Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. © 2012 Isin et al

A Computational Assay of Estrogen Receptor alpha Antagonists Reveals the Key Common Structural Traits of Drugs Effectively Fighting Refractory Breast Cancers

Somatic mutations of the Estrogen Receptor alpha (ER alpha) occur with an up to 40% incidence in ER sensitive breast cancer (BC) patients undergoing prolonged endocrine treatments. These polymorphisms are implicated in acquired resistance, disease relapse, and increased mortality rates, hence representing a current major clinical challenge. Here, multi-microseconds (12.5 mu s) molecular dynamics simulations revealed that recurrent ER alpha. polymorphisms (i.e. L536Q, Y5375, Y537N, D538G) (mER alpha) are constitutively active in their apo form and that they prompt the selection of an agonist (active)-like conformation even upon antagonists binding. Interestingly, our simulations rationalize, for thefirst time, the efficacy profile of (pre)clinically used Selective Estrogen Receptor Modulators/Downregulators (SERMs/SERDs) against these variants, enlightening, at atomistic level of detail, the key common structural traits needed by drugs able to effectively fight refractory BC types. This knowledge represents a key advancement for mechanism-based therapeutics targeting resistant ER alpha isoforms, potentially allowing the community to move a step closer to 'precision medicine' calibrated on patients' genetic profiles and disease progression

A mathematical and computational review of Hartree-Fock SCF methods in Quantum Chemistry

We present here a review of the fundamental topics of Hartree-Fock theory in Quantum Chemistry. From the molecular Hamiltonian, using and discussing the Born-Oppenheimer approximation, we arrive to the Hartree and Hartree-Fock equations for the electronic problem. Special emphasis is placed in the most relevant mathematical aspects of the theoretical derivation of the final equations, as well as in the results regarding the existence and uniqueness of their solutions. All Hartree-Fock versions with different spin restrictions are systematically extracted from the general case, thus providing a unifying framework. Then, the discretization of the one-electron orbitals space is reviewed and the Roothaan-Hall formalism introduced. This leads to a exposition of the basic underlying concepts related to the construction and selection of Gaussian basis sets, focusing in algorithmic efficiency issues. Finally, we close the review with a section in which the most relevant modern developments (specially those related to the design of linear-scaling methods) are commented and linked to the issues discussed. The whole work is intentionally introductory and rather self-contained, so that it may be useful for non experts that aim to use quantum chemical methods in interdisciplinary applications. Moreover, much material that is found scattered in the literature has been put together here to facilitate comprehension and to serve as a handy reference.Comment: 64 pages, 3 figures, tMPH2e.cls style file, doublesp, mathbbol and subeqn package

Protonation States of Remote Residues Affect Binding-Release Dynamics of the Ligand but not the Conformation of apo Ferric Binding Protein

We have studied the apo (Fe3+ free) form of periplasmic ferric binding protein (FbpA) under different conditions and we have monitored the changes in the binding and release dynamics of H2PO4- that acts as a synergistic anion in the presence of Fe3+. Our simulations predict a dissociation constant of 2.2$\pm$0.2 mM which is in remarkable agreement with the experimentally measured value of 2.3$\pm$0.3 mM under the same ionization strength and pH conditions. We apply perturbations relevant for changes in environmental conditions as (i) different values of ionic strength (IS), and (ii) protonation of a group of residues to mimic a different pH environment. Local perturbations are also studied by protonation or mutation of a site distal to the binding region that is known to mechanically manipulate the hinge-like motions of FbpA. We find that while the average conformation of the protein is intact in all simulations, the H2PO4- dynamics may be substantially altered by the changing conditions. In particular, the bound fraction which is 20$\%$ for the wild type system is increased to 50$\%$ with a D52A mutation/protonation and further to over 90$\%$ at the protonation conditions mimicking those at pH 5.5. The change in the dynamics is traced to the altered electrostatic distribution on the surface of the protein which in turn affects hydrogen bonding patterns at the active site. The observations are quantified by rigorous free energy calculations. Our results lend clues as to how the environment versus single residue perturbations may be utilized for regulation of binding modes in hFbpA systems in the absence of conformational changes.Comment: 26 pages, 4 figure

Small-Molecule Inhibitors of Dengue-Virus Entry

Flavivirus envelope protein (E) mediates membrane fusion and viral entry from endosomes. A low-pH induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal “stem" of the E ectodomain draw together the viral and cellular membranes. We found stem-derived peptides from dengue virus (DV) bind stem-less E trimer and mimic the stem-reconfiguration step in the fusion pathway. We adapted this experiment as a high-throughput screen for small molecules that block peptide binding and thus may inhibit viral entry. A compound identified in this screen, 1662G07, and a number of its analogs reversibly inhibit DV infectivity. They do so by binding the prefusion, dimeric E on the virion surface, before adsorption to a cell. They also block viral fusion with liposomes. Structure-activity relationship studies have led to analogs with submicromolar IC90s against DV2, and certain analogs are active against DV serotypes 1,2, and 4. The compounds do not inhibit the closely related Kunjin virus. We propose that they bind in a previously identified, E-protein pocket, exposed on the virion surface and although this pocket is closed in the postfusion trimer, its mouth is fully accessible. Examination of the E-trimer coordinates (PDB 1OK8) shows that conformational fluctuations around the hinge could open the pocket without dissociating the trimer or otherwise generating molecular collisions. We propose that compounds such as 1662G07 trap the sE trimer in a “pocket-open" state, which has lost affinity for the stem peptide and cannot support the final “zipping up" of the stem

Predictive Power of Molecular Dynamics Receptor Structures in Virtual Screening

Molecular dynamics (MD) simulation is a well-established method for understanding protein dynamics. Conformations from unrestrained MD simulations have yet to be assessed for blind virtual screening (VS) by docking. This study presents a critical analysis of the predictive power of MD snapshots to this regard, evaluating two well-characterized systems of varying flexibility in ligand-bound and unbound configurations. Results from such VS predictions are discussed with respect to experimentally determined structures. In all cases, MD simulations provide snapshots that improve VS predictive power over known crystal structures, possibly due to sampling more relevant receptor conformations. Additionally, MD can move conformations previously not amenable to docking into the predictive range