused after immunodepletion, possibly leading to cancer regression. In the last decade, major progress has been achieved by the introduction of engineered lypmhocytes. In the meantime, the understanding of the molecular aspects of the TCRpMHC interaction has become essential to guide in vitro and in vivo studies. In 1996, the determination of the first structure of a TCRpMHC complex by X-ray crystallography revealed the molecular basis of the interaction. Since then, molecular modeling techniques have taken advantage of crystal structures to study the conformational space of the complex, and understand the specificity of the recognition of the pMHC by the TCR. In the meantime, experimental techniques used to determine the sequences of TCR that bind to a pMHC complex have been used intensively, leading to the collection of large repertoires of TCR sequences that are specific for a given pMHC. There is a growing need for computational approaches capable of predicting the molecular interactions that occur upon TCR/pMHC binding without relying on the time consuming resolution of a crystal structure. This work presents new approaches to analyze the molecular principles that govern the recognition of the pMHC by the TCR and the subsequent activation of the T-cell. We first introduce TCRep 3D, a new method to model and study the structural properties of TCR repertoires, based on homology and ab initio modeling. We discuss the methodology in details, and demonstrate that it outperforms state of the art modeling methods in predicting relevant TCR conformations. Two successful applications of TCRep 3D that supported experimental studies on TCR repertoires are presented. Second, we present a rigid body study of TCRpMHC complexes that gives a fair insight on the TCR approach towards pMHC. We show that the binding mode of the TCR is correctly described by long-distance interactions. Finally, the last section is dedicated to a detailed analysis of an experimental hydrogen exchange study, which suggests that some regions of the constant domain of the TCR are subject to conformational changes upon binding to the pMHC. We propose a hypothesis of the structural signaling of TCR molecules leading to the activation of the T-cell. It is based on the analysis of correlated motions in the TCRpMHC structure. - L'activation de la réponse immunitaire spécifique dirigée contre les cellules tumorales est basée sur la reconnaissance par les Lymphocytes Τ Cytotoxiques (CTL), d'un peptide antigénique (p) présenté à la suface de la cellule par le complexe majeur d'histocompatibilité de classe I (MHC). La capacité des récepteurs des lymphocytes (TCR) à distinguer les peptides endogènes des peptides étrangers constitue le mécanisme de contrôle le plus important dirigé contre les cellules infectées. L'interaction entre le TCR et le pMHC est le sujet de beaucoup d'attention dans la thérapie du cancer, depuis la conception de la méthode de transfer adoptif: les lymphocytes capables d'une réponse importante contre les cellules tumorales sont extraits du patient, amplifiés in vitro, et réintroduits après immunosuppression. Il peut en résulter une régression du cancer. Ces dix dernières années, d'importants progrès ont été réalisés grâce à l'introduction de lymphocytes modifiés par génie génétique. En parallèle, la compréhension du TCRpMHC au niveau moléculaire est donc devenue essentielle pour soutenir les études in vitro et in vivo. En 1996, l'obtention de la première structure du complexe TCRpMHC à l'aide de la cristallographie par rayons X a révélé les bases moléculaires de l'interaction. Depuis lors, les techniques de modélisation moléculaire ont exploité les structures expérimentales pour comprendre la spécificité de la reconnaissance du pMHC par le TCR. Dans le même temps, de nouvelles techniques expérimentales permettant de déterminer la séquence de TCR spécifiques envers un pMHC donné, ont été largement exploitées. Ainsi, d'importants répertoires de TCR sont devenus disponibles, et il est plus que jamais nécessaire de développer des approches informatiques capables de prédire les interactions moléculaires qui ont lieu lors de la liaison du TCR au pMHC, et ce sans dépendre systématiquement de la résolution d'une structure cristalline. Ce mémoire présente une nouvelle approche pour analyser les principes moléculaires régissant la reconnaissance du pMHC par le TCR, et l'activation du lymphocyte qui en résulte. Dans un premier temps, nous présentons TCRep 3D, une nouvelle méthode basée sur les modélisations par homologie et ab initio, pour l'étude de propriétés structurales des répertoires de TCR. Le procédé est discuté en détails et comparé à des approches standard. Nous démontrons ainsi que TCRep 3D est le plus performant pour prédire des conformations pertinentes du TCR. Deux applications à des études expérimentales des répertoires TCR sont ensuite présentées. Dans la seconde partie de ce travail nous présentons une étude de complexes TCRpMHC qui donne un aperçu intéressant du mécanisme d'approche du pMHC par le TCR. Finalement, la dernière section se concentre sur l'analyse détaillée d'une étude expérimentale basée sur les échanges deuterium/hydrogène, dont les résultats révèlent que certaines régions clés du domaine constant du TCR sont sujettes à un changement conformationnel lors de la liaison au pMHC. Nous proposons une hypothèse pour la signalisation structurelle des TCR, menant à l'activation du lymphocyte. Celle-ci est basée sur l'analyse des mouvements corrélés observés dans la structure du TCRpMHC.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ‟closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization.

