Molecular simulations of lipid-mediated protein-protein interactions

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.     

Wetting and capillary condensation as means of protein organization in membranes.

Wetting and capillary condensation are thermodynamic phenomena in which the special affinity of interfaces to a thermodynamic phase, relative to the stable bulk phase, leads to the stabilization of a wetting phase at the interfaces. Wetting and capillary condensation are here proposed as mechanisms that in membranes may serve to induce special lipid phases in between integral membrane proteins leading to long-range lipid-mediated joining forces acting between the proteins and hence providing a means of protein organization. The consequences of wetting in terms of protein aggregation and protein clustering are derived both within a simple phenomenological theory as well as within a concrete calculation on a microscopic model of lipid-protein interactions that accounts for the lipid bilayer phase equilibria and direct lipid-protein interactions governed by hydrophobic matching between the lipid bilayer hydrophobic thickness and the length of the hydrophobic membrane domain. The theoretical results are expected to be relevant for optimizing the experimental conditions required for forming protein aggregates and regular protein arrays in membranes.     

Lipid-mediated interactions between intrinsic membrane proteins: a theoretical study based on integral equations

This study of lipid-mediated interactions between proteins is based on a theory recently developed by the authors for describing the structure of the hydrocarbon chains in the neighborhood of a protein inclusion embedded in a lipid membrane [Lagüe et al., Farad. Discuss. 111:165-172, 1998]. The theory involves the hypernetted chain integral equation formalism for liquids. The exact lateral density-density response function of the hydrocarbon core, extracted from molecular dynamics simulations of a pure dipalmitoylphosphatidylcholine bilayer based on an atomic model, is used as input. For the sake of simplicity, protein inclusions are modeled as hard repulsive cylinders. Numerical calculations were performed with three cylinder sizes: a small cylinder of 2.5-A radius, corresponding roughly to an aliphatic chain; a medium cylinder of 5-A radius, corresponding to a alpha-helical polyalanine protein; and a large cylinder of 9-A radius, representing a small protein, such as the gramicidin channel. The calculations show that the average hydrocarbon density is perturbed over a distance of 20-25 A from the edge of the cylinder for every cylinder size. The lipid-mediated protein-protein effective interaction is calculated and is shown to be nonmonotonic. In the case of the small and the medium cylinders, the lipid-mediated effective interaction of two identical cylinders is repulsive at an intermediate range but attractive at short range. At contact, there is a free energy of -2k(B)T for the 2.5-A-radius cylinder and -9k(B)T for the 5-A-radius cylinder, indicating that the association of two alpha-helices of both sizes is favored by the lipid matrix. In contrast, the effective interaction is repulsive at all distances in the case of the large cylinder. Results were obtained with two integral equations theories: hypernetted chain and Percus-Yevick. For the two theories, all results are qualitatively identical.     

Simulation studies of protein-induced bilayer deformations, and lipid-induced protein tilting, on a mesoscopic model for lipid bilayers with embedded proteins

Biological membranes are complex and highly cooperative structures. To relate biomembrane structure to their biological function it is often necessary to consider simpler systems. Lipid bilayers composed of one or two lipid species, and with embedded proteins, provide a model system for biological membranes. Here we present a mesoscopic model for lipid bilayers with embedded proteins, which we have studied with the help of the dissipative particle dynamics simulation technique. Because hydrophobic matching is believed to be one of the main physical mechanisms regulating lipid-protein interactions in membranes, we considered proteins of different hydrophobic length (as well as different sizes). We studied the cooperative behavior of the lipid-protein system at mesoscopic time- and lengthscales. In particular, we correlated in a systematic way the protein-induced bilayer perturbation, and the lipid-induced protein tilt, with the hydrophobic mismatch (positive and negative) between the protein hydrophobic length and the pure lipid bilayer hydrophobic thickness. The protein-induced bilayer perturbation was quantified in terms of a coherence length, xi(P), of the lipid bilayer hydrophobic thickness profile around the protein. The dependence on temperature of xi(P), and the protein tilt-angle, were studied above the main-transition temperature of the pure system, i.e., in the fluid phase. We found that xi(P) depends on mismatch, i.e., the higher the mismatch is, the longer xi(P) becomes, at least for positive values of mismatch; a dependence on the protein size appears as well. In the case of large model proteins experiencing extreme mismatch conditions, in the region next to the so-called lipid annulus, there appears an undershooting (or overshooting) region where the bilayer hydrophobic thickness is locally lower (or higher) than in the unperturbed bilayer, depending on whether the protein hydrophobic length is longer (or shorter) than the pure lipid bilayer hydrophobic thickness. Proteins may tilt when embedded in a too-thin bilayer. Our simulation data suggest that, when the embedded protein has a small size, the main mechanism to compensate for a large hydrophobic mismatch is the tilt, whereas large proteins react to negative mismatch by causing an increase of the hydrophobic thickness of the nearby bilayer. Furthermore, for the case of small, peptidelike proteins, we found the same type of functional dependence of the protein tilt-angle on mismatch, as was recently detected by fluorescence spectroscopy measurements.     

