CHARMM-GUI-Based Induced Fit Docking Workflow to Generate Reliable Protein-Ligand Binding Modes.

Molecular docking is a preferred method to predict ligand binding modes and their binding energy to target protein receptors, which is critical in early phase structure-based drug discovery. However, there is a persistent challenge in docking that can be attributed to the induced fit effect, as receptor binding sites undergo induced fit conformational changes upon ligand binding to achieve better binding modes. In this work, based on CHARMM-GUI LBS Finder& Refiner and High-Throughput Simulator, we present a straightforward CHARMM-GUI induced fit docking (CGUI-IFD) workflow to generate reliable protein-ligand binding modes. The CGUI-IFD workflow generates an ensemble of receptor binding site conformations through ligand-binding site (LBS) refinement, runs rigid receptor docking, and performs high-throughput molecular dynamics (MD) simulations of protein-ligand complex structures in explicit solvents. The results are evaluated based on the ligand root-mean-square deviation (RMSD)-based binding stability and the molecular mechanics generalized Born surface area binding energy. For a benchmark test, we used 258 cross-docking protein-ligand pairs across 41 target proteins from the Schrodinger IFD-MD data set. The application of CGUI-IFD on this data set shows 80% success rate (within 2.5 Å RMSD from the experimental structures). We expect that the CGUI-IFD workflow can be useful to generate reliable ligand binding modes for cross-docking cases.

Molecular basis of polyspecific drug and xenobiotic recognition by OCT1 and OCT2.

A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition. In mammals, organic cation transporter (OCT) subtypes 1 and 2 (OCT1 and OCT2, also known as SLC22A1 and SLC22A2, respectively) are polyspecific transporters responsible for the uptake and clearance of structurally diverse cationic compounds in the liver and kidneys, respectively. Notably, it is well established that human OCT1 and OCT2 play central roles in the pharmacokinetics and drug-drug interactions of many prescription medications, including metformin. Despite their importance, the basis of polyspecific cationic drug recognition and the alternating access mechanism for OCTs have remained a mystery. Here we present four cryo-electron microscopy structures of apo, substrate-bound and drug-bound OCT1 and OCT2 consensus variants, in outward-facing and outward-occluded states. Together with functional experiments, in silico docking and molecular dynamics simulations, these structures uncover general principles of organic cation recognition by OCTs and provide insights into extracellular gate occlusion. Our findings set the stage for a comprehensive structure-based understanding of OCT-mediated drug-drug interactions, which will prove critical in the preclinical evaluation of emerging therapeutics.

Molecular basis of polyspecific drug binding and transport by OCT1 and OCT2.

A wide range of endogenous and xenobiotic organic ions require facilitated transport systems to cross the plasma membrane for their disposition 1, 2 . In mammals, organic cation transporter subtypes 1 and 2 (OCT1 and OCT2, also known as SLC22A1 and SLC22A2, respectively) are polyspecific transporters responsible for the uptake and clearance of structurally diverse cationic compounds in the liver and kidneys, respectively 3, 4 . Notably, it is well established that human OCT1 and OCT2 play central roles in the pharmacokinetics, pharmacodynamics, and drug-drug interactions (DDI) of many prescription medications, including metformin 5, 6 . Despite their importance, the basis of polyspecific cationic drug recognition and the alternating access mechanism for OCTs have remained a mystery. Here, we present four cryo-EM structures of apo, substrate-bound, and drug-bound OCT1 and OCT2 in outward-facing and outward-occluded states. Together with functional experiments, in silico docking, and molecular dynamics simulations, these structures uncover general principles of organic cation recognition by OCTs and illuminate unexpected features of the OCT alternating access mechanism. Our findings set the stage for a comprehensive structure-based understanding of OCT-mediated DDI, which will prove critical in the preclinical evaluation of emerging therapeutics.

Mechanisms of isoform-specific residue influence on GTP-bound HRas, KRas, and NRas.

HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.

CHARMM-GUI Ligand Designer for Template-Based Virtual Ligand Design in a Binding Site.

Rational drug design involves a task of finding ligands that would bind to a specific target protein. This work presents CHARMM-GUI Ligand Designer that is an intuitive and interactive web-based tool to design virtual ligands that match the shape and chemical features of a given protein binding site. Ligand Designer provides ligand modification capabilities with 3D visualization that allow researchers to modify and redesign virtual ligands while viewing how the protein-ligand interactions are affected. Virtual ligands can also be parameterized for further molecular dynamics (MD) simulations and free energy calculations. Using 8 targets from 8 different protein classes in the directory of useful decoys, enhanced (DUD-E) data set, we show that Ligand Designer can produce similar ligands to the known active ligands in the crystal structures. Ligand Designer also produces stable protein-ligand complex structures when tested using short MD simulations. We expect that Ligand Designer can be a useful and user-friendly tool to design small molecules in any given potential ligand binding site on a protein of interest.

CHARMM-GUI LBS Finder & Refiner for Ligand Binding Site Prediction and Refinement.

