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G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β(2)-adrenergic receptor (β(2)AR) and the cholecystokinin (CCK) receptor subfamily. The β(2)AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous β(2)AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK(1)R shows functional sensitivity to membrane cholesterol while CCK(2)R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β(2)AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-T(m)) shows that mutation of a conserved CRAC sequence on TM7 of the β(2)AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.

Transmembrane proteins are responsible for transporting ions and small molecules across the hydrophobic region of the cell membrane. We are reviewing the evidence for regulation of these transport processes by interactions with the lipids of the membrane. We focus on ion channels, including potassium channels, mechanosensitive and pentameric ligand gated ion channels, and active transporters, including pumps, sodium or proton driven secondary transporters and ABC transporters. For ion channels it has been convincingly shown that specific lipid-protein interactions can directly affect their function. In some cases, a combined approach of molecular and structural biology together with computer simulations has revealed the molecular mechanisms. There are also many transporters whose activity depends on lipids but understanding of the molecular mechanisms is only beginning.

Diverse classes of proteins function through large-scale conformational changes; sophisticated enhanced sampling methods have been proposed to generate these macromolecular transition paths. As such paths are curves in a high-dimensional space, they have been difficult to compare quantitatively, a prerequisite to, for instance, assess the quality of different sampling algorithms. The Path Similarity Analysis (PSA) approach [1] alleviates these difficulties without relying on low-dimensional projections by utilizing the full information in 3N-dimensional trajectories in configuration space. PSA employs the Hausdorff or Frechet path (distance) metrics—adopted from computational geometry—enabling us to quantify path (dis)similarity, while the new concept of a Hausdorff-pair map permits the extraction of atomic-scale determinants responsible for path differences. Combined with clustering techniques, PSA facilitates the comparison of many paths, including collections of transition ensembles. We use the closed-to-open transition of the enzyme adenylate kinase (AdK)—a commonly used testbed for the assessment enhanced sampling algorithms [2]—to examine multiple microsecond equilibrium molecular dynamics (MD) transitions of AdK in its substrate-free form alongside transition ensembles from the MD-based dynamic importance sampling (DIMS-MD) and targeted MD (TMD) methods, and a geometrical targeting algorithm (FRODA). A Hausdorff pairs analysis of these ensembles revealed, for instance, that differences in DIMS-MD and FRODA paths were mediated by a set of conserved salt bridges whose charge-charge interactions are fully modeled in DIMS-MD but not in FRODA. We illustrate how existing trajectory analysis methods relying on pre-defined collective variables (such as native contacts or geometric quantities) can be used synergistically with PSA, as well as how PSA can be applied to more complex systems such as membrane transporter proteins.[1] Seyler, Kumar, Thorpe, Beckstein. PLoS Comput Biol (2015), http://dx.doi.org/10.1371/journal.pcbi.1004568; [2] Seyler and Beckstein. Mol Simul 40:855-877 (2014), http://dx.doi.org/10.1080/08927022.2014.919497.

Na+/H+ antiporters are vital for maintaining homeostasis in bacterial cells, in particular for survival in high-salt environments. In humans, these transporters are important drug targets, because their dysfunction is linked to a variety of diseases, including cancer and cardiovascular pathophysiology. However, to date the molecular mechanism by which these proteins transport ions remains poorly understood. We recently solved the structure of the archaeal transporter Thermus Thermophilus NapA in an outward-facing conformation where the putative ion binding site is exposed to the cellular exterior [1]. Using disulfide links to trap the protein, we have now obtained an inward-facing structure of NapA, revealing a large conformational change consistent with the alternating access mechanism long proposed for these transporters. We performed molecular dynamics (MD) simulations demonstrating the disulfide-linked structure to be stable in the absence of the links. Simulations of both inward- and outward-facing conformations show that the dimerization domain anchors the protein in the membrane while the core domain translates the binding site 6-7 A in an elevator-like mechanism. Na+ binding is also dependent on protonation states of the conserved aspartates and lysine for both conformations. This binding is consistent with the hypothesis that the conserved lysine plays a direct role in binding protons in Na+/H+ antiporters that transport 2 H+ [2]. Taken together, structural and simulation data for two distinct conformations of the same transporter give an unambiguous view into the molecular mechanism underlying Na+/H+ antiport.[1] Lee et. al. Nature, 501:573-577, 09 2013. http://dx.doi.org/10.1038/nature12484.[2] Lee et. al. J Gen Physiol, 144:529-544, 2014. http://dx.doi.org/10.1085/jgp.201411219.