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.

EADock : design of a new molecular docking algorithm and some of its applications

3 Summary 3. 1 English The pharmaceutical industry has been facing several challenges during the last years, and the optimization of their drug discovery pipeline is believed to be the only viable solution. High-throughput techniques do participate actively to this optimization, especially when complemented by computational approaches aiming at rationalizing the enormous amount of information that they can produce. In siiico techniques, such as virtual screening or rational drug design, are now routinely used to guide drug discovery. Both heavily rely on the prediction of the molecular interaction (docking) occurring between drug-like molecules and a therapeutically relevant target. Several softwares are available to this end, but despite the very promising picture drawn in most benchmarks, they still hold several hidden weaknesses. As pointed out in several recent reviews, the docking problem is far from being solved, and there is now a need for methods able to identify binding modes with a high accuracy, which is essential to reliably compute the binding free energy of the ligand. This quantity is directly linked to its affinity and can be related to its biological activity. Accurate docking algorithms are thus critical for both the discovery and the rational optimization of new drugs. In this thesis, a new docking software aiming at this goal is presented, EADock. It uses a hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with .the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 R around the center of mass of the ligand position in the crystal structure, and conversely to other benchmarks, our algorithms was fed with optimized ligand positions up to 10 A root mean square deviation 2MSD) from the crystal structure. This validation illustrates the efficiency of our sampling heuristic, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best-ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures in this benchmark could be explained by the presence of crystal contacts in the experimental structure. EADock has been used to understand molecular interactions involved in the regulation of the Na,K ATPase, and in the activation of the nuclear hormone peroxisome proliferatoractivated receptors a (PPARa). It also helped to understand the action of common pollutants (phthalates) on PPARy, and the impact of biotransformations of the anticancer drug Imatinib (Gleevec®) on its binding mode to the Bcr-Abl tyrosine kinase. Finally, a fragment-based rational drug design approach using EADock was developed, and led to the successful design of new peptidic ligands for the a5ß1 integrin, and for the human PPARa. In both cases, the designed peptides presented activities comparable to that of well-established ligands such as the anticancer drug Cilengitide and Wy14,643, respectively. 3.2 French Les récentes difficultés de l'industrie pharmaceutique ne semblent pouvoir se résoudre que par l'optimisation de leur processus de développement de médicaments. Cette dernière implique de plus en plus. de techniques dites "haut-débit", particulièrement efficaces lorsqu'elles sont couplées aux outils informatiques permettant de gérer la masse de données produite. Désormais, les approches in silico telles que le criblage virtuel ou la conception rationnelle de nouvelles molécules sont utilisées couramment. Toutes deux reposent sur la capacité à prédire les détails de l'interaction moléculaire entre une molécule ressemblant à un principe actif (PA) et une protéine cible ayant un intérêt thérapeutique. Les comparatifs de logiciels s'attaquant à cette prédiction sont flatteurs, mais plusieurs problèmes subsistent. La littérature récente tend à remettre en cause leur fiabilité, affirmant l'émergence .d'un besoin pour des approches plus précises du mode d'interaction. Cette précision est essentielle au calcul de l'énergie libre de liaison, qui est directement liée à l'affinité du PA potentiel pour la protéine cible, et indirectement liée à son activité biologique. Une prédiction précise est d'une importance toute particulière pour la découverte et l'optimisation de nouvelles molécules actives. Cette thèse présente un nouveau logiciel, EADock, mettant en avant une telle précision. Cet algorithme évolutionnaire hybride utilise deux pressions de sélections, combinées à une gestion de la diversité sophistiquée. EADock repose sur CHARMM pour les calculs d'énergie et la gestion des coordonnées atomiques. Sa validation a été effectuée sur 37 complexes protéine-ligand cristallisés, incluant 11 protéines différentes. L'espace de recherche a été étendu à une sphère de 151 de rayon autour du centre de masse du ligand cristallisé, et contrairement aux comparatifs habituels, l'algorithme est parti de solutions optimisées présentant un RMSD jusqu'à 10 R par rapport à la structure cristalline. Cette validation a permis de mettre en évidence l'efficacité de notre heuristique de recherche car des modes d'interactions présentant un RMSD inférieur à 2 R par rapport à la structure cristalline ont été classés premier pour 68% des complexes. Lorsque les cinq meilleures solutions sont prises en compte, le taux de succès grimpe à 78%, et 92% lorsque la totalité de la dernière génération est prise en compte. La plupart des erreurs de prédiction sont imputables à la présence de contacts cristallins. Depuis, EADock a été utilisé pour comprendre les mécanismes moléculaires impliqués dans la régulation de la Na,K ATPase et dans l'activation du peroxisome proliferatoractivated receptor a (PPARa). Il a également permis de décrire l'interaction de polluants couramment rencontrés sur PPARy, ainsi que l'influence de la métabolisation de l'Imatinib (PA anticancéreux) sur la fixation à la kinase Bcr-Abl. Une approche basée sur la prédiction des interactions de fragments moléculaires avec protéine cible est également proposée. Elle a permis la découverte de nouveaux ligands peptidiques de PPARa et de l'intégrine a5ß1. Dans les deux cas, l'activité de ces nouveaux peptides est comparable à celles de ligands bien établis, comme le Wy14,643 pour le premier, et le Cilengitide (PA anticancéreux) pour la seconde.

