SAMPL6: calculation of macroscopic pKa values from ab initio quantum mechanical free energies

International audienceMacroscopic pKa values were calculated for all compounds in the SAMPL6 blind prediction challenge, based on quantum chemical calculations with a continuum solvation model and a linear correction derived from a small training set. Microscopic pKa values were derived from the gas-phase free energy difference between protonated and deprotonated forms together with the Conductor-like Polarizable Continuum Solvation Model and the experimental solvation free energy of the proton. pH-dependent microstate free energies were obtained from the microscopic pKas with a maximum likelihood estimator and appropriately summed to yield macroscopic pKa values or microstate populations as function of pH. We assessed the accuracy of three approaches to calculate the microscopic pKas: direct use of the quantum mechanical free energy differences and correction of the direct values for short-comings in the QM solvation model with two different linear models that we independently derived from a small training set of 38 compounds with known pKa. The predictions that were corrected with the linear models had much better accuracy [root-mean-square error (RMSE) 2.04 and 1.95 pKa units] than the direct calculation (RMSE 3.74). Statistical measures indicate that some systematic errors remain, likely due to differences in the SAMPL6 data set and the small training set with respect to their interactions with water. Overall, the current approach provides a viable physics-based route to estimate macroscopic pKa values for novel compounds with reasonable accuracy

Analyse du contrôle allostérique et prédiction de structure pour une toxine de pathogène : l'apport des simulations de dynamique moléculaire

The protein CyaA is an important virulence factor of Bordetella pertussis involved in the whooping cough disease. The AC domain of CyaA is activated in eukaryotic host cells as an adenyl-cyclase by the ubiquitous and calcium sensitive protein calmodulin (CaM). This activation transformes the ATP into cAMP in an uncontrolled way, leading to severe cellular dysfunction. Only the structure of AC complexed with calcium-loaded calmodulin has been resolved by X-ray crystallography. Starting from this structure, molecular dynamics simulations of AC free and in complex with calmodulin enabled to characterize the effect of the calmodulin and calcium ions on the conformational plasticity of the complex. Conformational tendencies of free AC were also studied. The joint analysis of energetic influences and hydrogen bonds revealed a network of interactions between AC and calmodulin, in which three key residues, that may play an allosteric role on the catalytic activity of the adenyl-cyclase, were modified by site-directed mutagenesis. These experimental tests have led to the identification of an allosteric region which assures the communication of the conformational transition information between the binding site of the calmodulin and the catalytic site. A more thorough conformational exploration of the unbound AC state was undertaken by an innovative temperature accelerated molecular dynamics (TAMD) method. It led to a prediction of some sampled conformations of AC in its unbound state. These predictions could be used in the future to stabilize the free state and to facilitate the experimental study of its structureLa protéine CyaA est un facteur de virulence majeur de Bordetella pertussis, impliqué dans la maladie de la coqueluche. Le domaine catalytique AC de CyaA est directement transféré dans la cellule hôte eucaryote, où il est activé comme adénylcyclase par la calmoduline, une protéine ubiquitaire et sensible aux ions calcium. Ainsi, AC transforme l'ATP en AMPc de manière incontrôlée, ce qui conduit à des dérèglements cellulaires. Seule la structure de AC complexé à la calmoduline chargée d'ions calcium avait été résolue par cristallographie aux rayons X. À partir de cette structure, des simulations de dynamique moléculaire de AC libre, et en complexe avec la calmoduline nous ont permis de caractériser l'effet de la calmoduline et des ions calcium sur la plasticité conformationnelle du complexe. Les tendances conformationelles de AC libre ont aussi été étudiées. L'analyse conjointe des influences énergétiques et des liaisons hydrogène a révélé un réseau d'interactions entre AC et la calmoduline, dans lequel trois résidus clés, susceptibles de jouer un rôle allostérique sur l'activité de l'adénylcyclase ont été modifiés par mutagenèse dirigée. Ces tests expérimentaux ont conduits à la mise en évidence d'une région allostérique qui assure la communication de l'information de transition conformationnelle entre le site de fixation de la calmoduline et le site catalytique. Une exploration conformationnelle plus approfondie de AC à l'état non-lié a été entreprise par une méthode innovante de dynamique accélérée par la température (TAMD). Elle nous a conduit à la prédiction de conformations échantillonnées de AC dans son état libre. Ces prédictions pourraient être utilisées à l'avenir pour stabiliser l'état libre et faciliter l'étude expérimentale de sa structur

Analyse du contrôle allostérique et prédiction de structure pour une toxine de pathogène (l'apport des simulations de dynamique moléculaire)

