Homology modeling and metabolism prediction of human carboxylesterase-2 using docking analyses by GriDock: a parallelized tool based on AutoDock 4.0.

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted to developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas less has been done to predict the activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES2. The study involved first a homology modeling of the hCES2 protein based on the model of hCES1 since the two proteins share a high degree of homology (congruent with 73%). A set of 40 known substrates of hCES2 was taken from the literature; the ligands were docked in both their neutral and ionized forms using GriDock, a parallel tool based on the AutoDock4.0 engine which can perform efficient and easy virtual screening analyses of large molecular databases exploiting multi-core architectures. Useful statistical models (e.g., r (2) = 0.91 for substrates in their unprotonated state) were calculated by correlating experimental pK(m) values with distance between the carbon atom of the substrate's ester group and the hydroxy function of Ser228. Additional parameters in the equations accounted for hydrophobic and electrostatic interactions between substrates and contributing residues. The negatively charged residues in the hCES2 cavity explained the preference of the enzyme for neutral substrates and, more generally, suggested that ligands which interact too strongly by ionic bonds (e.g., ACE inhibitors) cannot be good CES2 substrates because they are trapped in the cavity in unproductive modes and behave as inhibitors. The effects of protonation on substrate recognition and the contrasting behavior of substrates and products were finally investigated by MD simulations of some CES2 complexes.

Improved prediction of coronary heart disease with markers of atherosclerosis and of inflammation in older adults?

Background: Several markers of atherosclerosis and of inflammation have been shown to predict coronary heart disease (CHD) individually. However, the utility of markers of atherosclerosis and of inflammation on prediction of CHD over traditional risk factors has not been well established, especially in the elderly. Methods: We studied 2202 men and women, aged 70-79, without baseline cardiovascular disease over 6-year follow-up to assess the risk of incident CHD associated with baseline noninvasive measures of atherosclerosis (ankle-arm index [AAI], aortic pulse wave velocity [aPWV]) and inflammatory markers (interleukin-6 [IL-6], C-reactive protein [CRP], tumor necrosis factor-a [TNF-a]). CHD events were studied as either nonfatal myocardial infarction or coronary death ("hard" events), and "hard" events plus hospitalization for angina, or the need for coronary-revascularization procedures (total CHD events). Results: During the 6-year follow-up, 283 participants had CHD events (including 136 "hard" events). IL-6, TNF-a and AAI independently predicted CHD events above Framingham Risk Score (FRS) with hazard ratios [HR] for the highest as compared with the lowest quartile for IL-6 of 1.95 (95%CI: 1.38-2.75, p for trend <0.001), TNF-a of 1.45 (95%CI: 1.04-2.02, p for trend 0.03), of 1.66 (95%CI: 1.19-2.31) for AAI 0.9, as compared to AAI 1.01-1.30. CRP and aPWV were not independently associated with CHD events. Results were similar for "hard" CHD events. Addition of IL-6 and AAI to traditional cardiovascular risk factors yielded the greatest improvement in the prediction of CHD; C-index for "hard"/total CHD events increased from 0.62/0.62 for traditional risk factors to 0.64/0.64 for IL-6 addition, 0.65/0.63 for AAI, and 0.66/0.64 for IL-6 combined with AAI. Being in the highest quartile of IL-6 combined with an AAI 0.90 or >1.40 yielded an HR of 2.51 (1.50-4.19) and 4.55 (1.65-12.50) above FRS, respectively. With use of CHD risk categories, risk prediction at 5 years was more accurate in models that included IL-6, AAI or both, with 8.0, 8.3 and 12.1% correctly reclassified, respectively. Conclusions: Among older adults, markers of atherosclerosis and of inflammation, particularly IL-6 and AAI, are independently associated with CHD. However, these markers only modestly improve cardiovascular risk prediction beyond traditional risk factors.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Regulation of the cardiac voltage-gated sodium channel Nav1.5 : regulation of Nav1.5 by ubiquitin-protein ligases of the Nedd4/Nedd4-like family and characterisation of two novel mutations in the gene encoding Nav1.5 (SCN5A) implicated in the Brugada syndrome triggered by fever