Coarse-grained strategy for modeling protein stability in concentrated solutions. III: directional protein interactions

We extend our coarse-grained modeling strategy described in parts I and II of this investigation to account for nonuniform spatial distributions of hydrophobic residues on the solvent-exposed surfaces of native proteins. Within this framework, we explore how patchy surfaces can influence the solvent-mediated protein-protein interactions, and the unfolding and self-assembly behaviors of proteins in solution. In particular, we compare the equilibrium unfolding and self-assembly trends for three model proteins that share the same overall sequence hydrophobicity, but exhibit folded configurations with different solvent-exposed native-state surface morphologies. Our model provides new insights into how directional interactions can affect native-state protein stability in solution. We find that strongly-directional attractions between native molecules with patchy surfaces can help stabilize the folded conformation through the formation of self-assembled clusters. In contrast, native proteins with more uniform surfaces are destabilized by protein-protein attractions involving the denatured state. Finally, we discuss how the simulation results provide insights into the experimental solution behaviors of several proteins that display directional interactions in their native states.     

Effect of lipid structural modifications on their intermolecular hydrogen bonding interactions and membrane functions

The large number of different membrane lipids with various structural modifications and properties and the characteristic lipid composition of different types of membranes suggest that different lipids have specific functions in the membrane. Many of the varying properties of lipids with different polar head groups and in different ionization states can be attributed to the presence of interactive or repulsive forces between the head groups in the bilayer. The interactive forces are hydrogen bonds between hydrogen bond donating groups such as --P--OH,--OH, and--NH3+ and hydrogen bond accepting groups such as --P--O- and --COO-. These interactions increase the lipid phase transition temperature and can account for the tendency of certain lipids to go into the hexagonal phase and the dependence of this tendency on the pH and ionization state of the lipid. The presence or absence of these interactions can also affect the penetration of hydrophobic substances into the bilayer, including hydrophobic residues of membrane proteins. Evidence for this suggestion has been gathered from studies of the myelin basic protein, a water-soluble protein with a number of hydrophobic residues. In this way the lipid composition can affect the conformation and activity of membrane proteins. Since hydrogen-bonding interactions depend on the ionization state of the lipid, they can be altered by changes in the environment which affect the pK of the ionizable groups. The formation of the hexagonal phase or inverted micelles, the conformation and activity of membrane proteins, and other functions mediated by lipids could thus be regulated in this way.     

Molecular theory of lipid-protein interaction and the Lalpha-HII transition.

