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Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM).sup.1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules.sup.2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms.sup.3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset. A localization algorithm is applied to datasets obtained with conventional and high-speed atomic force microscopy to increase image resolution beyond the limits set by the radius of the tip used., Author(s): George R. Heath [sup.1] [sup.4] , Ekaterina Kots [sup.2] , Janice L. Robertson [sup.3] , Shifra Lansky [sup.1] , George Khelashvili [sup.2] , Harel Weinstein [sup.2] , Simon Scheuring [...]

Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na+-dependent uptake of ω−3 fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na+. Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na+ -bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a "trap-and-flip" mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle. Molecular mechanisms of MFSD2A-mediated lysolipid transport into the brain has been elusive. Here, using molecular dynamics, the authors uncover how initial stages of transport cycle are enabled by substrate-induced conformational changes in MFSD2A. [ABSTRACT FROM AUTHOR]

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, [Glt.sub.Ph], showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that [Glt.sub.Ph] bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold., Glutamate transporters, also termed excitatory amino acid transporters (EAATs), maintain glutamate concentration gradients across the cell membrane by coupling neurotransmitter uptake to symport of three sodium ([Na.sup.+]) ions and a [...]

Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities. Fine-tuning CAR affinity: Chimeric antigen receptor (CAR) T cells provide a potent therapeutic approach for some human cancers refractory to other treatment approaches. However, the suboptimal affinity of CAR binder molecules for their target antigens can contribute to impaired antitumor function. He et al. used cryo-electron microscopy to determine the protein structure of several CAR binders complexed with the extracellular portion of human CD19, a target antigen found on B-cell malignancies. These structural data combined with molecular dynamics simulations and in vitro protein-binding assays were used to engineer mutant anti-CD19 CAR binders with modified sensitivity for the target antigen. These findings represent a proof-of-concept demonstration that high-resolution structural data can be used to rationally design optimized CAR binders capable of improved functional characteristics. —IRW [ABSTRACT FROM AUTHOR]

Rapid flip-flop of phospholipids across the two leaflets of biological membranes is crucial for many aspects of cellular life. The transport proteins that facilitate this process are classified as pump-like flippases and floppases and channel-like scramblases. Unexpectedly, Class A G protein–coupled receptors (GPCRs), a large class of signaling proteins exemplified by the visual receptor rhodopsin and its apoprotein opsin, are constitutively active as scramblases in vitro. In liposomes, opsin scrambles lipids at a unitary rate of >100,000 per second. Atomistic molecular dynamics simulations of opsin in a lipid membrane reveal conformational transitions that expose a polar groove between transmembrane helices 6 and 7. This groove enables transbilayer lipid movement, conceptualized as the swiping of a credit card (lipid) through a card reader (GPCR). Conformational changes that facilitate scrambling are distinct from those associated with GPCR signaling. In this review, we discuss the physiological significance of GPCR scramblase activity and the modes of its regulation in cells. [ABSTRACT FROM AUTHOR]

Recent discoveries about functional mechanisms of proteins in the TMEM16 family of phospholipid scramblases have illuminated the dual role of the membrane as both the substrate and a mechanistically responsive environment in the wide range of physiological processes and genetic disorders in which they are implicated. This is highlighted in the review of recent findings from our collaborative investigations of molecular mechanisms of TMEM16 scramblases that emerged from iterative functional, structural, and computational experimentation. In the context of this review, we present new MD simulations and trajectory analyses motivated by the fact that new structural information about the TMEM16 scramblases is emerging from cryo‐EM determinations in lipid nanodiscs. Because the functional environment of these proteins in in vivo and in in vitro is closer to flat membranes, we studied comparatively the responses of the membrane to the TMEM16 proteins in flat membranes and nanodiscs. We find that bilayer shapes in the nanodiscs are very different from those observed in the flat membrane systems, but the function‐related slanting of the membrane observed at the nhTMEM16 boundary with the protein is similar in the nanodiscs and in the flat bilayers. This changes, however, in the bilayer composed of longer‐tail lipids, which is thicker near the phospholipid translocation pathway, which may reflect an enhanced tendency of the long tails to penetrate the pathway and create, as shown previously, a nonconductive environment. These findings support the correspondence between the mechanistic involvement of the lipid environment in the flat membranes, and the nanodiscs. © 2019 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We have developed a dynamic self-consistent mean-field model, based on molecular-dynamics simulations, to study lipid-cholesterol bilayers. In this model the lipid bilayer is represented as a two-dimensional lattice field in the lipid chain order parameters, while cholesterol molecules are represented by hard rods. The motion of rods in the system is continuous and is not confined to lattice cells. The statistical mechanics of chain ordering is described by a mean field derived from an extension of a model due to Marcˇelja. The time evolution of the system is governed by stochastic equations. The ensemble of chain configurations required in partition sums, and the energies of interaction, are taken from atomistic level molecular-dynamics simulations of lipid bilayers. The model allows us to simulate systems 500 nm in lateral size for 20 μs time scales, or greater. We have applied the model to dipalmitoyl-phosphatidylcholine–cholesterol (Chol) bilayers at 50 °C for Chol concentrations between 2% and 33%. At low concentrations of Chol (2%–4%), the model predicts the formation of isolated clusters of Chol surrounded by relatively ordered lipid chains, randomly dispersed in the disordered bilayer. With increasing Chol composition, regions of Chol-induced order begin to overlap. Starting from about 11% Chol this ordering effect becomes system wide and regions unaffected by Chol are no longer detectable. From the analysis of properties of the model we conclude that the change in lipid chain order with increasing Chol concentration is continuous over the 20-μs scale of the simulations. We also conclude that at 50 °C no large-scale Chol-rich and Chol-depleted coexisting phase-separated regions form at any concentration. At no point in any of the simulations do we observe a higher degree of lateral organization, such as Chol-based superlattice structures. [ABSTRACT FROM AUTHOR]

