Role of hydrogen bonding and helix-lipid interactions in transmembrane helix association

To explore the role of hydrogen bonding and helix-lipid interactions in transmembrane helix association, we have calculated the potential of mean force (PMF) as a function of helix-helix distance between two pVNVV peptides, a transmembrane model peptide based on the GCN4 leucine-zipper, in a dimyristoylphosphatidylcholine (DMPC) membrane. The peptide name pVNVV represents the interfacial residues in the heptad repeat of the dimer. The free energy decomposition reveals that the total PMF consists of two competing contributions from helix-helix and helix-lipid interactions. The direct, favorable helix-helix interactions arise from the specific contribution from the helix-facing residues and the generic contribution from the lipid-facing residues. The Asn residues in the middle of the helices show the most significant per-residue contribution to the PMF with various hydrogen bonding patterns as a function of helix-helix distance. Release of lipid molecules between the helices into bulk lipid upon helix association makes the helix-lipid interaction enthalpically unfavorable but entropically favorable. Interestingly, the resulting unfavorable helix-lipid contribution to the PMF correlates well with the cavity volume between the helices. The calculated PMF with an Asn-to-Val mutant (pVNVV --> pVVVV) shows a dramatic free energy change upon the mutation, such that the mutant appears not to form a stable dimer below a certain peptide concentration, which is in good agreement with available experimental data of a peptide with the same heptad repeat. A transmembrane helix association mechanism and its implications in membrane protein folding are also discussed.     

In silico partitioning and transmembrane insertion of hydrophobic peptides under equilibrium conditions

Nascent transmembrane (TM) polypeptide segments are recognized and inserted into the lipid bilayer by the cellular translocon machinery. The recognition rules, described by a biological hydrophobicity scale, correlate strongly with physical hydrophobicity scales that describe the free energy of insertion of TM helices from water. However, the exact relationship between the physical and biological scales is unknown, because solubility problems limit our ability to measure experimentally the direct partitioning of hydrophobic peptides across lipid membranes. Here we use microsecond molecular dynamics (MD) simulations in which monomeric polyleucine segments of different lengths are allowed to partition spontaneously into and out of lipid bilayers. This approach directly reveals all states populated at equilibrium. For the hydrophobic peptides studied here, only surface-bound and transmembrane-inserted helices are found. The free energy of insertion is directly obtained from the relative occupancy of these states. A water-soluble state was not observed, consistent with the general insolubility of hydrophobic peptides. The approach further allows determination of the partitioning pathways and kinetics. Surprisingly, the transfer free energy appears to be independent of temperature, which implies that surface-to-bilayer peptide insertion is a zero-entropy process. We find that the partitioning free energy of the polyleucine segments correlates strongly with values from translocon experiments but reveals a systematic shift favoring shorter peptides, suggesting that translocon-to-bilayer partitioning is not equivalent but related to spontaneous surface-to-bilayer partitioning.     

Multidimensional umbrella sampling and replica‐exchange molecular dynamics simulations for structure prediction of transmembrane helix dimers

Structural information of a transmembrane (TM) helix dimer is useful in understanding molecular mechanisms of important biological phenomena such as signal transduction across the cell membrane. Here, we describe an umbrella sampling (US) scheme for predicting the structure of a TM helix dimer in implicit membrane using the interhelical crossing angle and the TM–TM relative rotation angles as the reaction coordinates. This scheme conducts an efficient conformational search on TM–TM contact interfaces, and its robustness is tested by predicting the structures of glycophorin A (GpA) and receptor tyrosine kinase EphA1 (EphA1) TM dimers. The nuclear magnetic resonance (NMR) structures of both proteins correspond to the global free‐energy minimum states in their free‐energy landscapes. In addition, using the landscape of GpA as a reference, we also examine the protocols of temperature replica‐exchange molecular dynamics (REMD) simulations for structure prediction of TM helix dimers in implicit membrane. A wide temperature range in REMD simulations, for example, 250–1000 K, is required to efficiently obtain a free‐energy landscape consistent with the US simulations. The interhelical crossing angle and the TM–TM relative rotation angles can be used as reaction coordinates in multidimensional US and be good measures for conformational sampling of REMD simulations. © 2013 Wiley Periodicals, Inc.     