We present a molecular-level theory for lipid-protein interaction and apply it to the study of lipid-mediated interactions between proteins and the protein-induced transition from the planar bilayer (Lalpha) to the inverse-hexagonal (HII) phase. The proteins are treated as rigid, membrane-spanning, hydrophobic inclusions of different size and shape, e.g., "cylinder-like," "barrel-like," or "vase-like." We assume strong hydrophobic coupling between the protein and its neighbor lipids. This means that, if necessary, the flexible lipid chains surrounding the protein will stretch, compress, and/or tilt to bridge the hydrophobic thickness mismatch between the protein and the unperturbed bilayer. The system free energy is expressed as an integral over local molecular contributions, the latter accounting for interheadgroup repulsion, hydrocarbon-water surface energy, and chain stretching-tilting effects. We show that the molecular interaction constants are intimately related to familiar elastic (continuum) characteristics of the membrane, such as the bending rigidity and spontaneous curvature, as well as to the less familiar tilt modulus. The equilibrium configuration of the membrane is determined by minimizing the free energy functional, subject to boundary conditions dictated by the size, shape, and spatial distribution of inclusions. A similar procedure is used to calculate the free energy and structure of peptide-free and peptide-rich hexagonal phases. Two degrees of freedom are involved in the variational minimization procedure: the local length and local tilt angle of the lipid chains. The inclusion of chain tilt is particularly important for studying noncylindrical (for instance, barrel-like) inclusions and analyzing the structure of the HII lipid phase; e.g., we find that chain tilt relaxation implies strong faceting of the lipid monolayers in the hexagonal phase. Consistent with experiment, we find that only short peptides (large negative mismatch) can induce the Lalpha --> HII transition. At the transition, a peptide-poor Lalpha phase coexists with a peptide-rich HII phase.     

Investigation on the mechanism of crystallization of soluble protein in the presence of nonionic surfactant

The mechanism of crystallization of soluble, globular protein (lysozyme) in the presence of nonionic surfactant C8E4 (tetraoxyethylene glycol monooctyl ether) was examined using both static and dynamic light scattering. The interprotein interaction was found to be attractive in solution conditions that yielded crystals and repulsive in the noncrystallizing solution conditions. The validity of the second virial coefficient as a criterion for predicting protein crystallization could be established even in the presence of nonionic surfactants. Our experiments indicate that the origin of the change in interactions can be attributed to the adsorption of nonionic surfactant monomers on soluble proteins, which is generally assumed to be the case with only membrane proteins. This adsorption screens the hydrophobic attractive force and enhances the hydration and electrostatic repulsive forces between protein molecules. Thus at low surfactant concentration, the effective protein-protein interaction remains repulsive. Large surfactant concentrations promote protein crystallization, possibly due to the attractive depletion force caused by the intervening free surfactant micelles.     

Clustering of non-polar contacts in proteins.

MOTIVATION: Hydrophobic or non-polar contacts in proteins are important for protein folding, protein stability and protein-protein interactions. In particular, in the interior of a protein, in the hydrophobic core, a large number of such contacts are found. The residues involved in these contacts often form a tightly packed cluster of atoms. It is useful for the understanding of protein structure to be able to identify and analyse such clusters. RESULTS: Tools for hierarchical cluster analysis of non-polar contacts in proteins are described. These tools allow for efficient identification of clusters of non-polar interactions in proteins, both internal clusters and clusters involved in protein-protein contacts. The non-polar contacts are represented by a dendrogram structure, which is a simple approach for flexible identification of clusters by visual inspection. The tools are demonstrated on the structure of crambin, the structure of the complex between human growth hormone and the human growth hormone binding protein, and a pair of lipase/esterase structures. Availability: On request from the author.     

A theoretical model for the association of amphiphilic transmembrane peptides in lipid bilayers

