Electrostatic interactions and hydrogen bond dynamics in chloride pumping by halorhodopsin

Translocation of negatively charged ions across cell membranes by ion pumps raises the question as to how protein interactions control the location and dynamics of the ion. Here we address this question by performing extensive molecular dynamics simulations of wild type and mutant halorhodopsin, a seven-helical transmembrane protein that translocates chloride ions upon light absorption. We find that inter-helical hydrogen bonds mediated by a key arginine group largely govern the dynamics of the protein and water groups coordinating the chloride ion.     

On the satisfaction of backbone‐carbonyl lone pairs of electrons in protein structures

Protein structures are stabilized by a variety of noncovalent interactions (NCIs), including the hydrophobic effect, hydrogen bonds, electrostatic forces and van der Waals’ interactions. Our knowledge of the contributions of NCIs, and the interplay between them remains incomplete. This has implications for computational modeling of NCIs, and our ability to understand and predict protein structure, stability, and function. One consideration is the satisfaction of the full potential for NCIs made by backbone atoms. Most commonly, backbone‐carbonyl oxygen atoms located within α‐helices and β‐sheets are depicted as making a single hydrogen bond. However, there are two lone pairs of electrons to be satisfied for each of these atoms. To explore this, we used operational geometric definitions to generate an inventory of NCIs for backbone‐carbonyl oxygen atoms from a set of high‐resolution protein structures and associated molecular‐dynamics simulations in water. We included more‐recently appreciated, but weaker NCIs in our analysis, such as n→π* interactions, Cα‐H bonds and methyl‐H bonds. The data demonstrate balanced, dynamic systems for all proteins, with most backbone‐carbonyl oxygen atoms being satisfied by two NCIs most of the time. Combinations of NCIs made may correlate with secondary structure type, though in subtly different ways from traditional models of α‐ and β‐structure. In addition, we find examples of under‐ and over‐satisfied carbonyl‐oxygen atoms, and we identify both sequence‐dependent and sequence‐independent secondary‐structural motifs in which these reside. Our analysis provides a more‐detailed understanding of these contributors to protein structure and stability, which will be of use in protein modeling, engineering and design.     

Interaction of arginine,lysine, and guanidine with surface residues of lysozyme: implication to protein stability

Additives are widely used to suppress aggregation of therapeutic proteins. However, the molecular mechanisms of effect of additives to stabilize proteins are still unclear. To understand this, we herein perform molecular dynamics simulations of lysozyme in the presence of three commonly used additives: arginine, lysine, and guanidine. These additives have different effects on stability of proteins and have different structures with some similarities; arginine and lysine have aliphatic side chain, while arginine has a guanidinium group. We analyze atomic contact frequencies to study the interactions of the additives with individual residues of lysozyme. Contact coefficient, quantified from contact frequencies, is helpful in analyzing the interactions with the guanidine groups as well as aliphatic side chains of arginine and lysine. Strong preference for contacts to the additives (over water) is seen for the acidic followed by polar and the aromatic residues. Further analysis suggests that the hydration layer around the protein surface is depleted more in the presence of arginine, followed by lysine and guanidine. Molecular dynamics simulations also reveal that the internal dynamics of protein, as indicated by the lifetimes of the hydrogen bonds within the protein, changes depending on the additives. Particularly, we note that the side-chain hydrogen-bonding patterns within the protein differ with the additives, with several side-chain hydrogen bonds missing in the presence of guanidine. These results collectively indicate that the aliphatic chain of arginine and lysine plays a critical role in the stabilization of the protein.     

Modeling correlated main-chain motions in proteins for flexible molecular recognition

We describe a new method for modeling protein and ligand main-chain flexibility, and show its ability to model flexible molecular recognition. The goal is to sample the full conformational space, including large-scale motions that typically cannot be reached in molecular dynamics simulations due to the computational intensity, as well as conformations that have not been observed yet by crystallography or NMR. A secondary goal is to assess the degree of flexibility consistent with protein-ligand recognition. Flexibility analysis of the target protein is performed using the graph-theoretic algorithm FIRST, which also identifies coupled networks of covalent and noncovalent bonds within the protein. The available conformations of the flexible regions are then explored with ROCK by random-walk sampling of the rotatable bonds. ROCK explores correlated motions by only sampling dihedral angles that preserve the coupled bond networks in the protein and generates conformers with good stereochemistry, without using a computationally expensive potential function. A representative set of the conformational ensemble generated this way can be used as targets for docking with SLIDE, which handles the flexibility of protein and ligand side-chains. The realism of this protein main-chain conformational sampling is assessed by comparison with time-resolved NMR studies of cyclophilin A motions. ROCK is also effective for modeling the flexibility of large cyclic and polycyclic ligands, as demonstrated for cyclosporin and zearalenol. The use of this combined approach to perform docking with main-chain flexibility is illustrated for the cyclophilin A-cyclosporin complex and the estrogen receptor in complex with zearalenol, while addressing the question of how much flexibility is allowed without hindering molecular recognition.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.     

