Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

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The matrix 2 (M2) protein from influenza A virus is a proton channel that uses His37 as a selectivity filter. Here we report high-resolution (1.10 Å) cryogenic crystallographic structures of the transmembrane domain of M2 at low and high pH. These structures reveal that waters within the pore form hydrogen-bonded networks or “water wires” spanning 17 Å from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium via second-shell interactions involving bridging water molecules. In addition, room temperature crystallographic structures indicate that water becomes increasingly fluid with increasing temperature and decreasing pH, despite the higher electrostatic field. Complementary molecular dynamics simulations reveal a collective switch of hydrogen bond orientations that can contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle.

Section: An Abundance Of Hydrogen-bond Donors Relative To Acceptors Imentioning

confidence: 99%

High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction

Thomaston

Alfonso‐Prieto

Woldeyes

et al. 2015

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126

“…In this context, ion channels present the greatest challenges and opportunities: Their narrow pores are usually hydrated; thus, channel blockers effectively have to displace these pore waters. Particularly compelling in this regard are proton channels, such as the M2 proton channel of influenza A virus and Hv1, because waters represent the vehicle with which to achieve proton conduction (91)(92)(93)(94)(95)(96). Notably, in the case of M2, the drug amantadine and other hydrophobic scaffolds were shown to displace clusters of hydrogenbonded waters from main hydration sites (52,54).…”

Section: S1mentioning

confidence: 99%

On the role of water density fluctuations in the inhibition of a proton channel

Gianti

Delemotte

Klein

et al. 2016

Hv1 is a transmembrane four-helix bundle that transports protons in a voltage-controlled manner. Its crucial role in many pathological conditions, including cancer and ischemic brain damage, makes Hv1 a promising drug target. Starting from the recently solved crystal structure of Hv1, we used structural modeling and molecular dynamics simulations to characterize the channel's most relevant conformations along the activation cycle. We then performed computational docking of known Hv1 inhibitors, 2-guanidinobenzimidazole (2GBI) and analogs. Although salt-bridge patterns and electrostatic potential profiles are well-defined and distinctive features of activated versus nonactivated states, the water distribution along the channel lumen is dynamic and reflects a conformational heterogeneity inherent to each state. In fact, pore waters assemble into intermittent hydrogen-bonded clusters that are replaced by the inhibitor moieties upon ligand binding. The entropic gain resulting from releasing these conformationally restrained waters to the bulk solvent is likely a major contributor to the binding free energy. Accordingly, we mapped the water density fluctuations inside the pore of the channel and identified the regions of maximum fluctuation within putative binding sites. Two sites appear as outstanding: One is the already known binding pocket of 2GBI, which is accessible to ligands from the intracellular side; the other is a site located at the exit of the proton permeation pathway. Our analysis of the waters confined in the hydrophobic cavities of Hv1 suggests a general strategy for drug discovery that can be applied to any ion channel.Hv1 | drug design | pore waters | binding site discovery | confined waters

“…The interplay between the protonation state of the His37 tetrad and conformations of the protein backbone and Trp41 side chains is essential for the pH activation mechanism of the channel (9-12). Under high-pH conditions the Trp41 side chains close the C-terminal pore below His37, forming a gate that blocks proton flow through the channel (referred to below as the "C closed " state).As the pH decreases to ∼6, the protonation state of the His37 tetrad reaches a critical level [likely Q2 (7)] and the channel becomes activated, able to enter the primary proton conduction cycle (4,9,(13)(14)(15)(16)(17). (Herein the notation Qn denotes the state of the His37 tetrad, where n is the number of biprotonated histidines, ranging from 0 to 4.)…”

mentioning

confidence: 99%

“…In this cycle, a proton diffusing down its concentration gradient through the activated M2 channel transiently protonates His37, generating the Q3 state (assuming Q2 as the activated resting state). The high density of positive charge in the channel destabilizes the C closed state sufficiently to open an aqueous conduction path (8,9,16,18). Diffusion of a…”

mentioning

confidence: 99%

Acid activation mechanism of the influenza A M2 proton channel

Liang

Swanson

Madsen

et al. 2016

Self Cite

146

The homotetrameric influenza A M2 channel (AM2) is an acidactivated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.ion channel | proton conduction | multiscale modeling | QM/MM | free-energy sampling V isualizing the vectorial flow of protons through membrane proteins is a long-standing challenge in biophysics and chemistry, with manifold implications for understanding bioenergetics, active transport, and proton channel function. Multiscale modeling has become a full partner with experimental structural biology in addressing proton transport (PT), because it can connect the dots between the high-resolution but static pictures obtained from crystallography and the lower-resolution but dynamically informed structures seen in the ensemble averages by NMR and other spectroscopic approaches. In general, multiscale modeling connects three or more disparate but coupled scales of behavior. In the case of PT in proteins, there are usually four pertinent scales: (i) the quantum mechanical scale of bond breaking and bond making inherent in the Grotthuss proton shuttle mechanism; (ii) the molecular scale of the dynamical motions of the protein, membrane, ions, and water molecules; (iii) the "free energy" scale that arises from the statistical ensemble averaging of those molecular motions; and (iv) the "transport" scale that manifests as a macroscopic conductance, with associated gating and other possible behaviors tied to macroscopic experimental variables. The proper and rigorous connection of these various scales, in an overall multiscale computational simulation, is often a substantial challenge. Here, we turn our attention to understanding the me...