The Open State Gating Mechanism of Gramicidin A Requires Relative Opposed Monomer Rotation and Simultaneous Lateral Displacement

SummaryThe gating mechanism of the open state of the gramicidin A (gA) channel is studied by using a new Monte Carlo Normal Mode Following (MC-NMF) technique, one applicable even without a target structure. The results demonstrate that the lowest-frequency normal mode (NM) at ∼6.5 cm−1 is the crucial mode that initiates dissociation. Perturbing the gA dimer in either direction along this NM leads to opposed, nearly rigid-body rotations of the gA monomers around the central pore axis. Tracking this NM by using the eigenvector-following technique reveals the channel's gating mechanism: dissociation via relative opposed monomer rotation and simultaneous lateral displacement. System evolution along the lowest-frequency eigenvector shows that the large-amplitude motions required for gating (dissociation) are not simple relative rigid-body motions of the monomers. Gating involves coupling intermonomer hydrogen bond breaking, backbone realignment, and relative monomer tilt with complex side chain reorganization at the intermonomer junction

Gating Gramicidin Channels in Lipid Bilayers: Reaction Coordinates and the Mechanism of Dissociation

The dissociation of gramicidin A (gA) channels into monomers is the simplest example of a channel gating process. The initial steps in this process are studied via a computational model that simulates the reaction coordinate for dimer-monomer dissociation. The nonbonded interaction energy between the monomers is determined, allowing for their free relative translational and rotational motion. Lowest energy pathways and reaction coordinates of the gating process are determined. Partial rupture of the six hydrogen bonds (6HB) at the dimer junction takes place by coupling monomer rotation and lateral displacement. Coupling rotation with axial separation is far more expensive energetically. The transition state for channel dissociation occurs when monomers are displaced laterally by ∼4–6 Å, separated by ∼1.6–2 Å, and rotated by ∼120°, breaking two hydrogen bonds. In membranes with significant hydrophobic mismatch there is a much greater likelihood of forming 4HB and possibly even 2HB states. In the 4HB state the pore remains fully open and conductive. However, transitions from the 6HB to 4HB and 4HB to 2HB states take place via intermediates in which the gA pore is closed and nonconductive. These lateral monomer displacements give rise to transitory pore occlusion at the dimer junction, which provides a rationale for fast closure events (flickers). Local dynamics of gA monomers also leads to lateral and rotational diffusion of the whole gA dimer, giving rise to diffusional rotation of the dimer about the channel axis

Anion Pathway and Potential Energy Profiles along Curvilinear Bacterial ClC Cl(−) Pores: Electrostatic Effects of Charged Residues

X-ray structures permit theoretical study of Cl(−) permeation along bacterial ClC Cl(−) pores. We determined the lowest energy curvilinear pathway, identified anion-coordinating amino acids, and calculated the electrostatic potential energy profiles. We find that all four bacterial ClC Cl(−) crystal structures correspond to closed states. E148 and S107 side chains form steric barriers on both sides of the crystal binding site in the StClC wild-type and EcClC wild-type crystals; both the EcClC(E148A) and EcClC(E148Q) mutants are blocked at the S107 site. We studied the effect that mutating the charge of some strongly conserved pore-lining amino acids has on the electrostatic potential energy profiles. When E148 is neutralized, it creates an electrostatic trap, binding the ion near midmembrane. This suggests a possible electrostatic mechanism for controlling anion flow: neutralize E148, displace the side chain of E148 from the pore pathway to relieve the steric barrier, then trap the anion at midmembrane, and finally either deprotonate E148 and block the pore (pore closure) or bring a second Cl(−) into the pore to promote anion flow (pore conductance). Side-chain displacement may arise by competition for the binding site between the oxygens of E148 and the anion moving down the electrostatic energy gradient. We also find that the charge state of E111 and E113 may electrostatically control anion conductance and occupancy of the binding site within the cytoplasmic pore

Antiport Mechanism for Cl−/H+ in ClC-ec1 from Normal-Mode Analysis

ClC chloride channels and transporters play major roles in cellular excitability, epithelial salt transport, volume, pH, and blood pressure regulation. One family member, ClC-ec1 from Escherichia coli, has been structurally resolved crystallographically and subjected to intensive mutagenetic, crystallographic, and electrophysiological studies. It functions as a Cl−/H+ antiporter, not a Cl− channel; however, the molecular mechanism for Cl−/H+ exchange is largely unknown. Using all-atom normal-mode analysis to explore possible mechanisms for this antiport, we propose that Cl−/H+ exchange involves a conformational cycle of alternating exposure of Cl− and H+ binding sites of both ClC pores to the two sides of the membrane. Both pores switch simultaneously from facing outward to facing inward, reminiscent of the standard alternating-access mechanism, which may have direct implications for eukaryotic Cl−/H+ transporters and Cl− channels

Water and Ion Permeation in bAQP1 and GlpF Channels: A Kinetic Monte Carlo Study

The kinetic Monte Carlo reaction-path-following technique is applied to determine the lowest-energy water pathway and the coordinating amino acids in bAQP1 and GlpF channels, both treated as rigid. In bAQP1, water molecules pass through the pore between the asparagine-proline-alanine (NPA) and selectivity filter (SF) sites one at a time. The water chain is interrupted at the SF where one water forms three stable hydrogen bonds with protein atoms. In this SF, water's conformation depends on the protonation locus of H182. In GlpF, two water molecules bond simultaneously to the NPA asparagines and pass through the SF in zigzag fashion. No water single-file forms in rigid GlpF. To accommodate a single file of waters requires narrowing the GlpF pore. Our results reveal that in both proteins a proposed bipolar water arrangement is thermally disrupted in the NPA region, especially in the cytoplasmic part of the pore. The equilibrium hydrogen-bonded chain is occasionally interrupted in the hydrophobic zones adjacent to the NPA motifs. The permeation of alkali cations through bAQP1 and GlpF is barred due to a large free-energy barrier in the NPA region as well as a large energy barrier blocking entry from the cytoplasm. Permeation of halides is prevented due to two large energy barriers in the cytoplasmic and periplasmic pores as well as a large free-energy barrier barring entry from the periplasm. Our results, based on modeling charge permeation, support an electrostatic rather than orientational basis for proton exclusion. Binding within the aquaporin pore cannot compensate sufficiently for dehydration of the protonic charge; there is also an electrostatic barrier in the NPA region blocking proton transport. The highly ordered single file of waters, which is drastically interrupted at the SF of bAQP1, may also contribute to proton block