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

Revealing the processes of ligand–protein associations deepens our understanding of molecular recognition and binding kinetics. Hydrogen bonds (H‐bonds) play a crucial role in optimizing ligand–protein interactions and ligand specificity. In addition to the formation of stable H‐bonds in the final bound state, the formation of transient H‐bonds during binding processes contributes binding kinetics that define a ligand as a fast or slow binder, which also affects drug action. However, the effect of forming the transient H‐bonds on the kinetic properties is little understood. Guided by results from coarse‐grained Brownian dynamics simulations, we used classical molecular dynamics simulations in an implicit solvent model and accelerated molecular dynamics simulations in explicit waters to show that the position and distribution of the H‐bond donor or acceptor of a drug result in switching intermolecular and intramolecular H‐bond pairs during ligand recognition processes. We studied two major types of HIV‐1 protease ligands: a fast binder, xk263, and a slow binder, ritonavir. The slow association rate in ritonavir can be attributed to increased flexibility of ritonavir, which yields multistep transitions and stepwise entering patterns and the formation and breaking of complex H‐bond pairs during the binding process. This model suggests the importance of conversions of spatiotemporal H‐bonds during the association of ligands and proteins, which helps in designing inhibitors with preferred binding kinetics. Copyright © 2014 John Wiley & Sons, Ltd.     

Vibrational frequency shifts as a probe of hydrogen bonds: thermal expansion and glass transition of myoglobin in mixed solvents

The contribution of hydrogen bonds to protein-solvent interactions and their impact on structural flexibility and dynamics of myoglobin are discussed. The shift of vibrational peak frequencies with the temperature of myoglobin in sucrose/water and glycerol/water solutions is used to probe the expansion of the hydrogen bond network. We observe a characteristic change in the temperature slope of the O–H stretching frequency at the glass transition which correlates with the discontinuity of the thermal expansion coefficient. The temperature-difference spectra of the amide bands show the same tendency, indicating that stronger hydrogen bonding in the bulk affects the main-chain solvent interactions in parallel. However, the hydrogen bond strength decreases relative to the bulk solvent with increasing cosolvent concentration near the protein surface, which suggests preferential hydration. Weaker and/or fewer hydrogen bonds are observed at low degrees of hydration. The central O–H stretching frequency of protein hydration water is red-shifted by 40 cm^–1 relative to the bulk. The shift increases towards lower temperatures, consistent with contraction and increasing strength of the protein-water bonds. The temperature slope shows a discontinuity near 180 K. The contraction of the network has reached a critical limit which leads to frozen-in structures. This effect may represent the molecular mechanism underlying the dynamic transition observed for the mean square displacements of the protein atoms and the heme iron of myoglobin. Received: 10 July 1996 / Accepted: 10 April 1997     

The dominant interaction between peptide and urea is electrostatic in nature: a molecular dynamics simulation study

The conformational equilibrium of a blocked valine peptide in water and aqueous urea solution is studied using molecular dynamics simulations. Pair correlation functions indicate enhanced concentration of urea near the peptide. Stronger hydrogen bonding of urea-peptide compared to water-peptide is observed with preference for helical conformation. The potential of mean force, computed using umbrella sampling, shows only small differences between urea and water solvation that are difficult to quantify. The changes in solvent structure around the peptide are explained by favorable electrostatic interactions (hydrogen bonds) of urea with the peptide backbone. There is no evidence for significant changes in hydrophobic interactions in the two conformations of the peptide in urea solution. Our simulations suggest that urea denatures proteins by preferentially forming hydrogen bonds to the peptide backbone, reducing the barrier for exposing protein residues to the solvent, and reaching the unfolded state.     

