David D. Busath

Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer.

M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric proton channel in the viral envelope and is essential for viral replication. Two manuscripts shed light on the functional mechanism of this channel. Sharma et al. (p. 509; see the Perspective by Fiorin et al.) determined the structure of the conductance domain in a lipid bilayer and propose that a histidine and tryptophan from each monomer form a cluster that guides protons through the channel in a mechanism that involves forming and breaking hydrogen bonds between adjacent pairs of histidines. Hu et al. (p. 505; see the Perspective by Fiorin et al.) focused on the structure and dynamics of the proton-selective histidine at high and low pH, proposing that proton conduction involves histidine deprotonation and reprotonation. A tetrameric cluster of histidine and tryptophan residues, through its unique chemistry, shepherds protons through the M2 channel. The M2 protein from the influenza A virus, an acid-activated proton-selective channel, has been the subject of numerous conductance, structural, and computational studies. However, little is known at the atomic level about the heart of the functional mechanism for this tetrameric protein, a His37-Trp41 cluster. We report the structure of the M2 conductance domain (residues 22 to 62) in a lipid bilayer, which displays the defining features of the native protein that have not been attainable from structures solubilized by detergents. We propose that the tetrameric His37-Trp41 cluster guides protons through the channel by forming and breaking hydrogen bonds between adjacent pairs of histidines and through specific interactions of the histidines with the tryptophan gate. This mechanism explains the main observations on M2 proton conductance.

Noncontact dipole effects on channel permeation. I. Experiments with (5F-indole)Trp13 gramicidin A channels.

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.

Model channel ion currents in NaCl-extended simple point charge water solution with applied-field molecular dynamics.

Using periodic boundary conditions and a constant applied field, we have simulated current flow through an 8.125-A internal diameter, rigid, atomistic channel with polar walls in a rigid membrane using explicit ions and extended simple point charge water. Channel and bath currents were computed from 10 10-ns trajectories for each of 10 different conditions of concentration and applied voltage. An electric field was applied uniformly throughout the system to all mobile atoms. On average, the resultant net electric field falls primarily across the membrane channel, as expected for two conductive baths separated by a membrane capacitance. The channel is rarely occupied by more than one ion. Current-voltage relations are concentration dependent and superlinear at high concentrations.

M2 protein from influenza A: from multiple structures to biophysical and functional insights.

The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N-terminal and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel.

Monte Carlo simulations of ion selectivity in a biological Na channel: Charge–space competition

Na channels that produce the action potentials of nerve and muscle include a selectivity filter formed by both positively and negatively charged amino acid residues in a molecular pore. Here we present Monte Carlo simulations of equilibrium ion absorption in such a system. Ions are treated as charged hard spheres in a uniform dielectric. Tethered carboxylate and amino groups known to line the selectivity filter of the Na channel are represented as charged hard spheres and restricted to the filter region of the channel. Consistent with experiments, we find (1) that absorption of Ca2+ into the filter exceeds absorption of Na+ only when the concentration of Ca2+ is some tenfold larger than physiological; (2) the model channel absorbs smaller alkali metal ions preferentially compared to larger ones. The alkali metal selectivity involves volume exclusion of larger ions from the center of the filter region.

Noncontact dipole effects on channel permeation. III. Anomalous proton conductance effects in gramicidin.

Proton transport on water wires, of interest for many problems in membrane biology, is analyzed in side-chain analogs of gramicidin A channels. In symmetrical 0.1N HCl solutions, fluorination of channel Trp(11), Trp-(13), or Trp(15) side chains is found to inhibit proton transport, and replacement of one or more Trps with Phe enhances proton transport, the opposite of the effects on K(+) transport in lecithin bilayers. The current-voltage relations are superlinear, indicating that some membrane field-dependent process is rate limiting. The interfacial dipole effects are usually assumed to affect the rate of cation translocation across the channel. For proton conductance, however, water reorientation after proton translocation is anticipated to be rate limiting. We propose that the findings reported here are most readily interpreted as the result of dipole-dipole interactions between channel waters and polar side chains or lipid headgroups. In particular, if reorientation of the water column begins with the water nearest the channel exit, this hypothesis explains the negative impact of fluorination and the positive impact of headgroup dipole on proton conductance.

Monte Carlo study of the selectivity of calcium channels: improved geometrical model

An extended model of a calcium channel is described involving a channel with a finite length. With this new geometry the channel is still selective but less so than in an infinite cylinder geometry (Boda, D., Busath, D. D., Henderson, D., and Sokolowski, S., 2000, J. phys. Chem. B, 104, 8903). The selectivity of the channel depends on the width and length of the channel filter but is not significantly affected by changes in the size of the entry vestibules. Interestingly, changes in the size of the entry vestibules do affect the details of the concentration profiles of some of the ions.

Monte Carlo Simulation Study of a System with a Dielectric Boundary: Application to Calcium Channel Selectivity

Equilibrium Monte Carlo simulations are reported for a model of a biological calcium channel in which the region that includes the channel and the selectivity filter has a different dielectric coefficient than that of the bath. These regions are separated by a sharp dielectric boundary. This simple geometry makes it possible to use the image charge method to incorporate polarization effects. We also include a description of changes in solvation energy that arise when ions move between different dielectrics; we use the Born description of hydration with empirical ionic radii that yield experimental hydration energies. In calculations of Ca2+vs. Na+ selectivity analogous to those of earlier work (Boda et al., Molec. Phys., 100, 2361 (2002)), we find that reducing the dielectric coefficient in the channel to values as low as 10 renders the channel model less calcium selective. Thus, this continuum description of polarization effects does not capture a delicate balance that seems to exist in biological channels between the energy that ions require for dehydration and the energy that ions gain by interaction with the charged groups in the pore.

Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A.

A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

Method for Estimating the Internal Permittivity of Proteins Using Dielectric Spectroscopy

Protein charge organization is dependent on the low-permittivity region in the hydrophobic core of the molecule. We suggest a novel approach to estimate the dielectric constant of this region by comparing measured and simulated first- and second-order charge moments. Here, the dipole moment is measured as a function of pH using dielectric spectroscopy. The results are compared to dipole moments based on Poisson-Boltzmann estimates of pK(a) shifts calculated from structures in the Protein Data Bank. Structures are additionally refined using CHARMM molecular dynamics simulations. The best estimate for the internal permittivity is found by minimizing the root-mean-square residual between measured and predicted charge moments. Using the protein β-lactoglobulin, a core dielectric constant in the range of 6-7 is estimated.