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Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an "in silico" approach. Homology models of the catalytic domain of sGC in "inactive" or "active" conformations were constructed using the structure of previously described crystals of the catalytic domains of "inactive" sGCs (2WZ1, 3ET6) and of "active" adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 μs. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of "inactive" and "active" conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the "active" structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop β2 -β3 , does not overlap with the forskolin binding site on adenylate cyclases. Proteins 2016; 84:1534-1548. © 2016 Wiley Periodicals, Inc.

The prediction of protein-ligand binding free energies is an important goal of computational biochemistry, yet accuracy, reproducibility, and cost remain a problem. Nevertheless, these are essential requirements for computational methods to become standard binding prediction tools in discovery pipelines. Here, we present the results of an extensive search for an optimal method based on an ensemble of umbrella sampling all-atom molecular simulations tested on the phosphorylated tetrapeptide, pYEEI, binding to the SH2 domain, resulting in an accurate and converged binding free energy of -9.0 ± 0.5 kcal/mol (compared to an experimental value of -8.0 ± 0.1 kcal/mol). We find that a minimum of 300 ns of sampling is required for every prediction, a target easily achievable using new generation accelerated MD codes. Convergence is obtained by using an ensemble of simulations per window, each starting from different initial conformations, and by optimizing window-width, orthogonal restraints, reaction coordinate harmonic potentials, and window-sample time. The use of uncorrelated initial conformations in neighboring windows is important for correctly sampling conformational transitions from the unbound to bound states that affect significantly the precision of the calculations. This methodology thus provides a general recipe for reproducible and practical computations of binding free energies for a class of semirigid protein-ligand systems, within the limit of the accuracy of the force field used.

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an "in silico" approach. Homology models of the catalytic domain of sGC in "inactive" or "active" conformations were constructed using the structure of previously described crystals of the catalytic domains of "inactive" sGCs (2WZ1, 3ET6) and of "active" adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 μs. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of "inactive" and "active" conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the "active" structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop β2 -β3 , does not overlap with the forskolin binding site on adenylate cyclases. Proteins 2016; 84:1534-1548. © 2016 Wiley Periodicals, Inc.

Approximately 100 proteins in the human genome contain an SH2 domain recognizing small flexible phosphopeptides. It is therefore important to understand in atomistic detail the way these peptides bind and the conformational changes that take place upon binding. Here, we obtained several spontaneous binding events between the p56 lck SH2 domain and the pYEEI peptide within 2 Å RMSD from the crystal structure and with kinetic rates compatible with experiments using high-throughput molecular dynamics simulations. Binding is achieved in two phases, fast contacts of the charged phospho-tyrosine and then rearrangement of the ligand involving the stabilization of two important loops in the SH2 domain. These observations provide insights into the binding pathways and induced conformations of the SH2-phosphopeptide complex which, due to the characteristics of SH2 domains, should be relevant for other SH2 recognition peptides.

The understanding of protein–ligand binding is of critical importance for biomedical research, yet the process itself has been very difficult to study because of its intrinsically dynamic character. Here, we have been able to quantitatively reconstruct the complete binding process of the enzyme-inhibitor complex trypsin-benzamidine by performing 495 molecular dynamics simulations of free ligand binding of 100 ns each, 187 of which produced binding events with an rmsd less than 2 Å compared to the crystal structure. The binding paths obtained are able to capture the kinetic pathway of the inhibitor diffusing from solvent (S0) to the bound (S4) state passing through two metastable intermediate states S2 and S3. Rather than directly entering the binding pocket the inhibitor appears to roll on the surface of the protein in its transition between S3 and the final binding pocket, whereas the transition between S2 and the bound pose requires rediffusion to S3. An estimation of the standard free energy of binding gives Δ G ° = -5.2 ± 0.4 kcal/mol (cf. the experimental value -6.2 kcal/mol), and a two-states kinetic model k on = (1.5 ± 0.2) × 10 8 M -1 s -1 and k off = (9.5 ± 3.3) × 10 4 s -1 for unbound to bound transitions. The ability to reconstruct by simple diffusion the binding pathway of an enzyme-inhibitor binding process demonstrates the predictive power of unconventional high-throughput molecular simulations. Moreover, the methodology is directly applicable to other molecular systems and thus of general interest in biomedical and pharmaceutical research.

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction and myocardial infarction. Several drugs that increase the activity of this enzyme are now in clinical phase of development: some of them are heme-dependent and might interact with the catalytic domain and others are heme-independent and supposedly bind to the sensory domain. The absence of reliable structural information is one of the factors that have precluded knowledge of the precise site of interaction of these molecules and of the mechanism of activation of the enzyme.