Sayan Mondal

Quantitative Modeling of Membrane Deformations by Multihelical Membrane Proteins: Application to G-Protein Coupled Receptors

The interpretation of experimental observations of the dependence of membrane protein function on the properties of the lipid membrane environment calls for a consideration of the energy cost of protein-bilayer interactions, including the protein-bilayer hydrophobic mismatch. We present a novel (to our knowledge) multiscale computational approach for quantifying the hydrophobic mismatch-driven remodeling of membrane bilayers by multihelical membrane proteins. The method accounts for both the membrane remodeling energy and the energy contribution from any partial (incomplete) alleviation of the hydrophobic mismatch by membrane remodeling. Overcoming previous limitations, it allows for radially asymmetric bilayer deformations produced by multihelical proteins, and takes into account the irregular membrane-protein boundaries. The approach is illustrated by application to two G-protein coupled receptors: rhodopsin in bilayers of different thickness, and the serotonin 5-HT(2A) receptor bound to pharmacologically different ligands. Analysis of the results identifies the residual exposure that is not alleviated by bilayer adaptation, and its quantification at specific transmembrane segments is shown to predict favorable contact interfaces in oligomeric arrays. In addition, our results suggest how distinct ligand-induced conformations of G-protein coupled receptors may elicit different functional responses through differential effects on the membrane environment.

Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A) receptor determine its activation and membrane-driven oligomerization properties.

From computational simulations of a serotonin 2A receptor (5-HT2AR) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT2AR, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.

Membrane Driven Spatial Organization of GPCRs

Spatial organization of G-protein coupled receptors (GPCRs) into dimers and higher order oligomers has been demonstrated in vitro and in vivo. The pharmacological readout was shown to depend on the specific interfaces, but why particular regions of the GPCR structure are involved, and how ligand-determined states change them remains unknown. Here we show why protein-membrane hydrophobic matching is attained upon oligomerization at specific interfaces from an analysis of coarse-grained molecular dynamics simulations of the spontaneous diffusion-interaction of the prototypical beta2-adrenergic (β2AR) receptors in a POPC lipid bilayer. The energy penalty from mismatch is significantly reduced in the spontaneously emerging oligomeric arrays, making the spatial organization of the GPCRs dependent on the pattern of mismatch in the monomer. This mismatch pattern is very different for β2AR compared to the highly homologous and structurally similar β1AR, consonant with experimentally observed oligomerization patterns of β2AR and β1AR. The results provide a mechanistic understanding of the structural context of oligomerization.

Why GPCRs behave differently in cubic and lamellar lipidic mesophases.

Recent successes in the crystallographic determination of structures of transmembrane proteins in the G protein-coupled receptor (GPCR) family have established the lipidic cubic phase (LCP) environment as the medium of choice for growing structure-grade crystals by the method termed “in meso”. The understanding of in meso crystallogenesis is currently at a descriptive level. To enable an eventual quantitative, energy-based description of the nucleation and crystallization mechanism, we have examined the properties of the lipidic cubic phase system and the dynamics of the GPCR rhodopsin reconstituted into the LCP with coarse-grained molecular dynamics simulations with the Martini force-field. Quantifying the differences in the hydrophobic/hydrophilic exposure of the GPCR to lipids in the cubic and lamellar phases, we found that the highly curved geometry of the cubic phase provides more efficient shielding of the protein from unfavorable hydrophobic exposure, which leads to a lesser hydrophobic mismatch and less unfavorable hydrophobic–hydrophilic interactions between the protein and lipid–water interface in the LCP, compared to the lamellar phase. Since hydrophobic mismatch is considered a driving force for oligomerization, the differences in exposure mismatch energies between the LCP and the lamellar structures suggest that the latter provide a more favorable setting in which GPCRs can oligomerize as a prelude to nucleation and crystal growth. These new findings lay the foundation for future investigations of in meso crystallization mechanisms related to the transition from the LCP to the lamellar phase and studies aimed at an improved rational approach for generating structure-quality crystals of membrane proteins.

The cost of living in the membrane: a case study of hydrophobic mismatch for the multi-segment protein LeuT.

