Explicit Membrane Research Articles

Membrane/Toxin Interaction Energetics via Serial Multiscale Molecular Dynamics Simulations.

Computing free energies of complex biomolecular systems via atomistic (AT) molecular dynamics (MD) simulations remains a challenge due to the need for adequate sampling and convergence. Recent coarse-grained (CG) methodology allows simulations of significantly larger systems (∼10(6) to 10(8) atoms) over longer (μs/ms) time scales. Such CG models appear to be capable of making semiquantitative predictions. However, their ability to reproduce accurate thermodynamic quantities remains uncertain. We have recently used CG MD simulations to compute the potential of mean force (PMF) or free energy profile of a small peptide toxin interacting with a lipid bilayer along a 1D reaction coordinate. The toxin studied was VSTx1 (Voltage Sensor Toxin 1) from spider venom which inhibits the archeabacterial voltage-gated potassium (Kv) channel KvAP by binding to the voltage-sensor (VS) domains. Here, we re-estimate this PMF profile using (i) AT MD simulations with explicit membrane and solvent and (ii) an implicit membrane and solvent (generalized Born; GBIM) model where only the peptide was explicit. We used the CG MD free energy simulations to guide the setup of the corresponding AT MD simulations. The aim was to avoid local minima in the AT simulations which would be difficult over shorter AT time scales. A cross-comparison of the PMF profiles revealed a conserved topology, although there were differences in the magnitude of the free energies. The CG and AT simulations predicted a membrane/water interface free energy well of -27 and -23 kcal/mol, respectively (with respect to water). The GBIM model, however, gave a reduced interfacial free energy well (-12 kcal/mol). In addition, the CG and GBIM models predicted a free energy barrier of +61 and +96 kcal/mol, respectively, for positioning the toxin at the center of the bilayer, which was considerably smaller in the AT simulations (+26 kcal/mol). Thus, we present a framework for serially combining CG and AT simulations to estimate the free energy of peptide/membrane interactions. Such approaches for combining simulations at different levels of granularity will become increasingly important in future studies of complex membrane/protein systems.

Molecular Mechanisms Underlying the Early Stage of Protein Translocation through the Sec Translocon

The Sec translocon, a protein-conducting channel, consists of a heterotrimeric complex (SecYEG in bacteria and Sec61 alpha beta gamma in eukaryotes) that provides a pathway for secretary proteins to cross membranes, or for membrane proteins to integrate into the membrane. The Sec translocon alone is a passive channel, and association with channel partners, including the ribosome or SecA ATPase in bacteria, is needed for protein translocation. Three recently published crystal structures of SecY are considered to represent the closed (resting state), pre-open (transitional state determined with the bound Fab fragment mimicking SecA interaction), and SecA-bound forms. To elucidate mechanisms of transition between closed and pre-open forms, we performed all-atom molecular dynamics simulations for the pre-open form of Thermus thermophilus SecYE and the closed form of Methanococcus janaschii SecYE beta in explicit solvent and membranes. We found that the closed form of SecY is stable, while the pre-open form without the Fab fragment undergoes large conformational changes toward the closed form. The pre-open form of SecY with Fab remains unchanged, suggesting that the cytosolic interaction mimicking SecA binding stabilizes the pre-open form of SecY. Importantly, a lipid molecule at the lateral gate region appears to be required to maintain the pre-open form in the membrane. We propose that the conformational transition from closed to pre-open states of SecY upon association with SecA facilitates intercalation of phospholipids at the lateral gate, inducing initial entry of the positively charged signal peptide into the channel.

Protegrin-1 Orientation in Membrane Bilayers: Insights from Potential of Mean Force Calculations as A Function of Its Tilt and Rotation Angles