Convergence of Free Energy Profile of Coumarin in Lipid Bilayer

Atomistic molecular dynamics (MD) simulations of druglike molecules embedded in lipid bilayers are of considerable interest as models for drug penetration and positioning in biological membranes. Here we analyze partitioning of coumarin in dioleoylphosphatidylcholine (DOPC) bilayer, based on both multiple, unbiased 3 μs MD simulations (total length) and free energy profiles along the bilayer normal calculated by biased MD simulations (～7 μs in total). The convergences in time of free energy profiles calculated by both umbrella sampling and z-constraint techniques are thoroughly analyzed. Two sets of starting structures are also considered, one from unbiased MD simulation and the other from "pulling" coumarin along the bilayer normal. The structures obtained by pulling simulation contain water defects on the lipid bilayer surface, while those acquired from unbiased simulation have no membrane defects. The free energy profiles converge more rapidly when starting frames from unbiased simulations are used. In addition, z-constraint simulation leads to more rapid convergence than umbrella sampling, due to quicker relaxation of membrane defects. Furthermore, we show that the choice of RESP, PRODRG, or Mulliken charges considerably affects the resulting free energy profile of our model drug along the bilayer normal. We recommend using z-constraint biased MD simulations based on starting geometries acquired from unbiased MD simulations for efficient calculation of convergent free energy profiles of druglike molecules along bilayer normals. The calculation of free energy profile should start with an unbiased simulation, though the polar molecules might need a slow pulling afterward. Results obtained with the recommended simulation protocol agree well with available experimental data for two coumarin derivatives.     

Key issues in the computational simulation of GPCR function: representation of loop domains

Some key concerns raised by molecular modeling and computational simulation of functional mechanisms for membrane proteins are discussed and illustrated for members of the family of G protein coupled receptors (GPCRs). Of particular importance are issues related to the modeling and computational treatment of loop regions. These are demonstrated here with results from different levels of computational simulations applied to the structures of rhodopsin and a model of the 5-HT2A serotonin receptor, 5-HT2AR. First, comparative Molecular Dynamics (MD) simulations are reported for rhodopsin in vacuum and embedded in an explicit representation of the membrane and water environment. It is shown that in spite of a partial accounting of solvent screening effects by neutralization of charged side chains, vacuum MD simulations can lead to severe distortions of the loop structures. The primary source of the distortion appears to be formation of artifactual H-bonds, as has been repeatedly observed in vacuum simulations. To address such shortcomings, a recently proposed approach that has been developed for calculating the structure of segments that connect elements of secondary structure with known coordinates, is applied to 5-HT2AR to obtain an initial representation of the loops connecting the transmembrane (TM) helices. The approach consists of a simulated annealing combined with biased scaled collective variables Monte Carlo technique, and is applied to loops connecting the TM segments on both the extra-cellular and the cytoplasmic sides of the receptor. Although this initial calculation treats the loops as independent structural entities, the final structure exhibits a number of interloop interactions that may have functional significance. Finally, it is shown here that in the case where a given loop from two different GPCRs (here rhodopsin and 5-HT2AR) has approximately the same length and some degree of sequence identity, the fold adopted by the loops can be similar. Thus, in such special cases homology modeling might be used to obtain initial structures of these loops. Notably, however, all other loops in these two receptors appear to be very different in sequence and structure, so that their conformations can be found reliably only by ab initio, energy based methods and not by homology modeling.     