Many observations of the role of the membrane in the function and organization of transmembrane (TM) proteins have been explained in terms of hydrophobic mismatch between the membrane and the inserted protein. For a quantitative investigation of this mechanism in the lipid-protein interactions of functionally relevant conformations adopted by a multi-TM segment protein, the bacterial leucine transporter (LeuT), we employed a novel method, Continuum-Molecular Dynamics (CTMD), that quantifies the energetics of hydrophobic mismatch by combining the elastic continuum theory of membrane deformations with an atomistic level description of the radially asymmetric membrane-protein interface from MD simulations. LeuT has been serving as a model for structure-function studies of the mammalian neurotransmitter:sodium symporters (NSSs), such as the dopamine and serotonin transporters, which are the subject of intense research in the field of neurotransmission. The membrane models in which LeuT was embedded for these studies were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid, or 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids. The results show that deformation of the host membrane alone is not sufficient to alleviate the hydrophobic mismatch at specific residues of LeuT. The calculations reveal significant membrane thinning and water penetration due to the specific local polar environment produced by the charged K288 of TM7 in LeuT, that is membrane-facing deep inside the hydrophobic milieu of the membrane. This significant perturbation is shown to result in unfavorable polar-hydrophobic interactions at neighboring hydrophobic residues in TM1a and TM7. We show that all the effects attributed to the K288 residue (membrane thinning, water penetration, and the unfavorable polar-hydrophobic interactions at TM1a and TM7), are abolished in calculations with the K288A mutant. The involvement of hydrophobic mismatch is somewhat different in the functionally distinct conformations (outward-open, occluded, inward-open) of LeuT, and the differences are shown to connect to structural elements (e.g., TM1a) known to play key roles in transport. This finding suggests a mechanistic hypothesis for the enhanced transport activity observed for the K288A mutant, suggesting that the unfavorable hydrophobic-hydrophilic interactions hinder the motion of TM1a in the functionally relevant conformational transition to the inward-open state. Various extents of such unfavorable interactions, involving exposure to the lipid environment of adjacent hydrophobic and polar residues, are common in multi-segment transmembrane proteins, and must be considered to affect functionally relevant conformational transitions.

Not Just an Oil Slick: How the Energetics of Protein-Membrane Interactions Impacts the Function and Organization of Transmembrane Proteins

The membrane environment, its composition, dynamics, and remodeling, have been shown to participate in the function and organization of a wide variety of transmembrane (TM) proteins, making it necessary to study the molecular mechanisms of such proteins in the context of their membrane settings. We review some recent conceptual advances enabling such studies, and corresponding computational models and tools designed to facilitate the concerted experimental and computational investigation of protein-membrane interactions. To connect productively with the high resolution achieved by cognate experimental approaches, the computational methods must offer quantitative data at an atomistically detailed level. We show how such a quantitative method illuminated the mechanistic importance of a structural characteristic of multihelical TM proteins, that is, the likely presence of adjacent polar and hydrophobic residues at the protein-membrane interface. Such adjacency can preclude the complete alleviation of the well-known hydrophobic mismatch between TM proteins and the surrounding membrane, giving rise to an energy cost of residual hydrophobic mismatch. The energy cost and biophysical formulation of hydrophobic mismatch and residual hydrophobic mismatch are reviewed in the context of their mechanistic role in the function of prototypical members of multihelical TM protein families: 1), LeuT, a bacterial homolog of mammalian neurotransmitter sodium symporters; and 2), rhodopsin and the β1- and β2-adrenergic receptors from the G-protein coupled receptor family. The type of computational analysis provided by these examples is poised to translate the rapidly growing structural data for the many TM protein families that are of great importance to cell function into ever more incisive insights into mechanisms driven by protein-ligand and protein-protein interactions in the membrane environment.

Cholesterol modulates the membrane effects and spatial organization of membrane-penetrating ligands for G-protein coupled receptors

The ligands of certain G-protein coupled receptors (GPCRs) are membrane soluble and reach their target from the lipid bilayer. Lipid composition and dynamics will therefore modulate the activity of these receptors, but specific roles of lipid components, including the ubiquitous cholesterol (Chol), are not clear. We have probed the organization and dynamics of such a lipid-bilayer-penetrating ligand, the endogenous ligand for the κ-opioid receptor (KOR) dynorphin A (1-17) (DynA), using molecular dynamics (MD) simulations of DynA in cholesterol-depleted and cholesterol-enriched model membranes. DynA is found to penetrate deep inside fluid dimyristoylphosphatidylcholine (DMPC) bilayers, and resides with its N-terminal helix at ∼6 Å away from the bilayer midplane, in a tilted orientation, at an ∼50° angle with respect to the membrane normal. In contrast, DynA inside DMPC/Chol membranes with 20% cholesterol (DMPC/Chol) is situated with its helical segment ∼5 A higher, i.e., closer to the lipid/water interface and in a relatively vertical orientation. The DMPC membrane shows greater thinning around the insertion and permits a stronger influx of water inside the hydrocarbon core than the DMPC/Chol membranes. Relating these results to data about key GPCR residues that have been implicated in interactions with membrane-inserting GPCR ligands, we conclude that the position of DynA in DMPC/Chol, but not in pure DMPC, correlates with generally proposed GPCR ligand entry pathways. Our predictions provide a possible mechanistic explanation as to why DynA binding to KOR, and the subsequent activation of the receptor, is facilitated in cholesterol-enriched environments. A quantitative description of DynA-induced membrane deformations is obtained with a continuum theory of membrane deformations (CTMD) that is based on hydrophobic matching. Comparison with the MD data reveals the significance of the lipid tail packing energy contribution in the DMPC/Chol mixtures in predicting equilibrium membrane shape around DynA. On this basis, specific corrections are introduced to this energy term within the CTMD framework, thereby extending the applicability of the CTMD framework to lipid raft mixtures and their interactions with GPCR proteins and their ligands.