Protegrin-1 (PG-1) belongs to the family of antimicrobial peptides. It interacts specifically with the membrane of a pathogen and kills the pathogen by releasing its cellular contents. To fully understand the energetics governing the orientation of PG-1 in different membrane environments and its effects on the physicochemical properties of the peptide, we have calculated the potentials of mean force (PMF) of PG-1 as a function of its tilt angle in explicit membrane bilayers composed of either DLPC (1,2-dilauroylphosphatidylcholine) or POPC (1-palmitoyl-2-oleoylphosphatidylcholine) lipid molecules. The resulting PMFs in explicit lipid bilayers were then used to search for the optimal hydrophobic thickness of the implicit membrane, in which a two-dimensional PMF in the tilt and rotation space was calculated. The calculated PMFs in explicit membrane systems clearly reveal that the energetically favorable tilt angle is affected by both the membrane hydrophobic thickness and the PG-1 rotation angle. Local thinning of the membrane around PG-1 is observed upon PG-1 tilting. The thinning effect is caused by different arginines in regard to the rotation orientation of the peptide. The two-dimensional PMF calculated in implicit membrane at specified rotation angles is in good accordance with those from the explicit membrane simulations. The ensemble-averaged Val16 15N and 13CO chemical shifts calculated from the two-dimensional free energy distribution agree fairly well with the experimental values, suggesting the accuracy and reliability of the application of PMFs to understand important physicochemical properties of membrane peptides/proteins.

Atomistic Model For the Outward-Facing State of Lactose Permease and Quantification of Ligand Binding

The Major Facilitator Superfamily (MFS) is an important class of membrane transporters whose members are found in unicellular organism to complex systems like the human central nervous system. Lactose Permease (LacY) of E. coli, an MFS protein that transports various sugar molecules across plasma membrane, has been studied as a prototype of secondary transport proteins. Though the structure of LacY in the cytoplasmic open state (inward-facing) has been determined, the atomic-level details of the periplasmic open structure (outward-facing) are unknown. Using a two-step hybrid simulation approach that involves Self-Guided Langevin Dynamics (SGLD) simulations with an implicit membrane followed by molecular dynamics simulations with an explicit membrane, we have obtained an opening of LacY on the periplasmic side that is consistent with the Double Electron-Electron Resonance (DEER) experiments (Smirnova et al., PNAS, 2007). The inward-facing state is stabilized by several helix-helix hydrogen bonds involving side chains of the residues N245, S41, E374, K42 and Q242 and mutations in these residues may destabilize these interactions and help crystallize the outward-facing state. We believe that this hybrid simulation approach can be extended to determine the unknown structures and mechanisms of other secondary active transporters. In addition, sugar binding to LacY has been investigated and our simulations support the alternating access model of sugar transport (same binding site accessible from either side of the membrane). More extensive studies on binding involved alchemical free energy perturbation calculations on αβ-(Galp)2 and ββ-(Galp)2 to better understand the anomeric binding effect. Our methods are tested by comparing the computed binding free energy values for NPG (p-nitrophenyl α-D-galactopyranoside) with the experimental values (Nie et al., JBC, 2006). This work will aid in understanding the effect of substrate structure and affinity to LacY.

Transmembrane Helix Orientation and Dynamics Examined by Ensemble Dynamics with Solid-State NMR Observables and Potential of Mean Force Calculations

Solid-state NMR (SSNMR) experiment is a very powerful technique to describe the orientation of membrane proteins and peptides in their native membrane bilayer environments. Such orientational description is invaluable since the function of membrane proteins and peptides involves a conformational change that often associates with their orientational changes. However, the present models used to interpret the SSNMR observables are rather static, and, in consequence, important motional information can be missing. In this work, we have investigated the orientation of single-pass transmembrane domain of virus protein “u” (Vpu) from HIV-1 by determining ensemble of structures using multiple conformer models. The resulting ensemble of structures showed significantly larger fluctuations in their orientation while averaged orientation maintained the compatible value with the static model. Further, we have compared the SSNMR ensemble dynamics results with the Vpu orientation from the molecular dynamics simulations and helix-tilt free energy calculations in explicit membranes.

Molecular Modeling and Simulations of the Transmembrane Domain of Human Growth Hormone Receptor

How transmembrane (TM) domains of membrane proteins transmit the signal across the cell membrane has long been a subject of keen interest in biology. There is a recent paradigm shift in the mechanism of activation for the cytokine receptor superfamily. The role of cytokine hormone binding to the extracellular domain is now recognized as an “inducer” of the conformational change of pre-dimerized TM domains that triggers subsequent intracellular responses. This is drastically different from its traditional role as an “organizer” whose sole function was to initiate the receptor TM dimer formation. Toward quantitative understanding of the mechanisms and accompanying energetics of TM-induced signaling of various single-pass TM receptors, we have generated TM homodimer models of human growth hormone receptor (hGHR) from primary sequence information using the GBSW implicit membrane model and replica-exchange molecular dynamics (REX-MD) simulations. The conformational clustering shows that hGHR forms right-handed TM dimers with two different interfacial motifs, i.e., LFFQ and GxxG. To test such prediction, we first carried out TOXCAT experiments of two hGHR TM mutants: Gly256Ile and Gly259Ile. Mutation of either position to isoleucine disrupts dimer formation. These results suggest the involvement of the glycine residues in the TM helix interaction through the GxxG motif, although we need more extensive experiments to examine the involvement of other residues in the TM dimer interface, or the existence of an alternate dimerization point. In addition, we have performed MD simulations of various hGHR dimer models extracted from GBSW REX-MD in explicit POPC membranes. The stability and orientational changes of hGHR TM dimers as well as various helix-lipid interactions will be also presented and discussed.