The key event in force-induced unfolding of Titin's immunoglobulin domains

Steered molecular dynamics simulation of force-induced titin immunoglobulin domain I27 unfolding led to the discovery of a significant potential energy barrier at an extension of approximately 14 A on the unfolding pathway that protects the domain against stretching. Previous simulations showed that this barrier is due to the concurrent breaking of six interstrand hydrogen bonds (H-bonds) between beta-strands A' and G that is preceded by the breaking of two to three hydrogen bonds between strands A and B, the latter leading to an unfolding intermediate. The simulation results are supported by Angstrom-resolution atomic force microscopy data. Here we perform a structural and energetic analysis of the H-bonds breaking. It is confirmed that H-bonds between strands A and B break rapidly. However, the breaking of the H-bond between strands A' and G needs to be assisted by fluctuations of water molecules. In nanosecond simulations, water molecules are found to repeatedly interact with the protein backbone atoms, weakening individual interstrand H-bonds until all six A'-G H-bonds break simultaneously under the influence of external stretching forces. Only when those bonds are broken can the generic unfolding take place, which involves hydrophobic interactions of the protein core and exerts weaker resistance against stretching than the key event.     

Structural coupling between FKBP12 and buried water

Globular proteins often contain structurally well-resolved internal water molecules. Previously, we reported results from a molecular dynamics study that suggested that buried water (Wat3) may play a role in modulating the structure of the FK506 binding protein-12 (FKBP12) (Park and Saven, Proteins 2005; 60:450-463). In particular, simulations suggested that disrupting a hydrogen bond to Wat3 by mutating E60 to either A or Q would cause a structural perturbation involving the distant W59 side chain, which rotates to a new conformation in response to the mutation. This effectively remodels the ligand-binding pocket, as the side chain in the new conformation is likely to clash with bound FK506. To test whether the protein structure is in effect modulated by the binding of a buried water in the distance, we determined high-resolution (0.92-1.29 A) structures of wild-type FKBP12 and its two mutants (E60A, E60Q) by X-ray crystallography. The structures of mutant FKBP12 show that the ligand-binding pocket is indeed remodeled as predicted by the substitution at position 60, even though the water molecule does not directly interact with any of the amino acids of the binding pocket. Thus, these structures support the view that buried water molecules constitute an integral, noncovalent component of the protein structure. Additionally, this study provides an example in which predictions from molecular dynamics simulations are experimentally validated with atomic precision, thus showing that the structural features of protein-water interactions can be reliably modeled at a molecular level.     

SARS-CoV-2 spike binding to ACE2 is stronger and longer ranged due to glycan interaction

Highly detailed steered molecular dynamics simulations are performed on differently glycosylated receptor binding domains of the severe acute respiratory syndrome coronavirus-2 spike protein. The binding strength and the binding range increase with glycosylation. The interaction energy rises very quickly when pulling the proteins apart and only slowly drops at larger distances. We see a catch-slip-type behavior whereby interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode is hydrogen bonds, but Lennard-Jones and electrostatic interactions are relevant as well.     

Molecular dynamics simulations of the two disaccharides of hyaluronan in aqueous solution

Hyaluronan is an unusually stiff polymer when in aqueous solution,which has important consequences for its biological function.Molecular dynamics simulations of hyaluronan disaccharides havebeen performed, with explicit inclusion of water, to determinethe molecular basis of this stiffness, and to investigate thedynamics of the glycosidic linkages. Our simulations revealthat stable sets of hydrogen bonds frequently connect the neighboringresidues of hyaluronan. Water caging around the glycosidic linkagewas observed to increase the connectivity between sugars, andfurther constrain them. This, we propose, explains the unusualstiffness of polymeric hyaluronan. It would allow the polysaccharideto maintain local secondary structure, and occupy large solutiondomains consistent with the visco-elastic nature of hyaluronan.Simulations in water showed no significant changes on inclusionof the exo-anomeric effect. This, we deduced, was due to hyaluronandisaccharides ordering first shell water molecules. In somecases these waters were observed to transiently induce con-formationalchange, by breaking intramolecular hydrogen bonds. conformation hyaluronan hydrogen bonds molecular dynamics water     

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Theoretical Investigation of Interaction of Sorbitol Molecules with Alcohol Dehydrogenase in Aqueous Solution Using Molecular Dynamics Simulation

The nature of protein–sorbitol–water interaction in solution at the molecular level, has been investigated using molecular dynamics simulations. In order to do this task, two molecular dynamics simulations of the protein ADH in solution at room temperature have been carried out, one in the presence (about 0.9 M) and another in the absence of sorbitol. The results show that the sorbitol molecules cluster and move toward the protein, and form hydrogen bonds with protein. Also, coating by sorbitol reduces the conformational fluctuations of the protein compared to the sorbitol-free system. Thus, it is concluded that at moderate concentration of sorbitol solution, sorbitol molecules interact with ADH via many H-bonds that prevent the protein folding. In fact, at more concentrated sorbitol solution, water and sorbitol molecules accumulate around the protein surface and form a continuous space-filling network to reduce the protein flexibility. Namely, in such solution, sorbitol molecules can stabilize a misfolded state of ADH, and prevent the protein from folding to its native structure.     