Prediction of protein–protein complexes using replica exchange with repulsive scaling

The realistic prediction of protein–protein complex structures is import to ultimately model the interaction of all proteins in a cell and for the design of new protein–protein interactions. In principle, molecular dynamics (MD) simulations allow one to follow the association process under realistic conditions including full partner flexibility and surrounding solvent. However, due to the many local binding energy minima at the surface of protein partners, MD simulations are frequently trapped for long times in transient association states. We have designed a replica-exchange based scheme employing different levels of a repulsive biasing between partners in each replica simulation. The bias acts only on intermolecular interactions based on an increase in effective pairwise van der Waals radii (repulsive scaling (RS)-REMD) without affecting interactions within each protein or with the solvent. For a set of five protein test cases (out of six) the RS-REMD technique allowed the sampling of near-native complex structures even when starting from the opposide site with respect to the native binding site for one partner. Using the same start structures and same computational demand regular MD simulations sampled near native complex structures only for one case. The method showed also improved results for the refinement of docked structures in the vicinity of the native binding geometry compared to regular MD refinement.     

Implementation and application of helix-helix distance and crossing angle restraint potentials

Based on the definition of helix-helix distance and crossing angle introduced by Chothia et al. (J Mol Biol 1981, 145, 215), we have developed the restraint potentials by which the distance and crossing angle of two selected helices can be maintained around target values during molecular dynamics simulations. A series of assessments show that calculated restraint forces are numerically accurate. Since the restraint forces are only exerted on atoms which define the helical principal axes, each helix can rotate along its helical axis, depending on the helix-helix intermolecular interactions. Such a restraint potential enables us to characterize the helix-helix interactions at atomic details by sampling their conformational space around specific distance and crossing angle with (restraint) force-dependent fluctuations. Its efficacy is illustrated by calculating the potential of mean force as a function of helix-helix distance between two transmembrane helical peptides in an implicit membrane model.     

Artificial chemokines: combining chemistry and molecular biology for the elucidation of interleukin-8 functionality

How can we understand the contribution of individual parts or segments to complex structures? A typical strategy to answer this question is simulation of a segmental replacement followed by realization and investigation of the resulting effect in structure-activity studies. For proteins, this problem is commonly addressed by site-directed mutagenesis. A more general approach represents the exchange of whole secondary structure elements by rationally designed segments. For a demonstration of this possibility we identified the alpha-helix at the C-terminus of human interleukin-8 (hIL-8). Since this chemokine possesses four conserved cysteine residues, it can easily be altered by ligation strategies. A set of different segments, which are able to form amphiphilic helices, was synthesized to mimic the C-terminal alpha-helix. Beside sequences of alpha-amino acids, oligomers of non-natural beta(3)-amino acids with the side chains of canonical amino acids were introduced. Such beta-peptides form helices, which differ from the alpha-helix in handedness and dipole orientation. Variants of the semisynthetic hIL-8 proteins demonstrated clearly that the exact side chain orientation is of more importance than helix handedness and dipole orientation. The activity of a chimeric protein with a beta-peptide helix that mimics the side chain orientation of the native alpha-helix most perfectly is comparable to that of the native hIL-8. Concepts like this could be a first step toward the synthesis of proteins consisting of large artificial secondary structure elements.     

Molecular dynamics study of the inclusion of cholesterol into cyclodextrins

The interaction of three cyclodextrins (CDs), viz. beta-CD, heptakis (2,6-di-O-methyl)-beta-CD (DM-beta-CD), and 2-hydroxypropyl-beta-CD (HP-beta-CD), with cholesterol was investigated using molecular dynamics (MD) simulations. The free energy along the reaction pathway delineating the inclusion of cholesterol into each CD was computed using the adaptive biasing force method. The association constant and the corresponding association free energy were derived by integrating the potential of mean force (PMF) over a representative ordering parameter. The results show that the free energy profiles possess two local minima corresponding to roughly equally probable binding modes. Among the three CDs, DM-beta-CD exhibits the highest propensity to associate with cholesterol. Ranking for binding cholesterol, viz. DM-beta-CD > HP-beta-CD > beta-CD, agrees nicely with experiment. Partitioning of the PMF into free energy components illuminates that entering of cholesterol into the CD cavity is driven mainly by electrostatic interactions, whereas deeper inclusion results from van der Waals forces and solvation effects. Additional MD simulations were performed to investigate the structural stability of the host-guest complexes near the free energy minima. The present results demonstrate that association of cholesterol and CDs follows two possible binding modes. Although the latter are thermodynamically favorable for all CDs, one of the two inclusion complexes appears to be preferred kinetically in the case of DM-beta-CD.     

