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.

Comparative molecular dynamics simulations of pathogenic and non-pathogenic huntingtin protein monomers and dimers.

Polyglutamine expansion at the N-terminus of the huntingtin protein exon 1 (Htt-ex1) is closely associated with a number of neurodegenerative diseases, which result from the aggregation of the increased polyQ repeat. However, the underlying structures and aggregation mechanism are still poorly understood. We performed microsecond-long all-atom molecular dynamics simulations to study the folding and dimerization of Htt-ex1 (about 100 residues) with non-pathogenic and pathogenic polyQ lengths, and uncovered substantial differences. The non-pathogenic monomer adopts a long α-helix that includes most of the polyQ residues, which forms the interaction interface for dimerization, and a PPII-turn-PPII motif in the proline-rich region. In the pathogenic monomer, the polyQ region is disordered, leading to compact structures with many intra-protein interactions and the formation of short ß-sheets. Dimerization can proceed via different modes, where those involving the N-terminal headpiece bury more hydrophobic residues and are thus more stable. Moreover, in the pathogenic Htt-ex1 dimers the proline-rich region interacts with the polyQ region, which slows the formation of ß-sheets.

Molecular simulations of IDPs: From ensemble generation to IDP interactions leading to disorder-to-order transitions.

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure but do exhibit some dynamical and structural ordering. The structural plasticity of IDPs indicates that entropy-driven motions are crucial for their function. Many IDPs undergo function-related disorder-to-order transitions upon by their interaction with specific binding partners. Approaches that are based on both experimental and theoretical tools enable the biophysical characterization of IDPs. Molecular simulations provide insights into IDP structural ensembles and disorder-to-order transition mechanisms. However, such studies depend strongly on the chosen force field parameters and simulation techniques. In this chapter, we provide an overview of IDP characteristics, review all-atom force fields recently developed for IDPs, and present molecular dynamics-based simulation methods that allow IDP ensemble generation as well as the characterization of disorder-to-order transitions. In particular, we introduce metadynamics, replica exchange molecular dynamics simulations, and also kinetic models resulting from Markov State modeling, and provide various examples for the successful application of these simulation methods to IDPs.

Amyloid-ß peptide dimers undergo a random coil to ß-sheet transition in the aqueous phase but not at the neuronal membrane.

Mounting evidence suggests that the neuronal cell membrane is the main site of oligomer-mediated neuronal toxicity of amyloid-ß peptides in Alzheimer's disease. To gain a detailed understanding of the mutual interference of amyloid-ß oligomers and the neuronal membrane, we carried out microseconds of all-atom molecular dynamics (MD) simulations on the dimerization of amyloid-ß (Aß)42 in the aqueous phase and in the presence of a lipid bilayer mimicking the in vivo composition of neuronal membranes. The dimerization in solution is characterized by a random coil to ß-sheet transition that seems on pathway to amyloid aggregation, while the interactions with the neuronal membrane decrease the order of the Aß42 dimer by attenuating its propensity to form a ß-sheet structure. The main lipid interaction partners of Aß42 are the surface-exposed sugar groups of the gangliosides GM1. As the neurotoxic activity of amyloid oligomers increases with oligomer order, these results suggest that GM1 is neuroprotective against Aß-mediated toxicity.

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.

Role of Oxidized Gly25, Gly29, and Gly33 Residues on the Interactions of Aß1-42 with Lipid Membranes.

Oxidative stress is known to play an important role in the pathogenesis of Alzheimer's disease. Moreover, it is becoming increasingly evident that the plasma membrane of neurons plays a role in modulating the aggregation and toxicity of Alzheimer's amyloid-ß peptide (Aß). In this study, the combined and interdependent effects of oxidation and membrane interactions on the 42 residues long Aß isoform are investigated using molecular simulations. Hamiltonian replica exchange molecular dynamics simulations are utilized to elucidate the impact of selected oxidized glycine residues of Aß42 on the interactions of the peptide with a model membrane comprised of 70% POPC, 25% cholesterol, and 5% of the ganglioside GM1. The main findings are that, independent of the oxidation state, Aß prefers binding to GM1 over POPC, which is further enhanced by the oxidation of Gly29 and Gly33 and reduced the formation of ß-sheet. Our results suggest that the differences observed in Aß42 conformations and its interaction with a lipid bilayer upon oxidation originate from the position of the oxidized Gly residue with respect to the hydrophobic sequence of Aß42 involving the Gly29-XXX-Gly33-XXX-Gly37 motif and from specific interactions between the peptide and the terminal sugar groups of GM1.