Diverse classes of proteins such as molecular motors, enzymes, and active transmembrane transporters function through large-scale conformational changes. Computer simulations of these conformational transitions are challenging. A range of coarse-grained and biased simulation techniques have been used to generate individual transitions or ensembles of transition pathways but it has been difficult to compare pathways produced by different methods and so to assess their relative strengths. We introduce a comprehensive method (pathway similarity analysis, PSA) for quantitatively characterizing and comparing macromolecular pathways. The Hausdorff and Frechet metrics (known from computational geometry) are used to quantify the degree of similarity between piecewise-linear curves in configuration space. We tested PSA on a toy system to study the effect of temperature fluctuations (path roughness) and dimensionality. We compare a sample of publicly accessible transition pathway simulation servers and our own dynamic importance sampling (DIMS) MD method for the closed-to-open transitions of the apo enzyme adenylate kinase (AdK). PSA was applied to ensembles of hundreds of trajectories of the conformational transitions of the transporter Mhp1 and of AdK and diphtheria toxin, which were produced by DIMS MD and the Geometrical Pathways algorithm. Clustered PSA enabled the selection of a small subset of representative trajectories for further analysis. A strength of PSA is its use of the full information available from the 3N-dimensional configuration space trajectory, without requiring additional specific knowledge about the system. We show how trajectory analysis methods relying on pre-defined collective variables such as native contacts or geometric quantities can be used synergistically with PSA. We discuss the method's potential to enhance our understanding of transition path sampling methods, validate them, and ultimately help guide future research toward deeper physical insights into conformational transitions.

Boron is an essential micronutrient for plants and animals. Transport of boron regulates uptake and protects against high levels of boron in plants and yeast. The yeast boron transporter Bor1p belongs to the SLC4 Anion Exchanger family of transporters that also includes the human chloride/bicarbonate exchanger. We have overexpressed and purified the Bor1p orthologue from Saccharomyces mikatae using Saccharomyces cerevisiae as a host. Helical crystals were produced by reconstituting the transporter together with cardiolipin and electron micrographs of frozen-hydrated tubes were recorded with a field emission gun at 200 kV using a direct electron detector. Crystals adopted two different helical symmetries from which two independent reconstructions were produced from 75 particles at a resolution of ∼7 A using a Fourier-Bessel approach. In order to interpret the dimeric densities revealed by the reconstructions, we built a homology model for Bor1p based on the crystal structure of the uracil transporter (UraA), which is the closest relative to Anion Exchangers within the APC superfamily of transporters. Initial homology models were produced from 10 alternative alignments of these distantly related proteins. The alignments were refined by mapping sequence conservation onto these 3D structures. The best model was then fitted to the cryo-EM map by adjusting the position of individual helices and the resulting structure was equilibrated in a lipid bilayer for 200 ns using molecular dynamics. The resulting structure suggests conformational changes relative to UraA in which helices at the dimer interface are tilted relative to the transporter domain, thus providing access to the substrate binding site from the extracellular side of the membrane. Comparison of our Bor1p structure with that from UraA are likely to reflect the structural changes that accompany the alternating access mechanism employed by this family of transporters.

Sodium/proton antiporters are integral membrane proteins that are vital for cell homeostasis. In humans, they maintain cellular pH and their dysfunction is linked to a variety of complex diseases, including cancer, cardiovascular pathophysiology, and autism. Na+/H+ antiporters are secondary active transporters that utilize the electrochemical gradient of one ionic species to drive the energetically uphill transmembrane transport of the other species. They operate by the alternating access mechanism whereby the protein cycles between an outward facing and inward facing conformation to switch the exposure of substrate binding sites between the extracellular and the intracellular environment.

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 200against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C terminal transmembrane (TM) helix. In coarse grain molecular dynamics simulations self-assembly of the membrane occurred with the syb2 TM helix inserted as expected from experimental data. The free energy profile for the position of the TM domain in the membrane was determined applying harmonic potentials to the peptide in its unbiased position, pulling it towards new biased equilibrium positions. The energy profile determined in this way predicts the energy landscapes for pulling syb2 towards the extravesicular side as expected for SNARE complex zippering. Applying a constant pulling force of 160 pN detaches the synaptobrevin C terminus from the vesicle's inner leaflet lipid head groups within ∼100ns and pulls the C terminus deeper into the membrane. This C terminal movement should occur on the physiological millisecond time scale at ∼120 pN force. It is facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. These results suggest a mechanism where fusion pore formation is induced by movement of the charged syb2 C terminus into the hydrophobic core of the membrane in response to the force generated by C terminal zippering of the SNARE complex. This displacement of the charged C terminus is expected to destabilize the membrane providing a plausible pathway to fusion pore formation. Supported by NIH grants R01GM085808, R21NS072577, EDICT Project grant 201924, an MRC fellowship and the Wellcome Trust.