La protéine CyaA est un facteur de virulence majeur de Bordetella pertussis, impliqué dans la maladie de la coqueluche. Le domaine catalytique AC de CyaA est directement transféré dans la cellule hôte eucaryote, où il est activé comme adénylcyclase par la calmoduline, une protéine ubiquitaire et sensible aux ions calcium. Ainsi, AC transforme l'ATP en AMPc de manière incontrôlée, ce qui conduit à des dérèglements cellulaires. Seule la structure de AC complexé à la calmoduline chargée d'ions calcium avait été résolue par cristallographie aux rayons X. À partir de cette structure, des simulations de dynamique moléculaire de AC libre, et en complexe avec la calmoduline nous ont permis de caractériser l'effet de la calmoduline et des ions calcium sur la plasticité conformationnelle du complexe. Les tendances conformationelles de AC libre ont aussi été étudiées. L'analyse conjointe des influences énergétiques et des liaisons hydrogène a révélé un réseau d'interactions entre AC et la calmoduline, dans lequel trois résidus clés, susceptibles de jouer un rôle allostérique sur l'activité de l'adénylcyclase ont été modifiés par mutagenèse dirigée. Ces tests expérimentaux ont conduits à la mise en évidence d'une région allostérique qui assure la communication de l'information de transition conformationnelle entre le site de fixation de la calmoduline et le site catalytique. Une exploration conformationnelle plus approfondie de AC à l'état non-lié a été entreprise par une méthode innovante de dynamique accélérée par la température (TAMD). Elle nous a conduit à la prédiction de conformations échantillonnées de AC dans son état libre. Ces prédictions pourraient être utilisées à l'avenir pour stabiliser l'état libre et faciliter l'étude expérimentale de sa structureThe protein CyaA is an important virulence factor of Bordetella pertussis involved in the whooping cough disease. The AC domain of CyaA is activated in eukaryotic host cells as an adenyl-cyclase by the ubiquitous and calcium sensitive protein calmodulin (CaM). This activation transformes the ATP into cAMP in an uncontrolled way, leading to severe cellular dysfunction. Only the structure of AC complexed with calcium-loaded calmodulin has been resolved by X-ray crystallography. Starting from this structure, molecular dynamics simulations of AC free and in complex with calmodulin enabled to characterize the effect of the calmodulin and calcium ions on the conformational plasticity of the complex. Conformational tendencies of free AC were also studied. The joint analysis of energetic influences and hydrogen bonds revealed a network of interactions between AC and calmodulin, in which three key residues, that may play an allosteric role on the catalytic activity of the adenyl-cyclase, were modified by site-directed mutagenesis. These experimental tests have led to the identification of an allosteric region which assures the communication of the conformational transition information between the binding site of the calmodulin and the catalytic site. A more thorough conformational exploration of the unbound AC state was undertaken by an innovative temperature accelerated molecular dynamics (TAMD) method. It led to a prediction of some sampled conformations of AC in its unbound state. These predictions could be used in the future to stabilize the free state and to facilitate the experimental study of its structurePARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Blinded evaluation of cathepsin S inhibitors from the D3RGC3 dataset using molecular docking and free energy calculations

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Towards in silico prediction of beta-lactamase-mediated antibiotic resistance using free energy calculations

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Blind Pose Prediction, Scoring, and Affinity Ranking of the CSAR 2014 Dataset

International audienceThe 2014 CSAR Benchmark Exercise was focused on three protein targets: coagulation factor Xa, spleen tyrosine kinase, and bacterial tRNA methyltransferase. Our protocol involved a preliminary analysis of the structural information available in the Protein Data Bank for the protein targets, which allowed the identification of the most appropriate docking software and scoring functions to be used for the rescoring of several docking conformations datasets, as well as for pose prediction and affinity ranking. The two key points of this study were (i) the prior evaluation of molecular modeling tools that are most adapted for each target and (ii) the increased search efficiency during the docking process to better explore the conformational space of big and flexible ligands

In silico prediction of β-lactamase hydrolysis efficiency: Finding the right balance between kinetic and thermodynamic terms

International audienceDuring recent decades, an alarming worldwide spread and diversification of β-lactamases has been obsd. in Gram-neg. species, conferring resistance to β-lactam antibiotics. In this context, the prediction of future β-lactamase mutants becomes an essential issue and we have developed a protocol that allows the evaluation of the energetic cost (thermodn.) assocd. with a mutation in the active site of a β-lactamase, in the presence of the β-lactam substrate. The catalytic efficiency for the β-lactamase-mediated hydrolysis of β-lactam antibiotics is represented by the ratio of a kinetic term (kcat) and a thermodn. term (Km). Here we present an approach based on quantum calcns. and mol. dynamics simulations allowing to est. the kcat term by evaluating β-lactamases from different classes, in the presence of several β-lactam substrates. Therefore, we are now able to predict in silico the overall catalytic efficiency for a large panel of β-lactamases. The precision of this prediction, which depends in turn on the precision for predicting the individual terms kcat and Km, will be discussed and compared with the precision of exptl. values. These results will ultimately provide essential information for the fight against resistance to β-lactam antibiotics

Temperature-accelerated molecular dynamics gives insights into globular conformations sampled in the free state of the AC catalytic domain.

International audienceThe catalytic domain of the adenyl cyclase (AC) toxin from Bordetella pertussis is activated by interaction with calmodulin (CaM), resulting in cAMP overproduction in the infected cell. In the X-ray crystallographic structure of the complex between AC and the C terminal lobe of CaM, the toxin displays a markedly elongated shape. As for the structure of the isolated protein, experimental results support the hypothesis that more globular conformations are sampled, but information at atomic resolution is still lacking. Here, we use temperature-accelerated molecular dynamics (TAMD) simulations to generate putative all-atom models of globular conformations sampled by CaM-free AC. As collective variables, we use centers of mass coordinates of groups of residues selected from the analysis of standard molecular dynamics (MD) simulations. Results show that TAMD allows extended conformational sampling and generates AC conformations that are more globular than in the complexed state. These structures are then refined via energy minimization and further unrestrained MD simulations to optimize inter-domain packing interactions, thus resulting in the identification of a set of hydrogen bonds present in the globular conformations