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.

Hotspot Identification on Protein Surfaces Using Probe-Based MD Simulations: Successes and Challenges.

Molecular Dynamics (MD) based computational co-solvent mapping methods involve the generation of an ensemble of MD-sampled target protein conformations and using selected small molecule fragments to identify and characterize binding sites on the surface of a target protein. This approach incorporates atomic-level solvation effects and protein mobility. It has shown great promise in the identification of conventional competitive and allosteric binding sites. It is also currently emerging as a useful tool in the early stages of drug discovery. This review summarizes efforts as well as discusses some methodological advances and challenges in binding site identification process through these co-solvent mapping methods.

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.

Computational Equilibrium Thermodynamic and Kinetic Analysis of K-Ras Dimerization through an Effector Binding Surface Suggests Limited Functional Role.

Dimer formation is believed to have a substantial impact on regulating K-Ras function. However, the evidence for dimerization and the molecular details of the process are scant. In this study, we characterize a K-Ras pseudo-C2-symmetric dimerization interface involving the effector interacting ß2-strand. We used structure matching and all-atom molecular dynamics (MD) simulations to predict, refine, and investigate the stability of this interface. Our MD simulation suggested that the ß2-dimer is potentially stable and remains relatively close to its initial conformation due to the presence of a number of hydrogen bonds, ionic salt bridges, and other favorable interactions. We carried out potential of mean force calculations to determine the relative binding strength of the interface. The results of these calculations indicated that the ß2 dimerization interface provides a weak binding free energy in solution and a dissociation constant that is close to 1 mM. Analyses of Brownian dynamics simulations suggested an association rate kon ≈ 10(5)-10(6) M(-1) s(-1). Combining these observations with available literature data, we propose that formation of auto-inhibited ß2 K-Ras dimers is possible but its fraction in cells is likely very small under normal physiologic conditions.

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.

Investigation of Changes in Tetracycline Repressor Binding upon Mutations in the Tetracycline Operator.

The tetracycline operon is an important gene network component, commonly used in synthetic biology applications because of its switch-like character. At the heart of this system is the highly specific interaction of the tet repressor protein (TetR) with its cognate DNA sequence (tetO). TetR binding on tetO practically stops expression of genes downstream of tetO by excluding RNA polymerase from binding the promoter and initiating transcription. Mutating the tetO sequence alters the strength of TetR-tetO binding and thus provides a tool to synthetic biologists to manipulate gene expression levels. We employ molecular dynamics (MD) simulations coupled with the free energy perturbation method to investigate the binding affinity of TetR to different tetO mutants. We also carry out in vivo tests in Escherichia coli for a series of promoters based on these mutants. We obtain reasonable agreement between experimental green fluorescent protein (GFP) repression levels and binding free energy differences computed from molecular simulations. In all cases, the wild-type tetO sequence yields the strongest TetR binding, which is observed both experimentally, in terms of GFP levels, and in simulation, in terms of free energy changes. Two of the four tetO mutants we tested yield relatively strong binding, whereas the other two mutants tend to be significantly weaker. The clustering and relative ranking of this subset of tetO mutants is generally consistent between our own experimental data, previous experiments with different systems and the free energy changes computed from our simulations. Overall, this work offers insights into an important synthetic biological system and demonstrates the potential, as well as limitations of molecular simulations to quantitatively explain biologically relevant behavior.

Intracellular crowding effects on the self-association of the bacterial cell division protein FtsZ.

The dimerization rate of the bacterial cell division protein FtsZ is strongly affected by the intracellular crowding. Yet the complexity of the intracellular environment makes it difficult to investigate via all-atom molecular dynamics or other detailed theoretical methods. We study the crowding effect on FtsZ dimerization which is the first step of an oligomerization process that results in more elaborate supramolecular structures. In particular, we consider the effect of intracellular crowding on the reaction rates, and their dependence on the different concentrations of crowding agents. We achieved this goal by using Brownian dynamics (BD) simulation techniques and a modified post-processing approach in which we decompose the rate constant in crowded media as a product of the rate constant in the dilute solution times a factor that incorporates the crowding effect. The latter factor accounts for the diffusion reduction and crowder induced energy. In addition we include the crowding effects on water viscosity in the BD simulations of crowded media. We finally show that biomolecular crowding has a considerable effect on the FtsZ dimerization by increasing the dimerization rate constant from 2.6×10(7)M(-1)s(-1) in the absence of crowders to 1.0×10(8)M(-1)s(-1) at crowding level of 0.30.