Stability and sugar recognition ability of ricin-like carbohydrate binding domains

Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two β-trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained a detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1α and site 2γ recognition.     

Conformational transitions in eosinophil cationic protein: a molecular dynamics study in aqueous environment

Extensive molecular dynamics simulations have been performed on eosinophil cationic protein (ECP). The two structures found in the crystallographic dimer (ECPA and ECPB) have been independently simulated. A small difference in the pattern of the sidechain hydrogen bonds in the starting structure has resulted in interesting differences in the conformations accessed during the simulations. In one simulation (ECPB), a stable equilibrium conformation was obtained and in the other (ECPA), conformational transitions at the level of sidechain interactions were observed. The conformational transitions exhibit the involvement of the solvent (water) molecules with a pore-like construct in the equilibrium conformation and an opening for a large number of water molecules during the transition phase. The details of these transitions are examined in terms of intra-protein hydrogen bonds, protein-water networks and the residence times of water molecules on the polar atoms of the protein. These properties show some significant differences in the region between the N-terminal helix and the loop before the C-terminal strand as a function of different conformations accessed during the simulations. However, the stable hydrogen bonds, the protein-water networks, and the hydration patterns in most part of the protein including the active site are very much similar in both the simulations, indicating the fact that these are intrinsic properties of proteins.     

A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein

The crystallographic dimer of the C-terminal fragment (CTF) of the L7/L12 ribosomal protein has been subjected to molecular dynamics (MD) simulations. A 90 picosecond (ps) trajectory for the protein dimer, 19 water molecules and two counter ions has been calculated at constant temperature. Effects of intermolecular interactions on the structure and dynamics have been studied. The exact crystallographic symmetry is lost and the atomic fluctuations differ from one monomer to the other. The average MD structure is more stable than the X-ray one, as judged by accessible surface area and energy calculations. Crystal (non-dimeric) interactions have been simulated in another 40 ps trajectory by using harmonic restraints to represent intermolecular hydrogen bonds. The conformational changes with respect ot the X-ray structure are then virtually suppressed.The unrestrained dimer trajectory has been scanned for cooperative motions involving secondary structure elements. The intrinsic collective motions of the monomer are transmitted via intermolecular contacts to the dimer structure.The existence of a stable dimeric form of CTF, resembling the crystallographic one, has been documented. At the cost of fairly small energy expenditure the dimer has considerable conformational flexibility. This flexibility may endow the dimer with some functional potential as an energy transducer.     

Stability and rigidity/flexibility—Two sides of the same coin?

Protein molecules require both flexibility and rigidity for functioning. The fast and accurate prediction of protein rigidity/flexibility is one of the important problems in protein science. We have determined flexible regions for four homologous pairs from thermophilic and mesophilic organisms by two methods: the fast FoldUnfold which uses amino acid sequence and the time consuming MDFirst which uses three-dimensional structures. We demonstrate that both methods allow determining flexible regions in protein structure. For three of the four thermophile–mesophile pairs of proteins, FoldUnfold predicts practically the same flexible regions which have been found by the MD/First method. As expected, molecular dynamics simulations show that thermophilic proteins are more rigid in comparison to their mesophilic homologues. Analysis of rigid clusters and their decomposition provides new insights into protein stability. It has been found that the local networks of salt bridges and hydrogen bonds in thermophiles render their structure more stable with respect to fluctuations of individual contacts. Such network includes salt bridge triads Agr-Glu-Lys and Arg-Glu-Arg, or salt bridges (such as Arg-Glu) connected with hydrogen bonds. This ionic network connects alpha helices and rigidifies the structure. Mesophiles can be characterized by stand alone salt bridges and hydrogen bonds or small ionic clusters. Such difference in the network of salt bridges results in different flexibility of homologous proteins. Combining both approaches allows characterizing structural features in atomic detail that determine the rigidity/flexibility of a protein structure. This article is a part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.