We have carried out atomic level molecular dynamics and Monte Carlo simulations of hydrated 18:0 sphingomyelin (SM)-cholesterol (CHOL) bilayers at temperatures of 20 and 50°C. The simulated systems each contained 266 SM, 122 CHOL, and 11861 water molecules. Each simulation was run for 10 ns under semi-isotropic pressure boundary conditions. The particle-mesh Ewald method was used for long-range electrostatic interactions. Properties of the systems were calculated over the final 3 ns. We compare the properties of 20 and 50°C bilayer systems with each other, with experimental data, and with experimental and simulated properties of pure SM bilayers and dipalmitoyl phospatidyl choline (DPPC)-CHOL bilayers. The simulations reveal an overall similarity of both systems, despite the 30°C temperature difference which brackets the pure SM main phase transition. The area per molecule, lipid chain order parameter profiles, atom distributions, and electron density profiles are all very similar for the two simulated systems. Consistent with simulations from our lab and others, we find strong intramolecular hydrogen bonding in SM molecules between the phosphate ester oxygen and the hydroxyl hydrogen atoms. We also find that cholesterol hydroxyl groups tend to form hydrogen bonds primarily with SM carbonyl, methyl, and amide moieties and to a lesser extent methyl and hydroxyl oxygens. [ABSTRACT FROM AUTHOR]

Both lipid and ion translocation by Ca2+-regulated TMEM16 transmembrane proteins utilizes a membrane-exposed hydrophilic groove. Several conformations of the groove are observed in TMEM16 protein structures, but how these conformations form, and what functions they support, remains unknown. From analyses of atomistic molecular dynamics simulations of Ca2+-bound nhTMEM16 we find that the mechanism of a conformational transition of the groove from membrane-exposed to occluded from the membrane involves the repositioning of transmembrane helix 4 (TM4) following its disengagement from a TM3/TM4 interaction interface. Residue L302 is a key element in the hydrophobic TM3/TM4 interaction patch that braces the open-groove conformation, which should be changed by an L302A mutation. The structure of the L302A mutant determined by cryogenic electron microscopy (cryo-EM) reveals a partially closed groove that could translocate ions, but not lipids. This is corroborated with functional assays showing severely impaired lipid scrambling, but robust channel activity by L302A. A membrane-exposed groove in Ca2+-gated TMEM16 scramblases forms the translocation pathway for ions and lipids. Here authors combine molecular dynamics with cryo-EM and functional assays to uncover the conformational transitions of the groove leading to a non-selective ion channel pore. [ABSTRACT FROM AUTHOR]

Summary BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila , disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. [ABSTRACT FROM AUTHOR]

Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Substrate and Na+ are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics. [ABSTRACT FROM AUTHOR]

Ions play key mechanistic roles in the gating dynamics of neurotransmitter: sodium symporters (NSSs). In recent microsecond scale molecular dynamics simulations of a complete model of the dopamine transporter, a NSS protein, we observed a partitioning of K+ ions from the intracellular side toward the unoccupied Na2 site of dopamine transporter following the release of the Na2-bound Na+. Here we evaluate with computational simulations and experimental measurements of ion affinities under corresponding conditions, the consequences of K+ binding in the Na2 site of LeuT, a bacterial homolog of NSS, when bothNa+ ions and substrate have left, and the transporter prepares for a new cycle. We compare the results with the consequences of binding Na+ in the same apo system. Analysis of >50-µs atomistic molecular dynamics and enhanced sampling trajectories of constructs with Glu290, either charged or neutral, point to the Glu290 protonation state as a main determinant in the structural reconfiguration of the extracellular vestibule of LeuT in which a "water gate" opens through coordinated motions of residues Leu25, Tyr108, and Phe253. The resulting water channel enables the binding/dissociation of the Na+ and K+ ions that are prevalent, respectively, in the extracellular and intracellular environments. [ABSTRACT FROM AUTHOR]