Na þ /H þ antiporters are vital to cells for maintaining homeostasis, especially in high-salt environments. New crystal structures for two such antiporters, cytoplasmic-open Escherichia coli NhaA and periplasmic-open Thermus Thermophilus NapA, show high structural similarity despite low sequence identity. Among their common features are a set of highly conserved charged residues (three aspartates and one lysine) near the putative ion binding site. Using molecular dynamics simulations, we observe that interaction with sodium is dependent on the charge states of the conserved aspartates. The lysine and the nearest asparate also form a previously unidentified salt bridge. Under simulated physiological pH the presence of a sodium ion disrupts and breaks the salt bridge in NhaA. Given the presence of this salt bridge in both protein structures, its behavior under simulation, and the known importance of the conserved lysine, we hypothesize that this salt bridge is directly involved in ion binding and transport. To address the question of sodium binding, we performed an ensemble of all-atom equilibrium molecular dyanamics simulations (over 10 ms in total), varying the protonation states of the conserved residues and quantifying the resulting sodium interaction. To address the question of proton binding, we performed heuristic pKa calculations on this ensemble of simulations, which support the hypothesis that the lysine binds protons in a sodium-dependent manner. In order to elucidate the structural basis for alternating access in NhaA, we created an outward-facing model of NhaA, based on the periplasmic-open NapA structure, and simulated conformational transitions of NhaA from its cytoplasmic- to periplasmic-open state using dynamic importance sampling MD. Taken together, the combination of recent structural and dynamic simulation data suggests a new model of ion binding and transport for the CPA2 class of antiporters. 987-Plat

The transmembrane protein NhaA from Escherichia coli is a prototypical sodium/proton antiporter. It enables the bacterium to grow under high salt conditions while homologous proteins in eukaryotes are involved in pH and cell volume regulation. A number of acidic and basic residues have been shown to be essential for the transport of one sodium ion for two protons but the mechanistic details of their involvement have not been fully determined. Furthermore, the conformational changes involved in the transport mechanism were not known. We present an unpublished crystal structure of NhaA in the inward facing conformation and of the homolog NapA in the outward facing conformation [1]. Using modelling and computer simulations we show how NhaA can function according to the alternating access model, resulting in large relative domain motions that are incompatible with previous structural models for transport in NhaA. Our structure of NhaA contains a salt bridge between the two conserved residues Asp163 and Lys300. With the help of molecular dynamics simulations we critically examine competing models for the molecular mechanism of the stoichiometric transport of two protons for one sodium ion, including one in which Lys300 maintains an active role in proton transport.[1] Lee et al, Nature 501 (2013), 573.