Constrained peptidomimetics reveal detailed geometric requirements of covalent prolyl oligopeptidase inhibitors.

Prolyl oligopeptidases cleave peptides on the carboxy side of internal proline residues and their inhibition has potential in the treatment of human brain disorders. Using our docking program fitted, we have designed a series of constrained covalent inhibitors, built from a series of bicyclic scaffolds, to study the optimal shape required for these small molecules. These structures bear nitrile functional groups that we predicted to covalently bind to the catalytic serine of the enzyme. Synthesis and biological assays using human brain-derived astrocytic cells and endothelial cells and human fibroblasts revealed that these compounds act as selective inhibitors of prolyl oligopeptidase activity compared to prolyl-dipeptidyl-aminopeptidase activity, are able to penetrate the cells and inhibit intracellular activities in intact living cells. This integrated computational and experimental study shed light on the binding mode of inhibitors in the enzyme active site and will guide the design of future drug-like molecules.

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers.

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.

Blind docking of 260 protein-ligand complexes with EADock 2.0.

Molecular docking softwares are one of the important tools of modern drug development pipelines. The promising achievements of the last 10 years emphasize the need for further improvement, as reflected by several recent publications (Leach et al., J Med Chem 2006, 49, 5851; Warren et al., J Med Chem 2006, 49, 5912). Our initial approach, EADock, showed a good performance in reproducing the experimental binding modes for a set of 37 different ligand-protein complexes (Grosdidier et al., Proteins 2007, 67, 1010). This article presents recent improvements regarding the scoring and sampling aspects over the initial implementation, as well as a new seeding procedure based on the detection of cavities, opening the door to blind docking with EADock. These enhancements were validated on 260 complexes taken from the high quality Ligand Protein Database [LPDB, (Roche et al., J Med Chem 2001, 44, 3592)]. Two issues were identified: first, the quality of the initial structures cannot be assumed and a manual inspection and/or a search in the literature are likely to be required to achieve the best performance. Second the description of interactions involving metal ions still has to be improved. Nonetheless, a remarkable success rate of 65% was achieved for a large scale blind docking assay, when considering only the top ranked binding mode and a success threshold of 2 A RMSD to the crystal structure. When looking at the five-top ranked binding modes, the success rate increases up to 76%. In a standard local docking assay, success rates of 75 and 83% were obtained, considering only the top ranked binding mode, or the five top binding modes, respectively.