Abstract: The genesis of the cardiac action potential, which accounts for the cardiac contraction, is due to the sodium current INa mediated by the voltage-gated sodium channel Nav1.5. Several cardiac arrhythmias such as the Brugada syndrome are known te be caused by mutations in SCN5A, the gene encoding Nav1.5. Studies of these mutations allowed a better understanding of biophysical and functional properties of Nav1.5. However, only few investigations have been performed in order to understand the regulation of Nav1.5. During my thesis, I investigated different mechanisms of regulation of Nav1.5 using a heterologous expression system, HEK293 cells, coupled with a technique of sodium current recording: the patch clamp in whole cell configuration. In previous studies it has been shown that an enzyme of the Nedd4 family (Nedd4-2) regulates an epithelial sodium channel via the interaction with PY-motifs present in the latter. Interestingly, Nav1.5 contains a similar PY-motif, which motivated us to study the role of Nedd4-2 expressed in heart for the regulation of Nav1.5. In a second study, we investigated the implication of two Nav1.5 mutants, which were either less functional or net functional (Nav1.5 R535X and Nav1.5 L325R respectively) implied in the genesis of the Brugada syndrome by fever. Our results established two mechanisms implied in Nav1.5 regulation. The first one implies that following the interaction between the PY-motif of Nav1.5 and Nedd4- 2 Nav1.5 is ubiquitinated by Nedd4-2. This ubiquitination leads to the internalization of Nav1 .5. The second mechanism is a phenomenon called the "dominant negative" effect of Nav1.5 L325R on Nay1.5 where the decrease of 'Na is potentially due to the retention of Nav1.5 by Nav1.5 L325R in an undefined intracellular compartment. These studies defined two mechanisms of Nav1.5 regulation, which could play an important role for the genesis of cardiac arrhythmias where molecular processes are still poorly understood. Résumé La genèse du potentiel d'action cardiaque, permettant la contraction cardiaque, est due au courant sodique INa issu des canaux sodiques cardiaques dépendants du voltage Nav1.5. Nombreuses arythmies cardiaques telles que le syndrome de Brugada sont connues pour être liées à des mutations du gène SCN5A, codant pour Nav1.5. L'étude de ces mutations a permis une meilleure compréhension des propriétés structurelles et fonctionnelles de Nav1.5 et leurs implications dans la genèse de ces pathologies. Néanmoins peu d'études ont été menées afin de comprendre les mécanismes de régulation de Nav1.5. Mon travail de thèse a consisté à étudier des mécanismes de régulation de Nav1.5 en utilisant un système d'expression hétérologue, les cellules HEK293, couplé à une technique d'enregistrement des courants sodiques, le "patch clamp" en configuration cellule entière. La présence sur Nav1.5 d'un motif-PY similaire à ceux nécessaires pour la régulation d'un canal épithélial sodique par une enzyme de la famille de Nedd4, nous a amenée à étudier le rôle de ces ubiquitine-ligases, en particulier Nedd4-2, dans la régulation de Nav1.5. La seconde étude s'est intéressée aux conséquences de deux mutations de SCN5A codant pour deux mutants peu ou pas fonctionnels (Nav1.5 L325R et Nav1.5 R535X respectivement) retrouvées chez des patients présentant un syndrome de Brugada exacerbé par un état fébrile. Nos résultats ont permis d'établir deux mécanismes de régulation de Nav1.5 L'un par Nedd4-2 qui implique rubiquitination de Nav1.5 par cette ligase suite à l'interaction entre le motif-PY de Nav1.5 et Nedd4-2. Cette modification déclenche l'internalisation du canal impliquée dans la diminution d'INa. Le second mécanisme quant à lui est un effet "dominant négatif" de Nav1.5 L325R sur Nav1.5 aboutissant à une diminution d'INa suite à la séquestration intracellulaire potentielle de Nav1.5 par Nav1.5 L325R. Ces études ont mis en évidence deux mécanismes de régulation de Nav1.5 pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des arythmies cardiaques dont les processus moléculaires au sein des cardiomyocytes, impliquant des modifications du courant sodiques, sont encore mal compris. Résumé destiné à un large public La dépolarisation électrique de la membrane des cellules cardiaques permet la contraction du coeur. La génèse de cette activité électrique est due au courant sodique issu d'un type de canal à sodium situé dans la membrane des cellules cardiaques. De nombreuses pathologies provoquant des troubles du rythme cardiaque sont issues de mutations du gène qui code pour ce canal à sodium. Ces canaux mutants, entrainant diverses pathologies cardiaques telles que le syndrome de Brugada, ont été largement étudiées. Néanmoins, peu de travaux ont été réalisés sur les mécanismes de régulation de ce canal à sodium non muté. Mon travail de thèse a consisté à étudier certains des mécanismes de régulation de ce canal à sodium en utilisant une technique permettant l'enregistrement des courants sodiques issus de l'expression de ces canaux à sodium à la membrane de cellules mammifères. La présence sur ce canal à sodium d'une structure spécifique, similaire à celle nécessaire pour la régulation d'un canal épithélial à sodium par une enzyme appelée Nedd4-2, nous a amenée à étudier le rôle de cette enzyme dans la régulation de ce canal à sodium. La seconde étude s'est intéressée aux rôles de deux mutations du gène codant pour ce canal à sodium retrouvées chez des patients présentant un syndrome de Brugada exacerbé par la fièvre. Nos résultats nous ont permis d'établir deux mécanismes de régulation de ce canal à sodium diminuant le courant sodique l'un par l'action de l'enzyme Nedd4-2, suite à son interaction avec ce canal, qui modifie ce canal à sodium (ubiquitination) diminuant de ce fait la densité membranaire du canal. L'autre par un mécanisme suggérant un effet négatif de l'un des canaux mutants sur l'expression à la membrane du canal à sodium non muté. Ces études ont mis en évidence deux mécanismes de régulation de ce canal à sodium pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des troubles du rythme cardiaques dont les mécanismes cellulaires sont encore incompris.