Exploring the Permeation Mechanism of Valinomycin Across Lipid Membranes

Valinomycin is a potassium specific ionophore used to transport ions down an electrochemical gradient across lipid membranes. Its small size, high selectivity, and strong antibiotic activity make it an interesting target for molecular dynamics simulations. At the same time its conformational flexibility, which strongly depends on the polarity of its environment, poses a challenge. This work was undertaken in order to elucidate the mechanism of valinomycin mediated potassium transport across a lipid bilayer. We have explored several advanced sampling techniques, but chose to perform multi-dimensional free energy calculations that explore conformational space while computing the potential of mean force for membrane translocation. By computing free energy surfaces with and without a bound K+ ion and calculating the free energetics of the ion binding process, we can describe a cycle that reveals the overall permeation mechanism. Our results using implicit and explicit solvent and membrane models will be discussed.

Comparison of Lipid Monolayers and Bilayers by Comparative Molecular Dynamics Simulations of a Lipid-Like Dye Molecule

Using p-polarized internal reflection fluorescence microscopy in monolayer membrane systems, Livanec and Dunn have demonstrated that the order of dipalmitoylphosphatidylcholine (DPPC) lipid molecules decrease as the surface pressure increases (Livanec and Dunn (2008) Langmuir 24 (24), 14066-14073). Although monolayer experiments provide more convenient control of membrane properties such as surface pressure, temperature, and lipid compositions, bilayers can offer a more dependable representation of a true biological membrane. This raises the question whether monolayer membranes can be used to study bilayer membranes. In order to explore the correspondence between monolayer and bilayer membranes, molecular dynamics (MD) simulations with lipid reporter dye molecule BODIPY-PC in a DPPC explicit membrane were performed. The correspondence was measured in different surface pressures: high pressure (40 mN/m), normal pressure (25 mN/m), and low pressure (3 mN/m). Each pressure system consisted of 5 bilayer and 5 monolayer systems, totaling up to 30 different simulations. Each simulation ran for 3 ns of equilibration and 47 ns production. Using trajectories, BODIPY-PC molecule's orientation was characterized in terms of tilt, azimuthal, and rotation angles. The calculations were compared between bilayer and monolayer systems to examine structural similarities. In addition, non-bonded energies (or the enthalpic contribution) of the BODIPY-PC molecule in different pressure systems were compared in order to address the driving force governing tilting of the dye molecule in different pressures. The calculated tilt, azimuthal, and rotation angles suggest that monolayer and bilayer systems yield quite similar results. The energy calculations demonstrate that entropy dominates the tilting of a dye molecule in low and normal pressure whereas enthalpy plays a higher role in tilting of a dye molecule in high pressure.

Folding simulations of gramicidin A into the β-helix conformations: Simulated annealing molecular dynamics study

Gramicidin A is a linear hydrophobic 15-residue peptide which consists of alternating D- and L-amino acids and forms a unique tertiary structure, called the beta(6.3)-helix, to act as a cation-selective ion channel in the natural conditions. In order to investigate the intrinsic ability of the gramicidin A monomer to form secondary structures, we performed the folding simulation of gramicidin A using a simulated annealing molecular dynamics (MD) method in vacuum mimicking the low-dielectric, homogeneous membrane environment. The initial conformation was a fully extended one. From the 200 different MD runs, we obtained a right-handed beta(4.4)-helix as the lowest-potential-energy structure, and left-handed beta(4.4)-helix, right-handed and left-handed beta(6.3)-helix as local-minimum energy states. These results are in accord with those of the experiments of gramicidin A in homogeneous organic solvent. Our simulations showed a slight right-hand sense in the lower-energy conformations and a quite beta-sheet-forming tendency throughout almost the entire sequence. In order to examine the stability of the obtained right-handed beta(6.3)-helix and beta(4.4)-helix structures in more realistic membrane environment, we have also performed all-atom MD simulations in explicit water, ion, and lipid molecules, starting from these beta-helix structures. The results suggested that beta(6.3)-helix is more stable than beta(4.4)-helix in the inhomogeneous, explicit membrane environment, where the pore water and the hydrogen bonds between Trp side-chains and lipid-head groups have a role to further stabilize the beta(6.3)-helix conformation.