Efficient estimation of binding free energies between peptides and an MHC class II molecule using coarse‐grained molecular dynamics simulations with a weighted histogram analysis method

We estimate the binding free energy between peptides and an MHC class II molecule using molecular dynamics (MD) simulations with the weighted histogram analysis method (WHAM). We show that, owing to its more thorough sampling in the available computational time, the binding free energy obtained by pulling the whole peptide using a coarse‐grained (CG) force field (MARTINI) is less prone to significant error induced by inadequate‐sampling than using an atomistic force field (AMBER). We further demonstrate that using CG MD to pull 3–4 residue peptide segments while leaving the remaining peptide segments in the binding groove and adding up the binding free energies of all peptide segments gives robust binding free energy estimations, which are in good agreement with the experimentally measured binding affinities for the peptide sequences studied. Our approach thus provides a promising and computationally efficient way to rapidly and reliably estimate the binding free energy between an arbitrary peptide and an MHC class II molecule. © 2017 Wiley Periodicals, Inc.     

Thermo-driven chiroptical switching polysilane featuring 2-cyclopentylethyl side group

A new rod-like helical polysilane, poly{(S)-3,7-dimethyloctyl-(2-cyclopentylethyl)silane}, was found to undergo a thermo-driven, helix-helix transition at –33 ° C in isooctane associated with the discontinuous changes in the Si -Si *transition energy and intensity in the transition temperature region. This is the first example of a helix-helix transition polysilane with a cycloalkyl group. A similar rod-like polysilane derivative, poly{(S)-3,7-dimethyloctyl-(1-cyclopentylmethyl)silane}, however, did not undergo any helix-helix transition between –61 and 80 ° C.     

Passive transport of C60 fullerenes through a lipid membrane: a molecular dynamics simulation study

To investigate the implications of the unique properties of fullerenes on their interaction with and passive transport into lipid membranes, atomistic molecular dynamics simulations of a C60 fullerene in a fully hydrated di-myristoyl-phoshatidylcholine lipid membrane have been carried out. In these simulations the free energy and the diffusivity of the fullerene were obtained as a function of its position within the membrane. These properties were utilized to calculate the permeability of fullerenes through the lipid membrane. Simulations reveal that the free energy decreases as the fullerene passes from the aqueous phase, through the head group layer and into the hydrophobic core of the membrane. This decrease in free energy is not due to hydrophobic interactions but rather to stronger van der Waals (dispersion) interactions between the fullerene and the membrane compared to those between the fullerene and (bulk) water. It was found that there is no free energy barrier for transport of a fullerene from the aqueous phase into the lipid core of the membrane. In combination with strong partitioning of the fullerenes into the lipidic core of the membrane, this "barrierless" penetration results in an astonishingly large permeability of fullerenes through the lipid membrane, greater than observed for any other known penetrant. When the strength of the dispersion interactions between the fullerene and its surroundings is reduced in the simulations, thereby emulating a nanometer sized hydrophobic particle, a large free energy barrier for penetration of the head group layer emerges, indicating that the large permeability of fullerenes through lipid membranes is a result of their unique interaction with their surrounding medium.     

Role of water mediated interactions in protein-protein recognition landscapes

The energy landscape picture of protein folding and binding is employed to optimize a number of pair potentials for direct and water-mediated interactions in protein complex interfaces. We find that water-mediated interactions greatly complement direct interactions in discriminating against various types of trap interactions that model those present in the cell. We highlight the context dependent nature of knowledge-based binding potentials, as contrasted with the situation for autonomous folding. By performing a Principal Component Analysis (PCA) of the corresponding interaction matrixes, we rationalize the strength of the recognition signal for each combination of the contact type and reference trap states using the differential in the idealized "canonical" amino acid compositions of native and trap layers. The comparison of direct and water-mediated contact potential matrixes emphasizes the importance of partial solvation in stabilizing charged groups in the protein interfaces. Specific water-mediated interresidue interactions are expected to influence significantly the kinetics as well as thermodynamics of protein association.     