Analysis of a boundary protein delimiting chromatin domains in yeast

SUMMARY The expression state of a eukaryotic gene depends in part on its location in the chromosome. This position effect results from the organization of eukaryotic genomes into discrete functional domains, defined by local differences in chromatin structure. The expression of genes within each domain appears to be defined and maintained by the concerted action of regulatory elements such as promoters, enhancers, silencers and locus control regions. Individual domains may be bordered by boundary elements that separate regions of permissive and silent chromatin. When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres towards more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one of these transcription factors, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino-acid substitutions that similarly inhibited the boundary and histone-binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains. RESUME L'expression des gènes eucaryotes dépend en partie de leur localisation sur les chromosomes. Cet effet de position résulte de l'organisation des génomes eucaryotes en domaines fonctionnels, définis par des changements locaux au niveau de la structure de la chromatine. Dans chacun de ces domaines, l'expression des gènes est définie et maintenue par l'action concertée de différents éléments régulateurs tels que les promoteurs, les amplificateurs, les silenceurs et les locus control régions. Ces domaines peuvent être entourés par des éléments barrière, séparant les régions de chromatine répressive des régions permissive pour l'expression des gènes. Lorsqu'ils se situent à proximité d'éléments chromosomiques comme les telomères, les gènes peuvent être réprimés de manière épigénétique. Chez la levure, cette répression est établie par la propagation des protéines SIR depuis les télomères vers les régions centromériques. Certains facteurs de transcription peuvent empêcher la répression d'un gène, lorsqu'ils sont placés entre ce gène et le télomère. Nous avons étudié un de ces facteurs, CTF-1, qui a la particularité de lier directement l'histone H3. La délétion de certaines parties de CTF-1 a permis de déterminer que la région responsable de l'activité barrière correspond au domaine d'interaction avec H3. Plusieurs mutations points effectuées dans ce domaine inhibent à la fois l'activité barrière et la capacité de lier H3. Des expériences d'immuno-précipitation de la chromatine indiquent que la protéine barrière CTF-1 prévient efficacement la propagation des protéines SIR et sépare des domaines contenant des histones hypo-acétylées de ceux constitués d'histones hyper-acétylées. Ces résultats suggèrent que CTF-1 interagit directement avec l'histone H3 pour empêcher la propagation de la chromatine répressive, délimitant ainsi des domaines de chromatine permissive et des domaines de chromatine silencieuse.

Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

Structure-based drug design studies of the interactions ofent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparumsarco/endoplasmic reticulum Ca2+-ATPase PfATP6

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosawere tested in silico against the Plasmodium falciparumCa2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.

Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.

C-reactive protein, procalcitonin, lactate and score apache II in septic patients' prognosis.

Background: APACHE-II IS a score, based on several clinical and analytical measurements within 24 hours of admission in Intensive Care Unit (ICU). C-Reactive Protein (CRP), Lactate and recently Procalcitonin (PCT), also are biomarkers for the assessment of septic patients. The aim of this study was to find out if CRP, lactate and PCT during the first 24 hours from severe sepsis or septic shock onset, improved prediction of the APACHE II in terms of prognosis. Conclusions: CRP improves the prediction of patients with sepsis used in conjunction with the APACHE II score in severe sepsis and, lactate along with the CRP are the best precictors of survival in the cases of septic shock. The PCT did not show any predictive value.

SwissTargetPrediction: a web server for target prediction of bioactive small molecules.

Bioactive small molecules, such as drugs or metabolites, bind to proteins or other macro-molecular targets to modulate their activity, which in turn results in the observed phenotypic effects. For this reason, mapping the targets of bioactive small molecules is a key step toward unraveling the molecular mechanisms underlying their bioactivity and predicting potential side effects or cross-reactivity. Recently, large datasets of protein-small molecule interactions have become available, providing a unique source of information for the development of knowledge-based approaches to computationally identify new targets for uncharacterized molecules or secondary targets for known molecules. Here, we introduce SwissTargetPrediction, a web server to accurately predict the targets of bioactive molecules based on a combination of 2D and 3D similarity measures with known ligands. Predictions can be carried out in five different organisms, and mapping predictions by homology within and between different species is enabled for close paralogs and orthologs. SwissTargetPrediction is accessible free of charge and without login requirement at http://www.swisstargetprediction.ch.

A role of peripheral myelin protein 2 in lipid homeostasis of myelinating Schwann cells.

Peripheral myelin protein 2 (Pmp2, P2 or Fabp8), a member of the fatty acid binding protein family, was originally described together with myelin basic protein (Mbp or P1) and myelin protein zero (Mpz or P0) as one of the most abundant myelin proteins in the peripheral nervous system (PNS). Although Pmp2 is predominantly expressed in myelinated Schwann cells, its role in glia is currently unknown. To study its function in PNS biology, we have generated a complete Pmp2 knockout mouse (Pmp2(-/-) ). Comprehensive characterization of Pmp2(-/-) mice revealed a temporary reduction in their motor nerve conduction velocity (MNCV). While this change was not accompanied by any defects in general myelin structure, we detected transitory alterations in the myelin lipid profile of Pmp2(-/-) mice. It was previously proposed that Pmp2 and Mbp have comparable functions in the PNS suggesting that the presence of Mbp can partially mask the Pmp2(-/-) phenotype. Indeed, we found that Mbp lacking Shi(-/-) mice, similar to Pmp2(-/-) animals, have preserved myelin structure and reduced MNCV, but this phenotype was not aggravated in Pmp2(-/-) /Shi(-/-) mutants indicating that Pmp2 and Mbp do not substitute each other's functions in the PNS. These data, together with our observation that Pmp2 binds and transports fatty acids to membranes, uncover a role for Pmp2 in lipid homeostasis of myelinating Schwann cells.