Tilt and Azimuthal Angles of a Transmembrane Peptide: A Comparison between Molecular Dynamics Calculations and Solid-State NMR Data of Sarcolipin in Lipid Membranes

We report molecular dynamics simulations in the explicit membrane environment of a small membrane-embedded protein, sarcolipin, which regulates the sarcoplasmic reticulum Ca-ATPase activity in both cardiac and skeletal muscle. In its monomeric form, we found that sarcolipin adopts a helical conformation, with a computed average tilt angle of 28 ± 6° and azymuthal angles of 66 ± 22°, in reasonable accord with angles determined experimentally (23 ± 2° and 50 ± 4°, respectively) using solid-state NMR with separated-local-field experiments. The effects of time and spatial averaging on both 15N chemical shift anisotropy and 1H/ 15N dipolar couplings have been analyzed using short-time averages of fast amide out-of-plane motions and following principal component dynamic trajectories. We found that it is possible to reproduce the regular oscillatory patterns observed for the anisotropic NMR parameters (i.e., PISA wheels) employing average amide vectors. This work highlights the role of molecular dynamics simulations as a tool for the analysis and interpretation of solid-state NMR data.

Mechanism Of Ion-ion And Ion-substrate Coupling In Secondary Amino-acid Transporters

The x-ray structures of LeuT and Glt, bacterial homologues of Na+/Cl--dependent amino-acid transporters, provides a great opportunity to better understand the molecular basis of monovalent cation. Both proteins possess ion-binding sites selective for Na+ over K+ and Li+. Extensive QM/MM minimization combined with all-atom free energy molecular dynamics simulations of the LeuT and Glt transporters embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity, coupling between co-transporter substrate and ions occupying binding sites. In this work, we demonstrate that there is a collective effect of multiple binding sites on a total selectivity for Na+ over Li+ both in LeuT and Glt. We also will discuss functional roles of different ion binding sites in the transport cycle. The role of local connectivity, site rigidity, atomic polarization and partial charge transfer in monovalent cation selectivity is discussed.

Conformational transition of the Sec translocon induced by channel partner: A molecular dynamics study

Sec translocon is a highly conserved heteromeric membrane protein complex, which functions as a protein-conducting channel. In bacteria, the Sec translocon (SecYEG) achieves the translocation of polypeptides across the membrane by binding of the channel partner, SecA ATPase. However, little is known about the atomically detailed mechanism on the translocation. Recently, a new crystal structure of the SecYE translocon bound with an anti-SecY Fab fragment has been determined. It contains a large hydrophobic crevasse open to the cytoplasm (the pre-open form) and differ from the crystal structure of SecYEβ from Methanococcus jannaschii in the closed form, suggesting that the binding of a channel partner induces a large conformational change of the Sec translocon in the initial step of the polypeptide translocation. To investigate the role of channel-partner binding to the SecYE translocon, we performed all-atom molecular dynamics simulations of SecYE with and without a Fab fragment in explicit membrane. During a 100-ns simulation, SecYE undergoes a large conformational transition toward the closed form in the absence of a Fab fragment, whereas the structure keeps the widely opened crevasse in the simulation of SecYE with a Fab fragment. In the transition, protein-lipid interaction around the lateral gate region of SecYE is changed greatly, indicating that there is a competition of interactions between the protein and phospholipid molecules, which is controlled by the binding of the channel partner to the SecYE transolon.