A theoretical model is proposed for the association of trans-bilayer peptides in lipid bilayers. The model is based on a lattice model for the pure lipid bilayer, which accounts accurately for the most important conformational states of the lipids and their mutual interactions and statistics. Within the lattice formulation the bilayer is formed by two independent monolayers, each represented by a triangular lattice, on which sites the lipid chains are arrayed. The peptides are represented by regular objects, with no internal flexibility, and with a projected area on the bilayer plane corresponding to a hexagon with seven lattice sites. In addition, it is assumed that each peptide surface at the interface with the lipid chains is partially hydrophilic, and therefore interacts with the surrounding lipid matrix via selective anisotropic forces. The peptides would therefore assemble in order to shield their hydrophilic residues from the hydrophobic surroundings. The model describes the self-association of peptides in lipid bilayers via lateral and rotational diffusion, anisotropic lipid-peptide interactions, and peptide-peptide interactions involving the peptide hydrophilic regions. The intent of this model study is to analyse the conditions under which the association of trans-bilayer and partially hydrophilic peptides (or their dispersion in the lipid matrix) is lipid-mediated, and to what extent it is induced by direct interactions between the hydrophilic regions of the peptides. The model properties are calculated by a Monte Carlo computer simulation technique within the canonical ensemble. The results from the model study indicate that direct interactions between the hydrophilic regions of the peptides are necessary to induce peptide association in the lipid bilayer in the fluid phase. Furthermore, peptides within each aggregate are oriented in such a way as to shield their hydrophilic regions from the hydrophobic environment. The average number of peptides present in the aggregates formed depends on the degree of mismatch between the peptide hydrophobic length and the lipid bilayer hydrophobic thickness: The lower the degree of mismatch is the higher this number is. Received: 30 December 1996 / Accepted: 9 May 1997     

Membrane-Mediated Protein-Protein Interactions and Connection to Elastic Models: A Coarse-Grained Simulation Analysis of Gramicidin A Association

To further foster the connection between particle based and continuum mechanics models for membrane mediated biological processes, we carried out coarse-grained (CG) simulations of gramicidin A (gA) dimer association and analyzed the results based on the combination of potential of mean force (PMF) and stress field calculations. Similar to previous studies, we observe that the association of gA dimers depends critically on the degree of hydrophobic mismatch, with the estimated binding free energy of >10 kcal/mol in a distearoylphosphatidylcholine bilayer. Qualitative trends in the computed PMF can be understood based on the stress field distributions near a single gA dimer and between a pair of gA dimers. For example, the small PMF barrier, which is ∼1 kcal/mol independent of lipid type, can be captured nearly quantitatively by considering membrane deformation energy associated with the region confined by two gA dimers. However, the PMF well depth is reproduced poorly by a simple continuum model that only considers membrane deformation energy beyond the annular lipids. Analysis of lipid orientation, configuration entropy, and stress distribution suggests that the annular lipids make a significant contribution to the association of two gA dimers. These results highlight the importance of explicitly considering contributions from annular lipids when constructing approximate models to study processes that involve a significant reorganization of lipids near proteins, such as protein-protein association and protein insertion into biomembranes. Finally, large-scale CG simulations indicate that multiple gA dimers also form clusters, although the preferred topology depends on the protein concentration. Even at high protein concentrations, every gA dimer requires contact to lipid hydrocarbons to some degree, and at most three to four proteins are in contact with each gA dimer; this observation highlights another aspect of the importance of interactions between proteins and annular lipids.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Protein–lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring (Review)

Biological membranes are characterized by a heterogeneous composition, which is not only manifested in the wide variety of their components, but also in aspects like the lateral organization, topology, and conformation of proteins and lipids. In bringing about the correct membrane structure, protein–lipid interactions can be expected to play a prominent role. The extent of hydrophobic matching between transmembrane protein segments and lipids potentially constitutes a versatile director of membrane organization, because a tendency to avoid hydrophobic mismatch could result in compensating adaptations such as tilt of the transmembrane segment or segregation into distinct domains. Also, interfacial interactions between lipid headgroups and the aromatic and charged residues that typically flank transmembrane domains may act as an organizing element. In this review, we discuss the numerous model studies that have systematically explored the influence of hydrophobic matching and interfacial anchoring on membrane structure. Designed peptides consisting of a polyleucine or polyleucine/alanine hydrophobic stretch, which is flanked on both sides by tryptophan or lysine residues, reflect the general layout of transmembrane protein segments. It is shown for phosphatidylcholine bilayers and for other model membranes that these peptides adapt a transmembrane topology without extensive peptide or lipid adaptations under conditions of hydrophobic matching, but that significant rearrangements can result from hydrophobic mismatch. Moreover, these effects depend on the nature of the flanking residues, implying a modulation of the mismatch response by interfacial interactions of the flanking residues. The implications of these model studies for the organization of biomembranes are discussed in the context of recent experiments with more complex systems.     