Characterization and functional identification of a novel plant extradiol 4,5-dioxygenase involved in betalain pigment biosynthesis in Portulaca Grandiflora

RESUME Les bétalaïnes sont des pigments chromo-alcaloïdes violets et jaunes présents dans les plantes appartenant à l'ordre des Caryophyllales et dans les champignons des genres Amanita et Hygrocybe. Leur courte voie de biosynthèse est élucidée chimiquement depuis de nombreuses années, mais les enzymes impliquées dans cette biosynthèse chez les plantes ne sont toujours pas caractérisées. L'enzyme de la DOPA-dioxygénase d' Amanita muscaria a été identifiée (Girod et Zryd, 1991a), mais de nombreuses tentatives d'isolation d'un homologue chez les plantes ont échoué. Afin d'isoler les gènes spécifiques des bétalaïnes chez les plantes, nous avons construit des banques soustraites d'ADNc à partir d'ARN total de pétales immatures de Portulaca grandiflora (Pg) de génotypes jaunes et blancs, respectivement violets et blancs. Les clones couleur- spécifiques ont été détectés en premier par analyse Northem du RNA de pétales blancs et colorés. Les candidats positifs ont alors été soumis à une analyse de transcription au niveau des tiges colorées, vertes et des feuilles, afin d'établir leur expression spécifique. Deux ARNs messagers complets ont une expression corrélée avec l'accumulation des bétalaïnes dans les tissus. Le premier de ces clones, A.16, code pour une oxydase de l'acyl-Coenzyme A (ACX) putative, mais le domaine de liaison du FAD essentiel pour l'activité d'ACX est absent. Toutes nos tentatives pour démontrer sa fonction ont échoué. Le rôle de cette protéine dans la voie de synthèse des bétalaïnes reste inconnu. Le deuxième de ces clones spécifique aux bétalaïnes, L.6 (isolé par Zaiko, 2000), a été renommé DODA en raison de son homologie avec le domaine LigB (pfam02900) d'une 4,5-dioxygénase extradiol bactérienne. DODA a été identifié in silico comme une dioxygénase extradiol en raison de la conservation stricte, au niveau de sa séquence peptidique, des résidus catalytiques de LigB et de ceux liant le cofacteur fer. Une analyse de transfert Southem a montré que ce gène est unique dans Pg. L'expression transitoire de DODA par transformation biolistique dans des pétales blancs de Pg a produit des taches violettes ou jaunes dans des cellules transformées. Une analyse HPLC de ces taches a démontré leur identité avec les bétalaïnes présentes naturellement dans les pétales violets et jaunes de Pg, confirmant ainsi la complémentation par le gène Pg DODA de l'allèle récessif cc présent dans les pétales blancs de Pg. Des homologues de DODA (DOPA-dioxygénase) ont été identifiés dans de nombreuses espèces de plantes, y compris dans celles sans bétalaïne. L'alignement de ces homologues a permis l'identification d'un motif spécifique aux bétalaïnes à côté d'une histidine catalytique conservée. Ce motif [H-P-(S,A)-(N,D)-x-T-P] remplace le motif [H-N-L-R] conservé dans les plantes sans bétalaïne et le motif [H-N-L-x] présent dans tous les homologues bactériens et archaebactériens. Une modélisation tridimensionnelle préliminaire du site actif de Pg DODA et de son homologue dans la mousse Physcomitrella patens a montré l'importance de ce motif spécifique aux bétalaïnes pour l'accessibilité du substrat au site actif. L'analyse phylogénétique de DODA a confirmé l'évolution séparée de cette protéine chez les plantes à bétalaïnes par comparaison avec celle des plantes sans bétalaïne. Nous avons donc conclu que les bétalaïnes sont apparues par modification de l'affinité pour un substrat d'enzymes similaires à DODA, chez un ancêtre unique des Caryophyllales qui a perdu toute capacité de biosynthèse des anthocyanes. Finalement, Pg DODA n'a aucune similarité avec la protéine DODA d' Amanita muscaria, bien