Analyzing In Silico the Relationship Between the Activation of the Edema Factor and Its Interaction With Calmodulin.

Molecular dynamics (MD) simulations have been recorded on the complex between the edema factor (EF) of Bacilllus anthracis and calmodulin (CaM), starting from a structure with the orthosteric inhibitor adefovir bound in the EF catalytic site. The starting structure has been destabilized by alternately suppressing different co-factors, such as adefovir ligand or ions, revealing several long-distance correlations between the conformation of CaM, the geometry of the CaM/EF interface, the enzymatic site and the overall organization of the complex. An allosteric communication between CaM/EF interface and the EF catalytic site, highlighted by these correlations, was confirmed by several bioinformatics approaches from the literature. A network of hydrogen bonds and stacking interactions extending from the helix V of of CaM, and the residues of the switches A, B and C, and connecting to catalytic site residues, is a plausible candidate for the mediation of allosteric communication. The greatest variability in volume between the different MD conditions was also found for cavities present at the EF/CaM interface and in the EF catalytic site. The similarity between the predictions from literature and the volume variability might introduce the volume variability as new descriptor of allostery.

mkgridXf: Consistent Identification of Plausible Binding Sites Despite the Elusive Nature of Cavities and Grooves in Protein Dynamics.

We describe here a method to identify potential binding sites in ensembles of protein structures as obtained by molecular dynamics simulations. This is a highly important task in the context of structure-based drug discovery, and many methods exist for the much simpler case of static structures. However, during molecular dynamics, the cavities and grooves that are used to define binding sites merge, split, appear, and disappear, and cover a large volume. Combined with the large number of sites (∼105 and more), these characteristics hamper a consistent and comprehensive definition of binding sites. Our method is based on the calculation of instantaneous cavities and of the pockets delineating them. Classification of the pockets over the structure ensemble generates consensus pockets, which define sites. Sites are reported as lists of atoms or residues. This avoids the pitfalls of the classification of cavities by spatial overlap, used in most existing methods, which is bound to fail on nonordered or unaligned ensembles or as soon as significant molecular motions are involved. To achieve a robust and consistent classification, we thoroughly optimized and benchmarked the method. For this, we assembled from the literature a set of reference sites on systems involving significant functional molecular motions. We tested different descriptors, metrics, and clustering methods. The resulting method is able to perform a global analysis of potential sites efficiently. Tests on examples show that our approach can make predictions of potential sites on the whole surface of a protein and identify novel sites absent from static structures.

Designed mono- and di-covalent inhibitors trap modeled functional motions for Trypanosoma cruzi proline racemase in crystallography.

Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific-stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design.