A protein performs its task by binding a variety of ligands in its local region that is also known as the ligand-binding-site (LBS). Therefore, accurate prediction, characterization, and refinement of LBS can facilitate protein functional annotations and structure-based drug design. In this work, we present CHARMM-GUI LBS Finder & Refiner (https://www.charmm-gui.org/input/lbsfinder) that predicts potential LBS, offers interactive features for local LBS structure analysis, and prepares various molecular dynamics (MD) systems and inputs by setting up distance restraint potentials for LBS structure refinement. LBS Finder & Refiner supports 5 different commonly used simulation programs, such as NAMD, AMBER, GROMACS, GENESIS, and OpenMM, for LBS structure refinement together with hydrogen mass repartitioning. The capability of LBS Finder & Refiner is illustrated through LBS structure predictions and refinements of 48 modeled and 20 apo benchmark target proteins. Overall, successful LBS structure predictions and refinements are seen in our benchmark tests. We hope that LBS Finder & Refiner is useful to predict, characterize, and refine potential LBS on any given protein of interest.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.

Ligand-Binding-Site Refinement to Generate Reliable Holo Protein Structure Conformations from Apo Structures.

The first important step in a structure-based virtual screening is the judicious selection of a receptor protein. In cases where the holo protein receptor structure is unavailable, significant reduction in virtual screening performance has been reported. In this work, we present a robust method to generate reliable holo protein structure conformations from apo structures using molecular dynamics (MD) simulation with restraints derived from holo structure binding-site templates. We perform benchmark tests on two different datasets: 40 structures from a directory of useful decoy-enhanced (DUD-E) and 84 structures from the Gunasekaran dataset. Our results show successful refinement of apo binding-site structures toward holo conformations in 82% of the test cases. In addition, virtual screening performance of 40 DUD-E structures is significantly improved using our MD-refined structures as receptors with an average enrichment factor (EF), an EF1% value of 6.2 compared to apo structures with 3.5. Docking of native ligands to the refined structures shows an average ligand root mean square deviation (RMSD) of 1.97 Å (DUD-E dataset and Gunasekaran dataset) relative to ligands in the holo crystal structures, which is comparable to the self-docking (i.e., docking of the native ligand back to its crystal structure receptor) average, 1.34 Å (DUD-E dataset) and 1.36 Å (Gunasekaran dataset). On the other hand, docking to the apo structures yields an average ligand RMSD of 3.65 Å (DUD-E) and 2.90 Å (Gunasekaran). These results indicate that our method is robust and can be useful to improve virtual screening performance of apo structures.

Improving Protein-Ligand Docking Results with High-Throughput Molecular Dynamics Simulations.

Structure-based virtual screening relies on classical scoring functions that often fail to reliably discriminate binders from nonbinders. In this work, we present a high-throughput protein-ligand complex molecular dynamics (MD) simulation that uses the output from AutoDock Vina to improve docking results in distinguishing active from decoy ligands in a directory of useful decoy-enhanced (DUD-E) dataset. MD trajectories are processed by evaluating ligand-binding stability using root-mean-square deviations. We select 56 protein targets (of 7 different protein classes) and 560 ligands (280 actives, 280 decoys) and show 22% improvement in ROC AUC (area under the curve, receiver operating characteristics curve), from an initial value of 0.68 (AutoDock Vina) to a final value of 0.83. The MD simulation demonstrates a robust performance across all seven different protein classes. In addition, some predicted ligand-binding modes are moderately refined during MD simulations. These results systematically validate the reliability of a physics-based approach to evaluate protein-ligand binding interactions.

Ligand-Binding-Site Structure Refinement Using Molecular Dynamics with Restraints Derived from Predicted Binding Site Templates.

Accurate modeling of ligand-binding-site structures plays a critical role in structure-based virtual screening. However, the structures of the ligand-binding site in most predicted protein models are generally of low quality and need refinements. In this work, we present a ligand-binding-site structure refinement protocol using molecular dynamics simulation with restraints derived from predicted binding site templates. Our benchmark validation shows great performance for 40 diverse sets of proteins from the Astex list. The ligand-binding sites on modeled protein structures are consistently refined using our method with an average Cα RMSD improvement of 0.90 Å. Comparison of ligand binding modes from ligand docking to initial unrefined and refined structures shows an average of 1.97 Å RMSD improvement in the refined structures. These results demonstrate a promising new method of structure refinement for protein ligand-binding-site structures.

Predicting X-ray solution scattering from flexible macromolecules.

Wide-angle X-ray solution scattering (WAXS) patterns contain substantial information about the structure and dynamics of a protein. Solution scattering from a rigid protein can be predicted from atomic coordinate sets to within experimental error. However, structural fluctuations of proteins in solution can lead to significant changes in the observed intensities. The magnitude and form of these changes contain information about the nature and spatial extent of structural fluctuations in the protein. Molecular dynamics (MD) simulations based on a crystal structure and selected force field generate models for protein internal motions, and here we demonstrate that they can be used to predict the impact of structural fluctuations on solution scattering data. In cases where the observed and calculated intensities correspond, we can conclude that the X-ray scattering provides direct experimental validation of the structural and MD results. In cases where calculated and observed intensities are at odds, the inconsistencies can be used to determine the origins of these discrepancies. They may be because of overestimates or underestimates of structural fluctuations in MD simulations, under-sampling of the structural ensemble in the simulations, errors in the structural model, or a mismatch between the experimental conditions and the parameters used in carrying out the MD simulation.

Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD.

The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.