Markers of atherosclerosis and of inflammation for prediction of coronary heart disease in older adults

BACKGROUND: Several markers of atherosclerosis and of inflammation have been shown to predict coronary heart disease (CHD) individually. However, the utility of markers of atherosclerosis and of inflammation on prediction of CHD over traditional risk factors has not been well established, especially in the elderly. METHODS: We studied 2202 men and women, aged 70-79, without baseline cardiovascular disease over 6-year follow-up to assess the risk of incident CHD associated with baseline noninvasive measures of atherosclerosis (ankle-arm index [AAI], aortic pulse wave velocity [aPWV]) and inflammatory markers (interleukin-6 [IL-6], C-reactive protein [CRP], tumor necrosis factor-a [TNF-a]). CHD events were studied as either nonfatal myocardial infarction or coronary death ("hard" events), and "hard" events plus hospitalization for angina, or the need for coronary-revascularization procedures (total CHD events). RESULTS: During the 6-year follow-up, 283 participants had CHD events (including 136 "hard" events). IL-6, TNF-a and AAI independently predicted CHD events above Framingham Risk Score (FRS) with hazard ratios [HR] for the highest as compared with the lowest quartile for IL-6 of 1.95 (95%CI: 1.38-2.75, p for trend<0.001), TNF-a of 1.45 (95%CI: 1.04-2.02, p for trend 0.03), of 1.66 (95%CI: 1.19-2.31) for AAI £0.9, as compared to AAI 1.01-1.30. CRP and aPWV were not independently associated with CHD events. Results were similar for "hard" CHD events. Addition of IL-6 and AAI to traditional cardiovascular risk factors yielded the greatest improvement in the prediction of CHD; C-index for "hard"/total CHD events increased from 0.62/0.62 for traditional risk factors to 0.64/0.64 for IL-6 addition, 0.65/0.63 for AAI, and 0.66/0.64 for IL-6 combined with AAI. Being in the highest quartile of IL-6 combined with an AAI £ 0.90 or >1.40 yielded an HR of 2.51 (1.50-4.19) and 4.55 (1.65-12.50) above FRS, respectively. With use of CHD risk categories, risk prediction at 5 years was more accurate in models that included IL-6, AAI or both, with 8.0, 8.3 and 12.1% correctly reclassified respectively. CONCLUSIONS: Among older adults, markers of atherosclerosis and of inflammation, particularly IL-6 and AAI, are independently associated with CHD. However, these markers only modestly improve cardiovascular risk prediction beyond traditional risk factors. Acknowledgments: This study was supported by Contracts NO1-AG-6-2101, NO1-AG-6- 2103, and NO1-AG-6-2106 of the National Institute on Aging. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Fast docking using the CHARMM force field with EADock DSS.

The prediction of binding modes (BMs) occurring between a small molecule and a target protein of biological interest has become of great importance for drug development. The overwhelming diversity of needs leaves room for docking approaches addressing specific problems. Nowadays, the universe of docking software ranges from fast and user friendly programs to algorithmically flexible and accurate approaches. EADock2 is an example of the latter. Its multiobjective scoring function was designed around the CHARMM22 force field and the FACTS solvation model. However, the major drawback of such a software design lies in its computational cost. EADock dihedral space sampling (DSS) is built on the most efficient features of EADock2, namely its hybrid sampling engine and multiobjective scoring function. Its performance is equivalent to that of EADock2 for drug-like ligands, while the CPU time required has been reduced by several orders of magnitude. This huge improvement was achieved through a combination of several innovative features including an automatic bias of the sampling toward putative binding sites, and a very efficient tree-based DSS algorithm. When the top-scoring prediction is considered, 57% of BMs of a test set of 251 complexes were reproduced within 2 Å RMSD to the crystal structure. Up to 70% were reproduced when considering the five top scoring predictions. The success rate is lower in cross-docking assays but remains comparable with that of the latest version of AutoDock that accounts for the protein flexibility. © 2011 Wiley Periodicals, Inc. J Comput Chem, 2011.

Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities.

Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.