In living cells specialized water channels, the glycero-aquaporins, are responsible for rapid passive transport of water through the membrane. Crystal structures showed that these protein pores have diameters comparable to the size of a single water molecule (around 0.3 nm) and lengths around 2 to 3 nm. Despite being strongly confined, water moves rapidly through these and other narrow peptide pores such as gramicidin. Understanding how water behaves in confinement has not been easy, not the least because there is no experimental technique available to unambiguously measure water flux through a single water pore that would be comparable to single channel patch clamp recordings on ion channels. Thus, if we want to understand how a physiological function such as the water flux derives from the structure, namely the geometry and chemical character of the water pathway, we can do this only with models that relate macroscopic transport coefficients to microscopic properties of the pore. Although there is no satisfactory microscopic theory for water transport across all the pore dimensions relevant for biology (1,2) it is clear that the single most important parameter for water permeation is the radius of the pore. Biological, water-permeable pores have radii ranging from 0.14 nm (gramicidin A, K+-channel selectivity filter) through ∼0.8 nm (ligand gated ion channels in the open state) to around 10 nm (the open region of the nuclear pore complex) (3). Transport through pores that are wide compared to the typical interaction and correlation lengths in liquid water (≫ 1 nm or more than ∼4 water diameters) can be described well by continuum theories. There is also progress being made on narrow pores that can only accommodate a 1D chain of water molecules; here the important simplification is that water molecules cannot pass each other. However, as soon as we enter the “multi-pass” regime of R > 2rwater ≈ 0.28 nm no theories are available. The molecular dynamics simulations by Portella and de Groot (4) in this issue hint at what a new theory for the transition from the no-pass to the multi-pass regime could look like. In these simulations water can move freely through “toy model” pores of variable radius, length, and surface character. The pores are based on the architecture of the gramicidin A channel and embedded in a membrane mimetic. Computer simulations afford full control over all parameters of the simulation, including many at the atomic level that are not accessible to experimentalists in the real world. The authors use this to good effect; for instance, the degree of hydrophilicity of a pore is adjusted by simply editing the backbone dipole moment. The simulations measure the two experimentally accessible permeation coefficients that characterize water transport. The osmotic permeability coefficient pf expresses the observation that osmotic water flux Jw = pfΔcsolute is driven by a concentration difference of an impermeable solute between two compartments separated by a semipermeable membrane. For single-file pores, pf was shown to be proportional to the rate of translocation of the whole n-water chain by one hop. From a physiological point of view such a collective hop is the important observable: one water molecule enters the pore on one side, another one exits on the other and the net effect is the movement of one water molecule across the membrane. The second observable is the diffusive permeability coefficient pd. It is proportional to the number of translocation events of individual water molecules through the pore per unit time and thus primarily characterizes the pore-water interaction. It can be measured by following dilute isotope-tagged water molecules in solution that interact in the same way as the untagged ones. Because it takes n + 1 individual hops along the chain to achieve an individual translocation event there exists a relationship between the measurable permeabilities and the number of water molecules n in the single file chain, pfpd=n+1. (1) Equation 1 has been used successfully to determine the channel length of single-file pores such as gramicidin A from osmotic measurements (2); the length is simply calculated as n times the average water-water distance. However, the theoretical value of the pf/pd ratio for wider molecular pores than single file ones has not been established unequivocally and Alan Finkelstein's remark from 1987 is still true: “There is presently no satisfactory physical theory for diffusion and flow through pores with radii only a few times larger than that of the solvent molecule.” (2) One of the many intriguing findings of Portella and de Groot (4) is that a more general, although (at the moment) phenomenological, relation, pfpd=q(R)(〈n〉+1), (2) seems to hold for any water filled molecular pore (at least up to a radius of ∼0.6 nm). Here q(R) is the number of effective single file columns that fit into the pore of radius R; for a true single file pore q(R) = 1 holds. Equation 2 relaxes two important constraints that were required to derive Eq. 1: The “no-pass” and the “no-vacancy” condition. It seems that we can interpret flow through wider pores as consisting of effective 1D chains of water molecules. Second, by identifying the ratio of the permeabilities with the average occupancy of the single file it also becomes possible to apply permeability measurements in a meaningful way to hydrophobic pores that may only be partially occupied. Computer simulations indicated that sufficiently hydrophobic pores would be, depending on the degree of hydrophobicity, filled with water vapor, or fluctuate between liquid filled and vapor filled states. Although plausible on physical grounds—liquid water at ambient conditions is close to its liquid-vapor phase coexistence (5) and thus any influence such as a surface or a change in chemical potential may tilt the balance toward the vapor state—it is not clear that such liquid-vapor oscillations actually exist in nature. This work, however, suggests how to experimentally measure such behavior. By measuring pf/pd − 1 for a narrow pore one determines the average pore occupancy; choosing R so small that q = 1 simplifies the considerations. Because the liquid-vapor equilibrium is easily shifted in either direction by a change in pressure or temperature (6) one should only have to repeat the measurements under different conditions and look for a sudden change in 〈n(T, P) 〉. It should, however, also be kept in mind that simulations ultimately need to find justification in experiments (and experiments need a model, based on theory and simulation). Before Eq. 2 can be really used to interpret measurements it will require some additional experimental validation. This may be quite a formidable task that needs to be addressed by theoreticians and experimentalists together, similar to the ongoing attempts to explain the dependence of pf on the pore length from experiments (7) and simulations (8). Even so, the work of Portella and de Groot should already inspire the formulation of new theories and clarify the meaning of the experimental observables. For instance, together with the insight that water permeation can be described by a simple collective coordinate (9) it lets us qualitatively interpret pf and pd in a slightly different light: pd is related to the probability that a distinguishable water molecule completes a translocation whereas pf measures the translocation of indistinguishable particles. It seems that we may be able to learn more about the microscopic water structure in a pore from macroscopic measurements than thought previously.