Aggregation behavior of ibuprofen, cholic acid and dodecylphosphocholine micelles.

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID-BA or NSAID-BA-PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA-IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA-IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA-IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA-IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.

The role of conserved waters in conformational transitions of Q61H K-ras.

To investigate the stability and functional role of long-residence water molecules in the Q61H variant of the signaling protein K-ras, we analyzed all available Ras crystal structures and conformers derived from a series of independent explicit solvent molecular dynamics (MD) simulations totaling 1.76 µs. We show that the protein samples a different region of phase space in the presence and absence of several crystallographically conserved and buried water molecules. The dynamics of these waters is coupled with the local as well as the global motions of the protein, in contrast to less buried waters whose exchange with bulk is only loosely coupled with the motion of loops in their vicinity. Aided by two novel reaction coordinates involving the distance (d) between the C(α) atoms of G60 at switch 2 and G10 at the P-loop and the N-C(α)-C-O dihedral (ξ) of G60, we further show that three water molecules located in lobe1, at the interface between the lobes and at lobe2, are involved in the relative motion of residues at the two lobes of Q61H K-ras. Moreover, a d/ξ plot classifies the available Ras x-ray structures and MD-derived K-ras conformers into active GTP-, intermediate GTP-, inactive GDP-bound, and nucleotide-free conformational states. The population of these states and the transition between them is modulated by water-mediated correlated motions involving the functionally critical switch 2, P-loop and helix 3. These results suggest that water molecules act as allosteric ligands to induce a population shift among distinct switch 2 conformations that differ in effector recognition.

Structure and dynamics of cholic acid and dodecylphosphocholine-cholic acid aggregates.

Bile acids are powerful detergents that emulsify and solubilize lipids, vitamins, cholesterol and other molecules in the biliary tract and intestines. It has long been known that bile acids form soluble mixed micelles with lipids. However, the detailed thermodynamic and structural properties of these micelles are not fully understood. This study sheds light on this issue based on results from multiple molecular dynamics simulations of cholic acid (CA) and dodecylphosphocholine (DPC) mixed micelles. We found that CA molecules form aggregates of up to 12 monomers with a mean size of 5-6. In agreement with several previous simulations and earlier predictions, the overall shape of these CA clusters is oblate disk-like such that the methyl groups point toward the core of the aggregate and the hydroxyl groups point away from it. The self-aggregation behavior of the CA clusters in the DPC-CA mixture is similar to the pure CA. Furthermore, the sizes and aggregation numbers of the DPC-CA mixed micelles are linearly dependent on CA molarity. In agreement with the radial shell model of Nichols and Ozarowski [Nichols, J. W.; Ozarowski, J. Biochemistry 1990, 29, 4600], our results demonstrate that CA molecules form a wedge between the DPC molecules with their hydroxyl and carboxyl groups facing the aqueous phase while their methyl groups are buried in and face the hydrocarbon core of the DPC micelle. The DPC-CA micelles simulated here tend to be spherical to prolate in shape, with the deviation from spherical geometry significantly increasing with increasing CA:DPC ratio.

Determining the orientation of protegrin-1 in DLPC bilayers using an implicit solvent-membrane model.

Continuum models that describe the effects of solvent and biological membrane molecules on the structure and behavior of antimicrobial peptides, holds a promise to improve our understanding of the mechanisms of antimicrobial action of these peptides. In such methods, a lipid bilayer model membrane is implicitly represented by multiple layers of relatively low dielectric constant embedded in a high dielectric aqueous solvent, while an antimicrobial peptide is accounted for by a dielectric cavity with fixed partial charge at the center of each one of its atoms. In the present work, we investigate the ability of continuum approaches to predict the most probable orientation of the beta-hairpin antimicrobial peptide Protegrin-1 (PG-1) in DLPC lipid bilayers by calculating the difference in the transfer free energy from an aqueous environment to a membrane-water environment for multiple orientations. The transfer free energy is computed as a sum of two terms; polar/electrostatic and non-polar. They both include energetic and entropic contributions to the free energy. We numerically solve the Poisson-Boltzmann equation to calculate the electrostatic contribution to the transfer free energy, while the non-polar contribution to the free energy is approximated using a linear solvent accessible surface area relationships. The most probable orientation of PG-1 is that with the lowest relative transfer free energy. Our simulation results indicate that PG-1 assumes an oblique orientation in DLPC lipid bilayers. The predicted most favorable orientation was with a tilt angle of 19 degrees, which is in qualitative agreement with the experimentally observed orientations derived from solid-state NMR data.