How the Dynamic Properties and Functional Mechanisms of GPCRs Are Modulated by Their Coupling to the Membrane Environment

Experimental observations of the dependence of function and organization of G protein-coupled receptors (GPCRs) on their lipid environment have stimulated new quantitative studies of the coupling between the proteins and the membrane. It is important to develop such a quantitative understanding at the molecular level because the effects of the coupling are seen to be physiologically and clinically significant. Here we review findings that offer insight into how membrane-GPCR coupling is connected to the structural characteristics of the GPCR, from sequence to 3D structural detail, and how this coupling is involved in the actions of ligands on the receptor. The application of a recently developed computational approach designed for quantitative evaluation of membrane remodeling and the energetics of membrane-protein interactions brings to light the importance of the radial asymmetry of the membrane-facing surface of GPCRs in their interaction with the surrounding membrane. As the radial asymmetry creates adjacencies of hydrophobic and polar residues at specific sites of the GPCR, the ability of membrane remodeling to achieve complete hydrophobic matching is limited, and the residual mismatch carries a significant energy cost. The adjacencies are shown to be affected by ligand-induced conformational changes. Thus, functionally important organization of GPCRs in the cell membrane can depend both on ligand-determined properties and on the lipid composition of various membrane regions with different remodeling capacities. That this functionally important reorganization can be driven by oligomerization patterns that reduce the energy cost of the residual mismatch, suggests a new perspective on GPCR dimerization and ligand-GPCR interactions. The relation between the modulatory effects on GPCRs from the binding of specific cell-membrane components, e.g., cholesterol, and those produced by the non-local energetics of hydrophobic mismatch are discussed in this context.

Protein and Lipid Interactions Driving Molecular Mechanisms of in meso Crystallization

The recent advances in the in meso crystallization technique for the structural characterization of G-protein coupled receptor (GPCR) proteins have established the usefulness of the lipidic-cubic phases (LCPs) in the field of crystallography of membrane proteins. It is surprising that despite the success of the approach, the molecular mechanisms of the in meso method are still not well understood. Therefore, the approach must rely on extensive screening for a suitable protein construct, for host and additive lipids, and for the appropriate precipitants and temperature. To shed light on the in meso crystallization mechanisms, we used extensive coarse-grained molecular dynamics simulations to study, in molecular detail, LCPs under different conditions (compositions and temperatures relevant to crystallogenesis) and their interactions with different types of GPCR constructs. The results presented show how the modulation of the lattice constant of the LCP (triggered by the addition of precipitant during the in meso assay), or of the host lipid type, can destabilize monomeric proteins in the bilayer of the LCP and thus drive their aggregation into the stacked lamellae, where the residual hydrophobic mismatch between the protein and the membrane can drive the formation of lateral contacts leading to nucleation and crystal growth. Moreover, we demonstrate how particular protein designs (such as transmembrane proteins engineered to contain large polar regions) can promote protein stacking interactions in the third, out-of-plane, dimension. The insights provided by the new aspects of the specific molecular mechanisms responsible for protein–protein interactions inside the cubic phase presented here should be helpful in guiding the rational design of future in meso trials with successful outcomes.

9.12 Interactions of the Cell Membrane with Integral Proteins

The function and organization of several membrane proteins have been shown to depend on their lipid membrane environment. In the light of such evidence, the choice of the lipid composition has become a critical issue when designing model systems for experiments and simulations, as well as in extrapolating results from model systems to physiological conditions. Additionally, the diversity of lipid composition between the plasma membranes of different cell types, as well as between different lipid domains within the same cell membrane is expected to have physiological consequences. Many observations of the effect of lipid membrane on protein function have been largely explained in terms of the energy cost due to hydrophobic mismatch; that is, the difference between the hydrophobic thickness of the unperturbed membrane and that of the protein embedded in the membrane. This chapter provides a quantitative framework for biophysicists to analyze the hydrophobic mismatch-induced membrane deformations and energetics in a particular membrane-protein system of interest. The utility of the framework is illustrated, along with applications providing mechanistic insights into the activation and oligomerization of G-protein coupled receptors.