que celle-ci complémente aussi la pigmentation des pétales blancs de Pg. La biosynthèse des bétalaïnes est un exemple remarquable de convergence évolutive biochimique indépendante entre espèces de règnes différents. ABSTRACT Betalains are violet and yellow chromo-alkaloid pigments present in plants belonging to the order Caryophyllales and also in the fungal genera Amanita and Hygrocybe. Their short biosynthetic pathway is chemically well understood since many years, but enzymes involved in the plant pathway are still uncharacterized. The DOPA-dioxygenase from Amanita muscaria was identified (Girod and Zryd, 1991a), but numerous attempts to identify a plant homologue to the corresponding gene, failed. In order to isolate betalain-specific genes in plants, subtractive cDNA libraries were built with total RNA from white and yellow and respectively, violet immature petals from Portulaca grandiflora (Pg) genotypes. Colour-specific clones were first detected by Northern blot analysis using RNA from white and coloured petals. Positive candidates were submitted to further transcription analysis in coloured, green stems and leaves in order to assess their specific expression. Two full-length mRNAs showed a correlated expression with betalain accumulation in tissues. One of them, A.16, encodes a putative acyl-Coenzyme A oxidase (ACX), but missing the FAD binding domain essential for the ACX activity. Thus, all attempts to demonstrate its function failed. The role of this protein in the betalain biosynthesis pathway, if any, is still unknown. The second betalain-specific mRNA, L.6 (isolated by Zaiko, 2000) shows a homology with a LigB domain (pfam02900) from a bacterial extradiol 4,5-dioxygenase. It was then renamed DODA (DOPA-dioxygenase). DODA was identified in silico as a highly conserved extradiol dioxygenase due to the strict conservation of its peptidic sequence with LigB catalytic residues and iron-binding cofactor residues. Southern blot analysis showed that this gene is a single copy-gene in Pg. Transient expression of DODA protein through biolistic transformation of Pg white petals produced violet or yellow spots in individual cells. HPLC analysis of these spots showed an identity with betalain pigments present naturally in yellow and violet Pg petals, thus confirming the complementation of the recessive cc allele present in Pg white petals by Pg DODA gene. DODA homologues were identified in numerous plant species including those without betalain. Alignment of these homologues allowed the identification of a betalain-specific pattern beside a highly conserved catalytic histidine. This [H-P-(S,A)-(N,D)-x-T-P] pattern replaces a [H-N-L-R] pattern strictly conserved in non-betalain plants and a [H-N-L-x] pattern present in all bacterial and archaebacterial homologues. Preliminary three-dimensional modeling of the active site of Pg DODA and its Physcomitrella patens moss homologue revealed the importance of this betalain-specific pattern for the substrate accessibility to the DODA active site. DODA phylogenetic analysis confirmed the separate evolution of this protein in betalain-producing plants. We conclude that betalain pigments appeared in a unique ancestor of the Caryophyllales order in which anthocyanin biosynthetic pathway was impaired, by a modification of enzymes of the DODA family for substrate affinity. The Pg DODA protein has no sequence similarity with Amanita muscaria DODA, despite the fact that they both complement Pg white petals for their pigmentation. Betalain biosynthesis is an interesting example of independent biochemical evolutionary convergence between species from different kingdoms.

Attracting cavities for docking. Replacing the rough energy landscape of the protein by a smooth attracting landscape.

