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.     

A poke in the eye: inhibiting HIV-1 protease through its flap-recognition pocket

A novel mechanism of inhibiting HIV-1 protease (HIVp) is presented. Using computational solvent mapping to identify complementary interactions and the Multiple Protein Structure method to incorporate protein flexibility, we generated a receptor-based pharmacophore model of the flexible flap region of the semiopen, apo state of HIVp. Complementary interactions were consistently observed at the base of the flap, only within a cleft with a specific structural role. In the closed, bound state of HIVp, each flap tip docks against the opposite monomer, occupying this cleft. This flap-recognition site is filled by the protein and cannot be identified using traditional approaches based on bound, closed structures. Virtual screening and dynamics simulations show how small molecules can be identified to complement this cleft. Subsequent experimental testing confirms inhibitory activity of this new class of inhibitor. This may be the first new inhibitor class for HIVp since dimerization inhibitors were introduced 17 years ago.     

Antibody humanization by molecular dynamics simulations—in‐silico guided selection of critical backmutations

Monoclonal antibodies represent the fastest growing class of biotherapeutic proteins. However, as they are often initially derived from rodent organisms, there is a severe risk of immunogenic reactions, hampering their applicability. The humanization of these antibodies remains a challenging task in the context of rational drug design. “Superhumanization” describes the direct transfer of the complementarity determining regions to a human germline framework, but this humanization approach often results in loss of binding affinity. In this study, we present a new approach for predicting promising backmutation sites using molecular dynamics simulations of the model antibody Ab2/3H6. The simulation method was developed in close conjunction with novel specificity experiments. Binding properties of mAb variants were evaluated directly from crude supernatants and confirmed using established binding affinity assays for purified antibodies. Our approach provides access to the dynamical features of the actual binding sites of an antibody, based solely on the antibody sequence. Thus we do not need structural data on the antibody–antigen complex and circumvent cumbersome methods to assess binding affinities. © 2016 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.     

Coarse-grained dynamics of the receiver domain of NtrC: fluctuations, correlations and implications for allosteric cooperativity

Receiver domains are key molecular switches in bacterial signaling. Structural studies have shown that the receiver domain of the nitrogen regulatory protein C (NtrC) exists in a conformational equilibrium encompassing both inactive and active states, with phosphorylation of Asp54 allosterically shifting the equilibrium towards the active state. To analyze dynamical fluctuations and correlations in NtrC as it undergoes activation, we have applied a coarse-grained dynamics algorithm using elastic network models. Normal mode analysis reveals possible dynamical pathways for the transition of NtrC from the inactive state to the active state. The diagonalized correlation between the inactive and the active (phosphorylated) state shows that most correlated motions occur around the active site of Asp54 and in the region Thr82 to Tyr101. This indicates a coupled correlation of dynamics in the "Thr82-Tyr101" motion. With phosphorylation inducing significant flexibility changes around the active site and alpha3 and alpha4 helices, we find that this activation makes the active-site region and the loops of alpha3/beta4 and alpha4/beta5 more stable. This means that phosphorylation entropically favors the receiver domain in its active state, and the induced conformational changes occur in an allosteric manner. Analyses of the local flexibility and long-range correlated motion also suggest a dynamics criterion for determining the allosteric cooperativity of NtrC, and may be applicable to other proteins.     

Langevin dynamics simulations reveal biologically relevant folds arising from the incorporation of a torsional potential

The conformational behaviour of polymer chains has been examined using Langevin dynamics simulation techniques. Polymer chains were modelled as “beads” undergoing Brownian motion in a defined potential that accounted for stretching, bending and solvation energies. As expected, the competition between chain stiffness and solvent interactions was found to yield standard swollen or collapsed configurations in good or poor solvents, respectively. However, when a torsional term was introduced into the model, additional biologically relevant conformations such as helices, sheets, turns and hairpins naturally arose.     

Using affinity chromatography to engineer and characterize pH‐dependent protein switches

Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione‐S‐transferase to convert this protein into a pH‐dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH‐dependent GSH‐binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector‐specific allostery into a protein structure.     

Molecular dynamics of oligopeptides. A comparative study of residue interactions in dipeptides

The molecular dynamics of dipeptides of natural amino acids were examined using protocols that do not violate the principle of equal distribution of energy over the degrees of freedom. Comparative analysis involved autocorrelation functions of complex exponentials from dihedrals. The mutual influence of residues was classified by the effects on the dynamic properties of the neighbors.     

Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non-fluorinated parallel dimeric coiled-coils

The alpha-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of the coiled-coil motifs, including those modified by fluorination, several fluorinated and nonfluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiled-coil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER used a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, and ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly, better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller L-ornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled-coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine when compared with 5,5,5,5',5',5'-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5',5',5'-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may provide a driving force for coiled-coil dimer formation. Estimation of the ellipticity at 222 nm from a series of snapshots from the MD simulations with DicroCalc shows distinct increases in the ellipticity when the coiled-coil is fluorinated, which suggests that the helicity in the folded coiled-coils is greater when fluorinated.     

Mass spectrometry‐based methods to study protein architecture and dynamics

Mass spectrometry is now an indispensable tool in the armamentarium of molecular biophysics, where it is used for tasks ranging from protein sequencing and mapping of post‐translational modifications to studies of higher order structure, conformational dynamics, and interactions of proteins with small molecule ligands and other biopolymers. This mini‐review highlights several popular mass spectrometry‐based tools that are now commonly used for structural studies of proteins beyond their covalent structure with a particular emphasis on hydrogen exchange and direct electrospray ionization mass spectrometry.     

Self‐assembly and chiroptical properties in supramolecular complexes of adenosine phosphates and guanidinium‐bispyrene

Supramolecular systems that respond to the hydrolysis of adenosine phosphates (APs) are attractive for biosensing and to fabricate bioinspired self‐assembled materials. Here, we report on the formation of supramolecular complexes between an achiral guanidinium derivative bearing two pyrene moieties, with each of the three adenosine phosphates: AMP, ADP, and ATP. By combining results from circular dichroism spectroscopy and molecular modeling simulations, we explore the induced chirality, the dynamics of the complexes, and the interactions at play, which altogether provide insights into the supramolecular self‐assembly between APs and the guanidinium‐bispyrene. Finally, we identify the chiroptical signals of interest in mixtures of the guanidinium derivative with the three APs in different proportions. This study constitutes a basis to evolve toward a chiroptical detection of the hydrolysis of APs based on organic supramolecular probes.     

pH‐dependent conformational dynamics of beta‐secretase 1: A molecular dynamics study

Beta‐secretase 1 (BACE‐1) is an aspartyl protease implicated in the overproduction of β‐amyloid fibrils responsible for Alzheimer disease. The process of β‐amyloid genesis is known to be pH dependent, with an activity peak between solution pH of 3.5 and 5.5. We have studied the pH‐dependent dynamics of BACE‐1 to better understand the pH dependent mechanism. We have implemented support for graphics processor unit (GPU) accelerated constant pH molecular dynamics within the AMBER molecular dynamics software package and employed this to determine the relative population of different aspartyl dyad protonation states in the pH range of greatest β‐amyloid production, followed by conventional molecular dynamics to explore the differences among the various aspartyl dyad protonation states. We observed a difference in dynamics between double‐protonated, mono‐protonated, and double‐deprotonated states over the known pH range of higher activity. These differences include Tyr 71‐aspartyl dyad proximity and active water lifetime. This work indicates that Tyr 71 stabilizes catalytic water in the aspartyl dyad active site, enabling BACE‐1 activity.     

Self‐assembling study of sarcolipin and its mutants in multiple molecular dynamic simulations

The Sarcolipin (SLN) is a single trans‐membrane protein that can self‐assembly to dimer and oligomer for playing importantphysiological function. In this work, we addressed the dimerization of wild type SLN (wSLN) and its mutants (mSLNs) – I17A and I20A, using both coarse‐grained (CG) and atomistic (AT) molecular dynamics (MD) simulations. Our results demonstrated that wSLN homodimer assembled as a left‐handed helical complex, while mSLNs heterodimers assembled as right‐handed complexes. Analysis of residue‐residue contacts map indicated that isoleucine (Ile)‐leucione (Leu) zipper domain played an important role in dimerization. The potential of mean force (PMF) demonstrated that wSLN homodimer was more stable than mSLNs heterodimers. Meanwhile, the mSLNs heterodimers preferred right‐handed rather than left‐handed helix. AT‐MD simulations for wSLN and mSLNs were also in line with CG‐MD simulations. These results provided the insights for understanding the mechanisms of SLNs self‐assembling. Proteins 2017; 85:1065–1077. © 2017 Wiley Periodicals, Inc.     

Folding thermodynamics of the hybrid‐1 type intramolecular human telomeric G‐quadruplex

Guanine‐rich DNA sequences that may form G‐quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G‐quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G‐quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G‐rich sequences in the presence or the absence of their complementary C‐rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26‐meric human telomeric sequence d[A₃G₃(T₂AG₃)₃A₂]. In the presence of K⁺ ions, the latter adopts the hybrid‐1 G‐quadruplex conformation, a tightly packed structure with an unusually small number of solvent‐exposed atomic groups. The K⁺‐induced folding of the G‐quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G‐quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G‐quadruplex formation. Based on our volume data, 432 ± 19 water molecules become released to the bulk upon the G‐quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute–solvent interactions all over the surface of the folded structure. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 216–227, 2014.     