Agonist and Antagonist Interactions in Beta Adrenergic Receptors

G-protein coupled receptors (GPCRs) are a large family of integral membrane proteins involved in signal transduction pathways, making them appealing drug targets for a wide spectrum of diseases. The recently solved X-ray structures of beta1 (B1AR) and beta2 (B2AR) adrenergic receptors bound to inverse agonists/antagonists open up a large field of potential investigations to understand the binding modes and mechanisms of activation of GPCRs.To investigate their structural and dynamic properties under pseudo in vivo conditions, we performed extensive molecular dynamics simulations (in an explicit membrane) of adrenergic receptors in complex with partial inverse agonists and agonists as well as in their apoform. To this end, we applied MD-based enhanced sampling techniques (steered MD, metadynamics, …) to describe ligand binding and to elucidate the process of ligand entrance and release.In this contribution, we rationalize the differences in binding mode between B1AR and B2AR for both agonists and antagonists (focusing on a limited set of key residues surrounding the binding pocket that are different between B1AR and B2AR). We also discuss main structural changes upon agonist/antagonist binding also in comparison with the most thoroughly studied GPCR, rhodopsin.

Backbone Conformation and Dynamics of the Lipid-Modified Membrane Anchor of Human N-Ras Investigated by Solid-State NMR and Molecular Dynamics Simulations

Many proteins involved in signal transduction are anchored to membranes by covalently attached lipid modifications. In this study we investigated the conformation and dynamics of the backbone and side chains of the N-Ras membrane anchoring domain. Experimental solid-state NMR studies involved doubly lipid-modified uniformly 13C and 15N labeled heptapeptides representing the C-terminus of N-Ras, which were incorporated into DMPC bilayers. A structural model of the peptide was calculated on the basis of isotropic chemical shifts, explicit torsion angle measurements, and nuclear Overhauser effects determined by solid-state NMR. The amplitude of molecular motions was assessed by 1H-13C order parameter measurements using separated local field NMR. For determination of the correlation times of the motions, T1 and T2 relaxation times were measured and analyzed using a generalized relaxation approach. To further understand the dynamics of Ras, molecular dynamics simulations of the molecule in lipid bilayers were conducted. In generating starting conditions for the simulation, special attention was paid to the backbone conformation since transitions between conformations were found to be rare events in a previous simulation of 100 ns length on this system [1]. Therefore, the experimentally determined conformation of the peptide backbone was equilibrated using a replica exchange technique in an explicit membrane environment. This enabled us to identify different conformers and to assess their relative probability. The resulting distribution of conformations was used subsequently for a long conventional MD simulation that was analyzed with regard to the experimental data. The combined simulations and experimental approach enabled a detailed model of the dynamics of the peptide to be obtained.[1] Vogel, A. Tan, K.-T. Waldmann, H. Feller, S.E. Brown, M.F. Huster, D. Biophys. J. 2007, 93, 2697-2712.

Structural And Dynamic Effects Of Cholesterol At Preferred Sites Of Interaction With Rhodopsin Identified From Microsecond Length Molecular Dynamics Simulations

A key unresolved question about GPCR function is the role of membrane components in receptor stability and activation. In particular, cholesterol is known to affect the function of membrane proteins, but the details of its effect on GPCRs are still elusive. Here, we describe how cholesterol modulates the behavior of TM1-TM2-TM7-helix 8(H8) functional network, that comprises the highly conserved NPxxY(x)5,6F motif, through specific interactions with the receptor. The inferences are based on the analysis of microsecond molecular dynamics (MD) simulations of Rhodopsin in an explicit membrane environment.

An Atomic-level Model for the Periplasmic Open State of Lactose Permease

Membrane transport proteins play significant roles in human physiology, drug transport, bacterial resistance to antibiotics, and diseases. Lactose permease of E. coli (LacY) transports various disaccharides and is a member of the major facilitator superfamily of proteins that exists in a broad range of organisms from archaea to the human central nervous system. Since only the atomic-level structure for the cytoplasmic open state of LacY has been determined, it is our objective to obtain a structure (or set of structures) of LacY open to the periplasm by utilizing a two-step hybrid approach of molecular simulations. In the first step, self-guided Langevin dynamics (SGLD) with an implicit membrane but explicit water is used to enhance conformational sampling. SGLD was found to significantly enhance protein motions compared to identical implicit membrane molecular dynamics (MD) simulations. Significant periplasmic conformational changes are only observed in simulations with Glu269 protonated and a disaccharide in the binding site, which is based on several simulations with different initial structures. LacY helix-helix distances obtained from double electron-electron resonance (DEER) experiments (Smirnova et al., PNAS, 2007) are used to select protein conformations consistent with a periplasmic open state. In the final step, explicit membrane MD simulations with screened structures from the implicit membrane simulations converged to periplasmic open structures. This hybrid implicit/explicit bilayer approach results in LacY structures that transition from a periplasmic closed state (pore radius, Rp, of ∼1A) to one fully open the periplasm (Rp= 3A). The helices on the outside of the protein are the first to fan out (H-III/IV then H-VIII) before there is a concerted motion of the periplasmic half. This two-step simulation approach in conjunction with experiments may be successful in predicting conformational changes of other membrane proteins.

