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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-HT2A 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.

We use a self-consistent mean-field theory, designed to investigate membrane reshaping and lipid demixing upon interaction with proteins, to explore BAR domains interacting with large patches of lipid membranes of heterogeneous compositions. The computational model includes contributions to the system free energy from electrostatic interactions and elastic energies of the membrane, as well as salt and lipid mixing entropies. The results from our simulation of a single adsorbing Amphiphysin BAR dimer indicate that it is capable of stabilizing a significantly curved membrane. However, we predict that such deformations will occur only for membrane patches that have the inherent propensity for high curvature, reflected in the tendency to create local distortions that closely match the curvature of the BAR dimer itself. Such favorable preconditioning for BAR-membrane interaction may be the result of perturbations such as local lipid demixing induced by the interaction, or of a prior insertion of the BAR domain's amphiphatic N-helix. From our simulations it appears that local segregation of charged lipids under the influence of the BAR dimer cannot produce high enough asymmetry between bilayer leaflets to induce significant bending. In the absence of additional energy contributions that favor membrane asymmetry, the membrane will remain nearly flat upon single BAR dimer adsorption, relative to the undulation expected from thermal fluctuations. Thus, we conclude that the N-helix insertions have a critical mechanistic role in the local perturbation and curving of the membrane, which is then stabilized by the electrostatic interaction with the BAR dimer. We discuss how these results can be used to estimate the tendency of BARs to bend membranes in terms of a spatially nonisotropic spontaneous curvature.

The glutamate transporter GltPh is a homolog of mammalian excitatory amino acid transporters (EAATs) that mediate glutamate re-uptake after discharge at the neuronal synaptic cleft, thereby enabling repeated signaling cycles and preventing excitotoxicity. While the structure of EAATs is not yet known, several stages of the transport cycle have been captured in crystallographic studies of the homotrimeric GltPh, suggesting a mechanism of separate elevator-like motions of three transport domains relative to a scaffold composed of the trimerization domains from each monomer. Dynamic aspects of the transport cycle monitored in single-molecule FRET experiments, revealed quiescent phases in which GltPh appears to be “locked” in the inward- or outward-facing states. Significantly higher transport rates, typical for human EAATs, were recently reported for a GltPh construct in which key residues were mutated to mimic the sequence of mammalian EAAT1. The mutant adopted a novel “unlocked” conformation, in which the transport domains were separated from the trimerization domains. Molecular dynamics simulations of this GltPh mutant in lipid bilayers found the unlocked conformation to be unstable if the transport/trimerization domain interface is solvated by water only, but stabilized by insertion of one or several hydrophobic moieties such as lipid tails. Analysis of the effect of the mutations on the local dynamics in several known stages of the transport cycle showed that a charged side chain introduced at the protein interface with the membrane produces membrane deformation and a destabilizing energy cost due to residual hydrophobic mismatch. Free energy perturbation calculations were used to estimate the impact of the mutation in the explored stages of the transport cycle in order to gain insights on the key structural determinants of EAAT transport efficiency.

DAT is a transmembrane protein in the family of Neurotransmitter:Sodium Symporters (NSS). The NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. In contrast to its bacterial homologue, the leucine transporter (LeuT), mammalian DAT contains long intracellular N- and C-terminal domains that are strongly implicated in the transporter function. The N-terminus (N-term), in particular, regulates the process of efflux, i.e. the reverse transport of the substrate (dopamine) through DAT. Currently, the molecular mechanisms of the efflux remain largely elusive due to lack of structural information on the N-terminal segment. To seek a valid prediction of the three-dimensional (3D) fold of the N-terminus from the human DAT (hDAT) we used a combination of ab initio structure prediction tools and extensive atomistic MD simulations. The ab initio modeling revealed structured regions within the first 57 residues of the N-term. The subsequent long (2.2 μs in total) atomistic MD simulations of the N-terminal in complex with a lipid membrane showed that the identified secondary structure elements were stable on the simulation timescales and were involved in specific interactions with pertinent models of the cell membrane. The N-term engages with lipid membranes through direct electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5-biphosphate) and PS (phosphatidylserine). We identify specific motifs along the N-terminal domain that are implicated in such interactions and show that differential modes of N-term/membrane associations result in differential positioning of the structured segments on the membrane surface. The molecular insights we report regarding the preferred modes of interaction of the DAT N-term with model membranes provide a novel structural context for future explorations of the mechanistic questions related to the functional mechanisms of this transporter.

The 5-HT2A Serotonin receptor (5-HT2AR) is a G-protein coupled receptor (GPCR) targeted by therapeutic drugs as well as hallucinogens such as LSD. We have previously shown that different modes of receptor activation and distinct cellular signaling are produced by hallucinogens and nonhallucinogenic congeners acting on the 5-HT2AR (Weinstein, 2006). We characterize the ligand-dependent states of 5-HT2AR from MD simulations of an experimentally-validated homology model based on rhodopsin and β2AR structures complemented by cognate information about other GPCRs. At different stages of 0.3 μs simulations of 5-HT-bound 5-HT2AR we observe sequential conformational changes that produce structural characteristics of receptor activation: (1) the extracellular part of TM6 moves inwards; (2) the toggle switch W6.48 flips to become parallel to the membrane; (3) the intracellular part of TM6 moves outwards and (4) the ionic lock in the DRY motif breaks. In the parallel simulations of 5-HT2AR with LSD (currently at 0.12 μs), only some of the same active-like components are observed (e.g., TM7 moving away from TM2, and the breaking of the ionic lock). Comparing the dynamics of 5-HT2AR complexed with LSD and 5-HT, we found that H8 of the LSD-bound receptor undergoes less rearrangement during the same time period, and that the interaction of extracellular loop 2 with LSD is stronger than with 5-HT, but that local distortions in TMs (e.g., proline kinks) are similar in the two systems. Interestingly, correlation analysis indicates that some of the local changes in 5-HT2AR relate to the dynamics of cholesterol in the membrane, at a series of preferred sites of interaction with the receptor, in a manner similar to those reported previously from μs rhodopsin simulations (Grossfield et al., 2006; Khelashvili et al., 2009).