Structure refinement of protein model decoys requires accurate side‐chain placement

In this study, the application of temperature‐based replica‐exchange (T‐ReX) simulations for structure refinement of decoys taken from the I‐TASSER dataset was examined. A set of eight nonredundant proteins was investigated using self‐guided Langevin dynamics (SGLD) with a generalized Born implicit solvent model to sample conformational space. For two of the protein test cases, a comparison of the SGLD/T‐ReX method with that of a hybrid explicit/implicit solvent molecular dynamics T‐ReX simulation model is provided. Additionally, the effect of side‐chain placement among the starting decoy structures, using alternative rotamer conformations taken from the SCWRL4 modeling program, was investigated. The simulation results showed that, despite having near‐native backbone conformations among the starting decoys, the determinant of their refinement is side‐chain packing to a level that satisfies a minimum threshold of native contacts to allow efficient excursions toward the downhill refinement regime on the energy landscape. By repacking using SCWRL4 and by applying the RWplus statistical potential for structure identification, the SGLD/T‐ReX simulations achieved refinement to an average of 38% increase in the number of native contacts relative to the original I‐TASSER decoy sets and a 25% reduction in values of C_α root‐mean‐square deviation. The hybrid model succeeded in obtaining a sharper funnel to low‐energy states for a modeled target than the implicit solvent SGLD model; yet, structure identification remained roughly the same. Without meeting a threshold of near‐native packing of side chains, the T‐ReX simulations degrade the accuracy of the decoys, and subsequently, refinement becomes tantamount to the protein folding problem. Proteins 2013. 2012 Published by Wiley Periodicals, Inc.     

Structure and energy landscape of a photoswitchable peptide: a replica exchange molecular dynamics study

A replica exchange molecular dynamics (REMD) simulation of a bicyclic azobenzene peptide in explicit dimethyl sulfoxide solution is presented in order to characterize the conformational structures and energy landscape of a photoswitchable peptide. It is shown that an enhanced-sampling technique such as the REMD method is essential to obtain a converged conformational sampling of the peptide at room temperature. This is because conventional MD simulations of less than approximately 100-ns length are either trapped in local minima (at 295 K) or-if run at high temperature-do not resemble the room-temperature REMD results. Calculating various nuclear Overhauser effects (NOEs) and (3)J-couplings, a good overall agreement between the REMD simulations and the NMR experiments of Renner et al. (Biopolymers 2000;54:501-514) is found. In particular, the REMD study confirms the general picture drawn by Renner et al. that the trans-isomer of the azobenzene peptide exhibits a well-defined structure, while the cis-isomer is a conformational heterogeneous system; that is, the trans-isomer occurs in 2 well-defined conformers, while the cis-isomer represents an energetically frustrated system that leads to an ensemble of conformational structures. Employing a principal component analysis of the REMD data, the free energy landscape of the systems is studied at various temperatures. The implications for the folding and unfolding pathways of the system are discussed.     

Conformational dynamics of xylanase a from Streptomyces lividans: Implications for TIM‐barrel enzyme thermostability

The conformational dynamics of xylanase A from Streptomyces lividans (Sl‐XlnA) were studied using Molecular Dynamics (MD) simulation to identify the thermally sensitive regions. Sl‐XlnA begins to unfold at loop4 and this unfolding expands to the loops near the N‐terminus. The high flexibility of loop6 during the 300 K simulation is related to its function. The intense movements of the 3₁₀‐helices also affect the structural stability. The interaction between the α4β5‐loop and the neighboring α5β6‐loop plays a crucial role in stabilizing the region from the α4β5‐loop to α6. The most thermally sensitive region is from β3 to loop4. The high mobility of the long loop4 easily transfers to the adjacent β4 and α4 and causes β4 and α4 to fluctuate. And, salt bridges ASP124‐ARG79, ASP200‐ARG159, and ASP231‐LYS166 formed a “clamp” to stabilize the region including α4, β4, β5, β6, and β7. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 594–604, 2013.     

On the Atomic Velocities in Molecular and Langevin Dynamics Simulations of Soft-Sphere Systems

Abstract

Time dependent probability distributions of the changes of direction of atomic velocities are considered in order to examine in detail the shape of the trajectories obtained through molecular simulations. We have analysed the atomic motions obtained from molecular dynamics simulations of soft-sphere systems at three very different states, i.e. a dilute fluid, a liquid at high density, and a solid. The methodology has also been used to check the reliability of the velocity evolution obtained when it is assumed that a single particle obeys the generalized Langevin equation and the effect of the other particles is represented by friction and random forces.