Investigating the effects of cysteine-118 oxidation on G12D KRas structure and dynamics: insights from MD simulations.

Mutations of Ras proteins are believed to be among the most prominent causes of cancer. There is increasing evidence that the activity of Ras may be controlled by the redox state of cysteine residues located within the NKCD motif. This redox signaling is critical to both physiological and pathological processes and occurs when C118 is oxidized in a reversible manner. In this study, we used atomistic molecular dynamics simulations and Markov state models to investigate the structural and conformational effects of C118 oxidation on the oncogenic mutant KRas(G12D). While both mutants share common features and exhibit some distinct conformational states and fluctuations, we have found that the oxidized variant KRas(G12D/C118SOH) is more dynamic than the unoxidized counterpart, particularly in the switch II region. Additionally, C118 oxidation is found to alter the structure of the nucleotide-binding site and the switch regions as well as perturb the conformational equilibrium between Ras active and inactive states. These conformational preferences may alter the affinity to different effectors, resulting in selective downstream activation. Our results are anticipated to help future drug development efforts aimed at KRAS-related anticancer treatment.Communicated by Ramaswamy H. Sarma.

How to make an undruggable enzyme druggable: lessons from ras proteins.

Significant advances have been made toward discovering allosteric inhibitors for challenging drug targets such as the Ras family of membrane-associated signaling proteins. Malfunction of Ras proteins due to somatic mutations is associated with up to a quarter of all human cancers. Computational techniques have played critical roles in identifying and characterizing allosteric ligand-binding sites on these proteins, and to screen ligand libraries against those sites. These efforts, combined with a wide range of biophysical, structural, biochemical and cell biological experiments, are beginning to yield promising inhibitors to treat malignancies associated with mutated Ras proteins. In this chapter, we discuss some of these developments and how the lessons learned from Ras might be applied to similar other challenging drug targets.

Conformational and Dynamical Effects of Tyr32 Phosphorylation in K-Ras: Molecular Dynamics Simulation and Markov State Models Analysis.

Phosphorylation of tyrosine 32 in K-Ras has been shown to influence function by disrupting the GTPase cycle. To shed light on the underlying mechanism and atomic basis of this process, we carried out a comparative investigation of the oncogenic G12D K-Ras mutant and its phosphorylated variant (pTyr32) using all-atom molecular dynamics simulations and Markov state models. We show that, despite sharing a number of common features, G12D and pTyr32-G12D K-Ras exhibit some distinct conformational states and fluctuations. In addition to notable differences in conformation and dynamics of residues surrounding the GTP binding site, nonlocal changes were observed at a number of loops. Switch I is more flexible in pTyr32-G12D K-Ras while switch II is more flexible in G12D K-Ras. We also used time-lagged independent component analysis and k-means clustering to identify five metastable states for each system. We utilized transition path theory to calculate the transition probabilities for each state to build a Markov state model for each system. These models and other close inspections suggest that the phosphorylation of Tyr32 strongly affects protein dynamics and the active site conformation, especially with regards to the canonical switch conformations and dynamics.

Spatiotemporal Analysis of K-Ras Plasma Membrane Interactions Reveals Multiple High Order Homo-oligomeric Complexes.

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers.

Distinct dynamics and interaction patterns in H- and K-Ras oncogenic P-loop mutants.

Despite years of study, the structural or dynamical basis for the differential reactivity and oncogenicity of Ras isoforms and mutants remains unclear. In this study, we investigated the effects of amino acid variations on the structure and dynamics of wild type and oncogenic mutants G12D, G12V, and G13D of H- and K-Ras proteins. Based on data from µs-scale molecular dynamics simulations, we show that the overall structure of the proteins remains similar but there are important differences in dynamics and interaction networks. We identified differences in residue interaction patterns around the canonical switch and distal loop regions, and persistent sodium ion binding near the GTP particularly in the G13D mutants. Our results also suggest that different Ras variants have distinct local structural features and interactions with the GTP, variations that have the potential to affect GTP release and hydrolysis. Furthermore, we found that H-Ras proteins and particularly the G12V and G13D variants are significantly more flexible than their K-Ras counterparts. Finally, while most of the simulated proteins sampled the effector-interacting state 2 conformational state, G12V and G13D H-Ras adopted an open switch state 1 conformation that is defective in effector interaction. These differences have implications for Ras GTPase activity, effector or exchange factor binding, dimerization and membrane interaction. Proteins 2017; 85:1618-1632. © 2017 Wiley Periodicals, Inc.

Mixed-Probe Simulation and Probe-Derived Surface Topography Map Analysis for Ligand Binding Site Identification.

Membrane proteins represent a considerable fraction of pharmaceutical drug targets. A computational technique to identify ligand binding pockets in these proteins is therefore of great importance. We recently reported such a technique called pMD-membrane that utilizes small molecule probes to detect ligand binding sites and surface hotspots on membrane proteins based on probe-based molecular dynamics simulation. The current work extends pMD-membrane to a diverse set of small organic molecular species that can be used as cosolvents during simulation of membrane proteins. We also describe a projection technique for globally quantifying probe densities on the protein surface and introduce a technique to construct surface topography maps directly from the probe-binding propensity of surface residues. The map reveals surface patterns and geometric features that aid in filtering out high probe density hotspots lacking pocketlike characteristics. We demonstrate the applicability of the extended pMD-membrane and the new analysis tool by exploring the druggability of full-length G12D, G12V, and G13D oncogenic K-Ras mutants bound to a negatively charged lipid bilayer. Using data from 30 pMD-membrane runs conducted in the presence of a 2.8 M cosolvent made up of an equal proportion of seven small organic molecules, we show that our approach robustly identifies known allosteric ligand binding sites and other reactive regions on K-Ras. Our results also show that accessibility of some pockets is modulated by differential membrane interactions.

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers.

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.