Role of secondary structure in protein-phospholipid surface interactions: reconstitution and denaturation of apolipoprotein C-I:DMPC complexes

Binding of protein to a phospholipid surface is commonly mediated by amphipathic alpha-helices. To understand the role of alpha-helical structure in protein-lipid interactions, we used discoidal lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and human apolipoprotein C-I (apoC-I, 6 kDa) or its mutants containing single Pro substitutions along the sequence and differing in their alpha-helical content in solution (0-48%) and on DMPC (40-75%). Thermal denaturation revealed that lipoprotein stability correlates weakly with the protein helix content: proteins with higher alpha-helical content on DMPC may form more stable complexes. Lipoprotein reconstitution upon cooling from the heat-denatured state and DMPC clearance studies revealed that protein secondary structure in solution and on DMPC correlates strongly with the maximal temperature of lipoprotein reconstitution: more helical proteins can reconstitute lipoproteins at higher temperatures. Interestingly, at Tc = 24 degrees C of the DMPC gel-to-liquid crystal transition, the clearance rate is independent of the protein helical content. Consequently, if the packing defects at the phospholipid surface are readily available (e.g., at the lipid phase boundary), insertion of protein into these defects is independent of the secondary structure in solution. However, if hydrophobic defects are limited, protein binding and insertion are aided by other surface-bound proteins and depend on their helical propensity: the larger the propensity, the faster the binding and the broader its temperature range. This positive cooperativity in binding of alpha-helices to phospholipid surface, which may result from direct and/or lipid-mediated protein-protein interactions, may be important for lipoprotein metabolism and for protein-membrane binding.     

Effect of membrane characteristics on phase separation and domain formation in cholesterol-lipid mixtures

We examine, using an analytical mean-field model, the distribution of cholesterol in a lipid bilayer. The model accounts for the perturbation of lipid packing induced by the embedded cholesterol, in a manner similar to that of transmembrane proteins. We find that the membrane-induced interactions between embedded cholesterol molecules vary as a function of the cholesterol content. Thus, the effective lipid-cholesterol interaction is concentration-dependent. Moreover, it transitions from repulsive to attractive to repulsive as the cholesterol content increases. As the concentration of cholesterol in the bilayer exceeds a critical value, phase separation occurs. The coexistence between cholesterol-rich and cholesterol-poor domains is universal for any bilayer parameters, although the composition of the cholesterol-rich phase varies as a function of the lipid properties. Although we do not assume specific cholesterol-lipid interactions or the formation of a lipid-cholesterol cluster, we find that the composition of the cholesterol-rich domains is constant, independent of the cholesterol content in the bilayer.     

A synergistic effect between cholesterol and tryptophan-flanked transmembrane helices modulates membrane curvature

The aim of this study was to gain insight into the structural consequences of hydrophobic mismatch for membrane proteins in lipid bilayers that contain cholesterol. For this purpose, tryptophan-flanked peptides, designed to mimic transmembrane segments of membrane proteins, were incorporated in model membranes of unsaturated phosphatidylcholine bilayers of varying thickness and containing varying amounts of cholesterol. Analysis of the lipid organization by (31)P NMR and cryo-TEM demonstrated the formation of an isotropic phase, most likely representing a cubic phase, which occurred exclusively in mixtures containing lipids with relatively long acyl chains. Formation of this phase was inhibited by incorporation of lysophosphatidylcholine. These results indicate that the isotropic phase is formed as a consequence of negative hydrophobic mismatch and that its formation is related to a negative membrane curvature. When either peptide or cholesterol was omitted from the mixture, isotropic-phase formation did not occur, not even when the concentrations of these compounds were significantly increased. This suggests that formation of the isotropic phase is the result of a synergistic effect between the peptides and cholesterol. Interestingly, isotropic-phase formation was not observed when the tryptophans in the peptide were replaced by either lysines or histidines. We propose a model for the mechanism of this synergistic effect, in which its dependence on the flanking residues is explained by preferential interactions between cholesterol and tryptophan residues.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.