Sampling efficiency in explicit and implicit membrane environments studied by peptide folding simulations

Efficient exploration of the conformational space of peptides embedded in biological membranes is vital to extract converged thermodynamic and kinetic data from simulation studies. Recently developed implicit membrane models promise vast increases in sampling efficiency compared to explicit membrane simulations, allowing for ab initio structure prediction and functional studies. In this study, a previously developed implicit membrane model, based on the generalized Born method, is compared to an explicit di-palmitoyl-phosphatidyl-choline lipid bilayer and an octane slab membrane mimic. The complete folding process of a synthetic 16-residue peptide is compared using these three setups. Since the comparison requires the entire folding pathway to be captured, individual simulations ranged up to 3 mus of MD. A quantitative sampling comparison using a wide range of performance metrics reveals that the implicit membrane model is at least 2 orders of magnitude more efficient than the simplest explicit setups.

Mechanism of Signal Propagation upon Retinal Isomerization: Insights from Molecular Dynamics Simulations of Rhodopsin Restrained by Normal Modes

As one of the best studied members of the pharmaceutically relevant family of G-protein-coupled receptors, rhodopsin serves as a prototype for understanding the mechanism of G-protein-coupled receptor activation. Here, we aim at exploring functionally relevant conformational changes and signal transmission mechanisms involved in its photoactivation brought about through a cis-trans photoisomerization of retinal. For this exploration, we propose a molecular dynamics simulation protocol that utilizes normal modes derived from the anisotropic network model for proteins. Deformations along multiple low-frequency modes of motion are used to efficiently sample collective conformational changes in the presence of explicit membrane and water environment, consistent with interresidue interactions. We identify two highly stable regions in rhodopsin, one clustered near the chromophore, the other near the cytoplasmic ends of transmembrane helices H1, H2, and H7. Due to redistribution of interactions in the neighborhood of retinal upon stabilization of the trans form, local structural rearrangements in the adjoining H3–H6 residues are efficiently propagated to the cytoplasmic end of these particular helices. In the structures obtained by our simulations, all- trans retinal interacts with Cys 167 on H4 and Phe 203 on H5, which were not accessible in the dark state, and exhibits stronger interactions with H5, while some of the contacts made (in the cis form) with H6 are lost.

Molecular mechanism of substrate specificity in the bacterial neutral amino acid transporter LeuT

The recently published X-ray structure of LeuT, a Na(+)/Cl(-)-dependent neurotransmitter transporter, has provided fresh impetus to efforts directed at understanding the molecular principles governing specific neurotransmitter transport. The combination of the LeuT crystal structure with the results of molecular simulations enables the functional data on specific binding and transport to be related to molecular structure. All-atom FEP and molecular dynamics (MD) simulations of LeuT embedded in an explicit membrane were performed alongside a decomposition analysis to dissect the molecular determinants of the substrate specificity of LeuT. It was found that the ligand must be in a zwitterionic (ZW) form to bind tightly to the transporter. The theoretical results on the absolute binding-free energies for leucine, alanine, and glycine show that alanine can be a potent substrate for LeuT, although leucine is preferred, which is consistent with the recent experimental data (Singh et al., Nature 2007;448:952-956). Furthermore, LeuT displays robust specificity for leucine over glycine. Interestingly, the ability of LeuT to discriminate between substrates relies on the dynamics of residues that form its binding pocket (e.g., F253 and Q250) and the charged side chains (R30-D404) from a second coordination shell. The water-mediated R30-D404 salt bridge is thought to be part of the extracellular (EC) gate of LeuT. The introduction of a polar ligand such as glycine to the water-depleted binding pocket of LeuT gives rise to structural rearrangements of the R30-D404-Q250 hydrogen-bonding network and leads to increased hydration of the binding pocket. Conformational changes associated with the broken hydrogen bond between Q250 and R30 are shown to be important for tight and selective ligand binding to LeuT.

Control of Ion Selectivity in LeuT: Two Na+ Binding Sites with Two Different Mechanisms

The x-ray structure of LeuT, a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na+. Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na+ over K+ or Li+, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na+ over K+ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na+ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a “snug-fit” mechanism.