The cystic fibrosis transmembrane conductance regulator (CFTR) is the only known member of the ATP binding cassette transporter superfamily that functions as an ion channel. Its architecture is analogous to that of ABC exporters such as p-glycoprotein, MsbA and Sav1866, which export their substrates using an alternating access mechanism driven by catalytic cycles coupled to ATP hydrolysis. The opening and closing of the CFTR channel is also driven by ATP catalytic cycles. We studied the state-dependence of the reactivity of three engineered cysteines located at positions 334, 337 and 338 in transmembrane helix 6 (TM6) of CFTR toward externally-applied, thiol-directed reagents, in order to investigate the conformational changes associated with channel gating. The engineered cysteine at position 334 was more reactive in the closed state, consistent with the findings of Zhang et al. (2005; J. Biol. Chem. 280: 41997-42003), while the engineered cysteines at positions 337 and 338 were more reactive in the open state. These findings are consistent with the predictions of homology models of the CFTR channel based on the crystal structures of bacterial homologues captured in inward- and outward-facing states of the proteins. Funded by the National Institute of Diabetes and Digestive and Kidney Diseases, the Cystic Fibrosis Foundation, and the American Lung Association.

The transmembrane protein NhaA from Escherichia coli is a prototypical sodium/proton antiporter. It enables the bacterium to grow under high salt conditions while homologous proteins in eukaryotes are involved in pH and cell volume regulation. A number of acidic and basic residues have been shown to be essential for the transport of one sodium ion for two protons but the mechanistic details of their involvement have not been fully determined. In particular, a highly conserved lysine residue (Lys300) near the center of the membrane had so far been only given a possible indirect role in the transport mechanism. We present a new atomic resolution structure of the inward facing conformation that shows a novel salt bridge between Lys300 and the conserved Asp163. Microsecond molecular dynamics simulations indicate that the salt bridge is sensitive to the presence of a sodium ion that spontaneously binds to the conserved aspartate residue 164. The simulations show how binding of sodium ion can be coupled to a structural change which might trigger a conformational change to an outward facing conformation. Taken together, the structural and simulation data generate a new hypothesis for how Lys300 could be directly involved in proton transport.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated, anion-selective channel that is the product of the gene mutated in the inherited disease, cystic fibrosis. Chloride conductance via CFTR is a key component of the salt and water secretory system that maintains mucus hydration in the airway lumen. CFTR belongs to the large family of ABC transporter proteins, but it is the only member known to function as an ion channel. The channel is highly selective for anions over cations, but the structural basis for the observed charge selectivity is unknown. Individual point mutations generally do not seem to strongly affect charge selectivity, suggesting the possibility of a mechanism that is not highly dependent on local structural specialization such as that seen in the K-channel selectivity filter. We used a recent structural model of CFTR, based on the crystal structure of the Sav1866 ABC transporter and experimentally validated by cysteine scanning [1], to begin a computational investigation of the structural basis of the selectivity of the CFTR channel for anions over cations. Poisson-Boltzmann calculations suggest that an excess of basic residues in the transmembrane domain creates a large, anion-stabilizing region that includes the channel pore and extends towards the extracellular vestibule. The results indicate that electrostatic interactions affecting a large volume of the protein are a major contributor to anion over cation selectivity of the CFTR channel, similar to the situation observed in OprP [2].[1] C Alexander et al. Biochemistry 48 (2009) 10078-10088.[2] P Pongprayoon et al PNAS 106 (2009), 21614-21618.

The outer membrane protein OprP from Pseudomonas aeruginosa forms an anion-selective pore, especially selective for phosphate ions. The protein is homo-trimeric, with each pore lined by three positively charged loops (L3, L5, and T7) folded into its lumen. OprP plays a key role in high-affinity phosphate uptake under the condition of phosphate starvation. To better understand the mechanism of phosphate-selective permeation, we employed three simulation techniques: (i) equilibrium molecular dynamics simulations (MD); (ii) steered MD (SMD); (iii) umbrella sampling to calculate a potential of mean force (PMF) for phosphate and chloride ions. The PMFs reveal a deep energy well midway along the OprP channel. Two adjacent phosphate-binding sites (W1 and W2), each with a well depth of ∼8kT, are identified close to the L3 loop in the most constricted region of the pore. The transfer of phosphate between sites W1 and W2 is correlated with changes in conformation of the sidechain of K121, which serves as a ‘charged brush’ to facilitate phosphate passage between the two subsites. The PMF for chloride has also been computed and can be compared with that of phosphate. Our simulations suggest that OprP does not conform to the conventional picture of a channel with a relatively flat energy landscape for permeant ions, but rather resembles a membrane-inserted binding protein with a high specificity that allows access to a centrally located binding site from both the extracellular and the periplasmic spaces.