Molecular docking is a computational approach for predicting the most probable position of ligands in the binding sites of macromolecules and constitutes the cornerstone of structure-based computer-aided drug design. Here, we present a new algorithm called Attracting Cavities that allows molecular docking to be performed by simple energy minimizations only. The approach consists in transiently replacing the rough potential energy hypersurface of the protein by a smooth attracting potential driving the ligands into protein cavities. The actual protein energy landscape is reintroduced in a second step to refine the ligand position. The scoring function of Attracting Cavities is based on the CHARMM force field and the FACTS solvation model. The approach was tested on the 85 experimental ligand-protein structures included in the Astex diverse set and achieved a success rate of 80% in reproducing the experimental binding mode starting from a completely randomized ligand conformer. The algorithm thus compares favorably with current state-of-the-art docking programs. © 2015 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Role of Munc18c in SNARE-mediated exocytosis

Background: The SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptors) and SM (Sec1/Munc18) family of proteins form the core machinery that drives the fusion of vesicles in different membrane trafficking steps. They are highly conserved, implying a similar mode of binding and function. In vertebrates, Munc18a is essential for neuronal exocytosis. It binds to its partner syntaxin1a (Syx1a) at both its N-peptide and closed conformation, and thereby inhibits SNARE complex formation in vitro. By contrast, its close homolog Munc18c is thought to interact with only the N-peptide of its partner Syx4. Moreover, different effects of Munc18c on SNARE complex formation have been reported, suggesting that the two Munc18/Syx pairs act differently. Objective: The aim of the present study was to investigate whether the mechanism of action of Munc18c indeed deviates from that of Munc18a by using sensitive biochemical and biophysical methods. Results: I found that Munc18c does have a similar binding mode as Munc18a and interacts tightly with Syx4 at both the N-peptide and closed conformation. Moreover, I established, through a novel assay, that Munc18c inhibits SNARE complex assembly, with both the binding sites contributing to inhibition, similar to Munc18a. However, there were several subtle differences between the two Munc18/Syx pairs. Munc18a exerted stronger inhibition than Munc18c. Also their respective Syx partners were found to differ in the rate of binding to SNAP25, suggesting that the equilibrium of their open and closed conformations is different. Moreover, Munc18a was found to interact with Syx 1, 2, 3 but not 4, while Munc18c bound to Syx 2, 4 and 1 but not 3. By comparing the kinetics of interaction of Syx with either Munc18 or SNAP25, I found that the block of SNARE complex assembly by Munc18 is effective on a shorter time scale, but SNAP25 eventually binds to Syx resulting in SNARE complex formation. Nevertheless, these findings do not explain how Syx can escape the tight grip of Munc18, suggesting that other proteins or mechanisms are needed for this step. I also discovered that Munc18 is able to bind on the surface of the SNARE core complex; however, this observation needs to be tested more rigorously. Conclusion: Munc18c was found to be similar to Munc18a in its mode of binding to Syx and inhibition of SNARE complex assembly. However, differences in kinetics and interaction specificities were observed between the different Munc18/Syx pairs. -- Contexte : Les familles des protéines SNARE (Soluble N-ethylmaleimide-sensitive factor At- tachment protein Receptors) et SM (Sec1/Munc18) forment le coeur de la machinerie chargée de la fusion vésiculaire au cours des différentes étapes du trafic intracellulaire. Elles sont très conservées, suggérant un mode d'interaction et des fonctions semblables. Chez les Verté- brés, Munc18a est essentielle à l'exocytose neuronale. Elle se lie à sa partenaire d'interaction syntaxin1a (Syx1a) à la fois via un peptide N-terminal et la conformation fermée de celle-ci, inhibant ainsi la formation du complexe SNARE in vitro. Son homologue proche Munc18c au contraire, est supposée interagir seulement avec le peptide N-terminal de sa partenaire Syx4. En outre, différents effets de Munc18c sur la formation du complexe SNARE ont été décrits, suggérant que les deux paires Munc18/Syx fonctionnent différemment. Objectif : Le but de cette étude est de tester si les mécanismes de fonctionnement de Munc18c diffèrent vraiment de ceux de Munc18a par le biais de méthodes biochimiques et biophysiques très précises. Résultats : J'ai pu démontrer que Munc18c se comporte en effet de façon semblable à Munc18a, et interagit étroitement avec Syx4 à ses deux sites de liaison. J'ai pu de surcroît montrer par une nouvelle méthode que Munc18c inhibe l'assemblage du complexe SNARE en impliquant ces deux sites de liaison, comme le fait Munc18a. il existe cependant de subtiles différences entre les deux paires Munc18/Syx : Munc18a exerce une inhibition plus forte que Munc18c ; leurs Syx partenaires diffèrent également dans leur degré de liaison à SNAP25, ce qui suggère un équilibre different de leurs conformations ouverte et fermée. De plus, Munc18a interagit avec Syx 1, 2 et 3 mais pas Syx 4, alors que Munc18c se lie à Syx 2, 4 et 1 mais pas Syx 3. En comparant les cinétiques d'interaction de Syx avec Munc18 ou SNAP25, j'ai découvert que le blocage par Munc18 de l'assemblage du complexe SNARE est effectif de façon brève, bien que SNAP25 finisse par se lier à Syx et aboutir ainsi à la formation du complexe SNARE. Ces découvertes n'expliquent cependant pas comment Syx parvient à échapper à la solide emprise de Munc18, et suggèrent ainsi l'intervention nécessaire d'autres protéines ou mécanismes à cette étape. J'ai également découvert que Munc18 peut se lier à la surface de la partie centrale du complexe SNARE - cette observation reste à être testée de façon plus stringente. Conclusion : Il a pu être établi que Munc18c est semblable à Munc18a quant à son mode de liaison à Syx et d'inhibition de l'assemblage du complexe SNARE. Des différences de cinétique et de spécificité d'interaction entre les diverses paires Munc18/Syx ont cependant été identifiées.