Background: The importance of the material properties of membranes for diverse cellular processes is well established. Notably, the elastic properties of the membrane, which depend on its composition, can directly influence membrane reshaping and fusion processes as well as the organisation and function of membrane proteins. Determining these properties is therefore key for a mechanistic understanding of how the cell functions. Results: We have developed a method to determine the bending rigidity and tilt modulus, for lipidic assemblies of arbitrary lipid composition and shape, from molecular dynamics simulations. The method extracts the elastic moduli from the distributions of microscopic tilts and splays of the lipid components. We present here an open source implementation of the method as a set of Python modules using the computational framework OpenStructure. These modules offer diverse algorithms typically used in the calculatation the elastic moduli, including routines to align MD trajectories of complex lipidic systems, to determine the water/lipid interface, to calculate lipid tilts and splays, as well as to fit the corresponding distributions to extract the elastic properties. We detail the implementation of the method and give several examples of how to use the modules in specific cases. Conclusions: The method presented here is, to our knowledge, the only available computational approach allowing to quantify the elastic properties of lipidic assemblies of arbitrary shape and composition (including lipid mixtures). The implementation as python modules offers flexibility, which has already allowed the method to be applied to diverse lipid assembly types, ranging from bilayers in the liquid ordered and disordered phases to a study of the inverted-hexagonal phase, and with different force-fields (both all-atom and coarse grained representations). The modules are freely available through GitHub at https://github.com/njohner/ost_pymodules/ while OpenStructure can be obtained at http://www.openstructure.org. [ABSTRACT FROM AUTHOR]

Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl ß-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins. [ABSTRACT FROM AUTHOR]

The physiological functions of neurotransmitter:sodium symporters (NSS) in reuptake of neurotransmitters from the synapse into the presynaptic nerve have been shown to be complemented by their involvement, together with non-plasma membrane neurotransmitter transporters, in the reverse transport of substrate (efflux) in response to psychostimulants. Recent experimental evidence implicates highly anionic phosphatidylinositol 4,5-biphosphate (PIP 2 ) lipids in such functions of the serotonin (SERT) and dopamine (DAT) transporters. Thus, for both SERT and DAT, neurotransmitter efflux has been shown to be strongly regulated by the presence of PIP 2 lipids in the plasma membrane, and the electrostatic interaction of the N-terminal region of DAT with the negatively charged PIP 2 lipids. We examine the experimentally established phenotypes in a structural context obtained from computational modeling based on recent crystallographic data. The results are shown to set the stage for a mechanistic understanding of physiological actions of neurotransmitter transporters in the NSS family of membrane proteins. This article is part of a Special Issue entitled: Lipid–protein interactions. [ABSTRACT FROM AUTHOR]

ABSTRACT The dopamine transporter (DAT) is a transmembrane protein belonging to the family of neurotransmitter:sodium symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The 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 reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N-terminal segment. Here we report a computational model of the N-term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N-term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 μs). Combining MD simulations with continuum mean-field modeling we found that the N-term engages with lipid membranes through electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5-Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N-term implicated in such interactions and show that differential modes of N-term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function. Proteins 2015; 83:952-969. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Structural and functional properties of integral membrane proteins are often studied in detergent micellar environments (proteomicelles), but how such proteomicelles form and organize is not well understood. This makes it difficult to evaluate the relationship between the properties of the proteins measured in such a detergent-solubilized form and under native conditions. To obtain mechanistic information about this relationship for the leucine transporter (LeuT), a prokaryotic homologue of the mammalian neurotransmitter/sodium symporters (NSSs), we studied the properties of proteomicelles formed by n-dodecyl-β,Dmaltopyranoside (DDM) detergent. Extensive atomistic molecular dynamics simulations of different protein/detergent/water number ratios revealed the formation of a proteomicelle characterized by a constant-sized shell of detergents surrounding LeuT protecting its transmembrane segments from unfavorable hydrophobic/hydrophilic exposure. Regardless of the DDM content in the simulated system, this shell consisted of a constant number of DDM molecules (∼120 measured at a 4 Å cutoff distance from LeuT). In contrast, the overall number of DDMs in the proteomicelle (aggregation number) was found to depend on the detergent concentration, reaching a saturation value of 226±17 DDMs in the highest concentration regime simulated. Remarkably, we found that at high detergent-to-protein ratios we observed two independent ways of DDM penetration into LeuT, both leading to a positioning of the DDM molecule in the second substrate (S2) binding site of LeuT. Consonant with several recent experimental studies demonstrating changes in functional properties of membrane proteins due to detergent, our findings highlight how the environment in which the membrane proteins are examined may affect the outcome and interpretation of their mechanistic features [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]