Synthetic non-peptide mimetics of alpha-helices

Proteins in nature fold into native conformations in which combinations of peripherally projected aliphatic, aromatic and ionic functionalities direct a wide range of properties. Alpha-helices, one of the most common protein secondary structures, serve as important recognition regions on protein surfaces for numerous protein-protein, protein-DNA and protein-RNA interactions. These interactions are characterized by conserved structural features within the alpha-helical domain. Rational design of structural mimetics of these domains with synthetic small molecules has proven an effective means to modulate such protein functions. In this tutorial review we discuss strategies that utilize synthetic small-molecule antagonists to selectively target essential protein-protein interactions involved in certain diseases. We also evaluate some of the protein-protein interactions that have been or are potential targets for alpha-helix mimetics.     

Molecular dynamics simulation study of the structural features and inclusion capacities of cucurbit[6]uril derivatives in aqueous solutions

Molecular dynamics (MD) simulations were performed for cucurbit[6]uril (CB6) methyl and cyclohexyl derivatives in aqueous solutions. Furthermore, MD simulations have been conducted to study the inclusion complexes between each CB6 derivative with α,ω-pentane diammonium ion (NH₃⁺(CH₂)₅NH₃⁺) to estimate the binding free energies, the complex geometries and the intermolecular forces responsible for complex formation. Results show a complete inclusion of the guest molecule in the cavity of the host for all complexes. Results also indicate that the guest dynamics inside the cavity of the substituted host is similar to that for the unsubstituted host. This demonstrates that the molecular recognition of the host is not affected by the alkyl substitution at the equator. Also, there is an insignificant conformational change of the macrocyclic structure upon inclusion of the guest. Molecular mechanics/Poisson Boltzmann surface area method was used to estimate the binding free energy of each complex. Results indicate that host–guest electrostatic interactions make the largest contribution to the complex binding free energy. Moreover, van der Waals interactions add significantly to the complex stability. The guest molecules show more or less similar binding free energies with the substituted CB6 that exhibits slightly more negative values than unsubstituted CB6 which is proved also by umbrella sampling.     

The balance between side-chain and backbone-driven association in folding of the α-helical influenza A transmembrane peptide

The correct balance between attractive, repulsive and peptide hydrogen bonding interactions must be attained for proteins to fold correctly. To investigate these important contributors, we sought a comparison of the folding between two 25-residues peptides, the influenza A M2 protein transmembrane domain (M2TM) and the 25-Ala (Ala₂₅). M2TM forms a stable α-helix as is shown by circular dichroism (CD) experiments. Molecular dynamics (MD) simulations with adaptive tempering show that M2TM monomer is more dynamic in nature and quickly interconverts between an ensemble of various α-helical structures, and less frequently turns and coils, compared to one α-helix for Ala₂₅. DFT calculations suggest that folding from the extended structure to the α-helical structure is favored for M2TM compared with Ala₂₅. This is due to CH⋯O attractive interactions which favor folding to the M2TM α-helix, and cannot be described accurately with a force field. Using natural bond orbital (NBO) analysis and quantum theory atoms in molecules (QTAIM) calculations, 26 CH⋯O interactions and 22 NH⋯O hydrogen bonds are calculated for M2TM. The calculations show that CH⋯O hydrogen bonds, although individually weaker, have a cumulative effect that cannot be ignored and may contribute as much as half of the total hydrogen bonding energy, when compared to NH⋯O, to the stabilization of the α-helix in M2TM. Further, a strengthening of NH⋯O hydrogen bonding interactions is calculated for M2TM compared to Ala₂₅. Additionally, these weak CH⋯O interactions can dissociate and associate easily leading to the ensemble of folded structures for M2TM observed in folding MD simulations.