Functional dynamics of PDZ binding domains: a normal-mode analysis.

Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domains are relatively small (80-120 residues) protein binding modules central in the organization of receptor clusters and in the association of cellular proteins. Their main function is to bind C-terminals of selected proteins that are recognized through specific amino acids in their carboxyl end. Binding is associated with a deformation of the PDZ native structure and is responsible for dynamical changes in regions not in direct contact with the target. We investigate how this deformation is related to the harmonic dynamics of the PDZ structure and show that one low-frequency collective normal mode, characterized by the concerted movements of different secondary structures, is involved in the binding process. Our results suggest that even minimal structural changes are responsible for communication between distant regions of the protein, in agreement with recent NMR experiments. Thus, PDZ domains are a very clear example of how collective normal modes are able to characterize the relation between function and dynamics of proteins, and to provide indications on the precursors of binding/unbinding events.

Nucleic acid-binding properties of the RRM-containing protein RDM1.

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L119GF --> AAA mutation affects the mode of RDM1 binding to single-stranded DNA.

Cis-configured aziridines are new pseudo-irreversible dual-mode inhibitors of Candida albicans secreted aspartic protease 2

A series of cis-configured epoxides and aziridines containing hydrophobic moieties and amino acid esters,were synthesized as new potential inhibitors of the secreted aspartic protease 2 (SAP2) of Candida albicans. Enzyme assays revealed the N- benzyl-3-phenyl-substituted aziridines 11 and 17 as the most potent inhibitors, with second-order inhibition, rate constants (k(2)) between 56000 and 12-1000 M-1 min(-1). The compounds were shown to be pseudo-irreversible dual-mode, inhibitors: the interm ediate esterified enzyme resulting from nucleophilic ring opening was hydrolyzed and yielded amino alcohols as transition state-mimetic reversible inhibitors. The results of docking studies with the ring-closed aziridine forms of the inhibitors suggest binding modes mainly dominated by hydrophobic interactions with the S1, S1' S2, and S2' subsites of the protease, and docking studies with the processed amino alcohol forms predict additional hydrogen bonds of the new hydroxy group to the active site Asp residues. C. albicans growth assays showed the compounds to decrease SAP2-dependent growth while not affecting SAP2-independent growth.