Mark S.P. Sansom

HOLE: A program for the analysis of the pore dimensions of ion channel structural models

A method (HOLE) that allows the analysis of the dimensions of the pore running through a structural model of an ion channel is presented. The algorithm uses a Monte Carlo simulated annealing procedure to find the best route for a sphere with variable radius to squeeze through the channel. Results can be displayed in a graphical fashion or visualized with most common molecular graphical packages. Advances include a method to analyze the anisotropy within a pore. The method can also be used to predict the conductance of channels using a simple empirically corrected ohmic model. As an example the program is applied to the cholera toxin B-subunit pentamer. The compatibility of the crystal structure and conductance data is established.

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.

Liquid–vapor oscillations of water in hydrophobic nanopores

Water plays a key role in biological membrane transport. In ion channels and water-conducting pores (aquaporins), one-dimensional confinement in conjunction with strong surface effects changes the physical behavior of water. In molecular dynamics simulations of water in short (0.8 nm) hydrophobic pores the water density in the pore fluctuates on a nanosecond time scale. In long simulations (460 ns in total) at pore radii ranging from 0.35 to 1.0 nm we quantify the kinetics of oscillations between a liquid-filled and a vapor-filled pore. This behavior can be explained as capillary evaporation alternating with capillary condensation, driven by pressure fluctuations in the water outside the pore. The free-energy difference between the two states depends linearly on the radius. The free-energy landscape shows how a metastable liquid state gradually develops with increasing radius. For radii > ≈0.55 nm it becomes the globally stable state and the vapor state vanishes. One-dimensional confinement affects the dynamic behavior of the water molecules and increases the self diffusion by a factor of 2–3 compared with bulk water. Permeabilities for the narrow pores are of the same order of magnitude as for biological water pores. Water flow is not continuous but occurs in bursts. Our results suggest that simulations aimed at collective phenomena such as hydrophobic effects may require simulation times >50 ns. For water in confined geometries, it is not possible to extrapolate from bulk or short time behavior to longer time scales.

Acquisition of siderophores in Gram-negative bacteria

The outer membrane of Gram-negative bacteria constitutes a permeability barrier that protects the cell from exterior hazards, but also complicates the uptake of nutrients. In the case of iron, the challenge is even greater, because of the scarcity of this indispensable element in the cells surroundings. To solve this dilemma, bacteria have evolved sophisticated mechanisms whereby the concerted actions of receptor, transporter and energy-transducing proteins ensure that there is a sufficient supply of iron-containing compounds, such as siderophores.

Hinges, swivels and switches: the role of prolines in signalling via transmembrane α-helices

Abstract Extracellular signals are transduced across membranes via conformational changes in the transmembrane domains (TMs) of ion channels and G-protein-coupled receptors (GPCRs). Experimental and simulation studies indicate that such conformational switches in transmembrane α-helices can be generated by proline-containing motifs that form molecular hinges. Computational approaches tested on model channel-forming peptides (e.g. alamethicin) reveal functional mechanisms in gap-junction proteins (such as connexin) and voltage-gated K + channels. Similarly, functionally important roles for proline-based switches in TM6 and TM7 were identified in GPCRs. However, hinges in transmembrane helices are not confined to proline-containing sequence motifs, as evidenced by a non-proline hinge in the M2 helix of the nicotinic acetylcholine receptor. This helix lines the pore and plays a key role in the gating of this channel.

Membrane proteins: molecular dynamics simulations.

Molecular dynamics simulations of membrane proteins are making rapid progress, because of new high-resolution structures, advances in computer hardware and atomistic simulation algorithms, and the recent introduction of coarse-grained models for membranes and proteins. In addition to several large ion channel simulations, recent studies have explored how individual amino acids interact with the bilayer or snorkel/anchor to the headgroup region, and it has been possible to calculate water/membrane partition free energies. This has resulted in a view of bilayers as being adaptive rather than purely hydrophobic solvents, with important implications, for example, for interaction between lipids and arginines in the charged S4 helix of voltage-gated ion channels. However, several studies indicate that the typical current simulations fall short of exhaustive sampling, and that even simple protein-membrane interactions require at least ca. 1 micros to fully sample their dynamics. One new way this is being addressed is coarse-grained models that enable mesoscopic simulations on multi-micros scale. These have been used to model interactions, self-assembly and membrane perturbations induced by proteins. While they cannot replace all-atom simulations, they are a potentially useful technique for initial insertion, placement, and low-resolution refinement.

Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1

Triangulating to Mechanism Cellular uptake and release of a variety of substrates are mediated by secondary transporters, but no crystal structures are known for all three fundamental states of the transport cycle, which has limited explanations for their proposed mechanisms. Shimamura et al. (p. 470) report a 3.8-angstrom structure of the inward-facing conformation of the bacterial sodium-benzylhydantoin transport protein, Mhp1, complementing the other two available structures. Molecular modeling for the interconversions of these structures shows a simple rigid body rotation of four helices relative to the rest of the structure in which the protein switches reversibly from outward- to inward-facing. Three complementary crystal structures reveal the mechanism of a transport protein in molecular dynamics simulations. The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepTSo, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepTSo represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.

Functional analysis of a structural model of the ATP‐binding site of the KATP channel Kir6.2 subunit

ATP‐sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore‐forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP‐binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C‐terminus of one subunit, and with R50 in the N‐terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP‐dependent channel inhibition. The model also suggests that interactions between the C‐terminus of one subunit and the ‘slide helix’ of the adjacent subunit may be involved in ATP‐dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.

Alamethicin and related peptaibols — model ion channels

Peptaibols are considered as models of those ion channels which consist of a bundle of transbilayer helices surrounding a central pore. X-Ray diffraction and NMR studies have yielded high resolution structures for several peptaibols. In conjunction with other spectroscopic investigations and molecular dynamics simulations, these studies suggest that peptaibols form proline-kinked α-helices, and that there may be “hinge-bending” movement of the helix in the region of the central proline residue. The amphipathicity of peptaibol helices is analyzed in relation to their channel-forming properties. Studies of the interactions of peptaibols with lipid bilayers suggest that they are helical when in a membrane-like environment, and that the helix orientation relative to the bilayer is sensitive to the peptaibol: lipid ratio, and to the degree of hydration of the bilayer. Electrical studies reveal that many peptaibols form multiple-conductance level channels in a voltage-dependent fashion. Analysis of conductance levels provides support for the “barrel stave” model of channel formation, whereby different conductance levels correspond to different numbers of monomers in a helix bundle. Alternative models for voltage-activation are discussed, and the roles of molecular dipoles and of hinge-bending in this process are considered. Two molecular models for an N = 6 bundle of alamethicin helices are presented and their electrostatic properties analyzed. The relevance of studies of peptaibols to channel and transport proteins in general is considered.