Proton transport behavior through the influenza A M2 channel: insights from molecular simulation

Abstract

The structural properties of the influenza A virus M2 transmembrane channel in dimyristoylphosphatidylcholine bilayer for each of the four protonation states of the proton-gating His-37 tetrad and their effects on proton transport for this low-pH activated, highly proton-selective channel are studied by classical molecular dynamics with the multistate empirical valence-bond (MS-EVB) methodology. The excess proton permeation free energy profile and maximum ion conductance calculated from the MS-EVB simulation data combined with the Poisson-Nernst-Planck theory indicates that the triply protonated His-37 state is the most likely open state via a significant side-chain conformational change of the His-37 tetrad. This proposed open state of M2 has a calculated proton permeation free energy barrier of 7 kcal/mol and a maximum conductance of 53 pS compared to the experimental value of 6 pS. By contrast, the maximum conductance for Na(+) is calculated to be four orders of magnitude lower, in reasonable agreement with the experimentally observed proton selectivity. The pH value to activate the channel opening is estimated to be 5.5 from dielectric continuum theory, which is also consistent with experimental results. This study further reveals that the Ala-29 residue region is the primary binding site for the antiflu drug amantadine (AMT), probably because that domain is relatively spacious and hydrophobic. The presence of AMT is calculated to reduce the proton conductance by 99.8% due to a significant dehydration penalty of the excess proton in the vicinity of the channel-bound AMT.

FIGURE 1

Reduced system in the MS-EVB2 simulation. The M2 channel is highlighted and colored blue. The proton-gating His-37 and Trp-41 residues are colored orange and mauve, respectively. The C-terminal end is at the top. The figure was created with visual molecular dynamics software (87).

FIGURE 2

Comparison of the RMSF of the M2 protein backbone atoms between the full composite system and the reduced system in Fig. 1 for protonation states +1, +2, +3, and +4.

FIGURE 3

Distance of COM between any two of the four α-helices as a function of time during 6.0 ns classical MD simulations for protonation states +1, +2, +3, and +4.

FIGURE 4

Tilt angle of the four α-helices with respect to the DMPC bilayer normal as a functions of time during 6.0 ns classical MD simulations for protonation states +1, +2, +3, and +4.

FIGURE 5

Profile of the average pore radius during 6.0 ns classical MD simulations as calculated by the HOLE2 program (62) for protonation states +1, +2, +3, and +4. The water Van der Waals radius of 1.4 Å is marked by a straight dashed line. The locations of the His-37 and Trp-41 domains are indicated by rectangular bars with overhead legends “HIS37” and “TRP41”, respectively.

FIGURE 6

Torsion angle χ₁ of the four His-37 residues as a function of time during 6.0 ns classical MD simulations for protonation states +1, +2, +3, and +4.

FIGURE 7

Free energy profile of the excess proton CEC along the channel permeation pathway for protonation states +1 (solid), +2 (dotted), +3 (dot-dashed), and +4 (dashed). The locations of the His-37 and Trp-41 domains are indicated by rectangular bars with overhead legends “HIS37” and “TRP41”, respectively.

FIGURE 8

Profile of the position-dependent diffusion coefficient of the excess proton for protonation states +1, +2, +3, and +4.

FIGURE 9

Free energy profile along the channel permeation pathway of excess proton (dashed) and proton with the presence of AMT (dotted) and sodium cation (solid) for the proposed open state +3. The locations of the Ala-29, His-37, and Trp-41 domains are indicated by rectangular bars with overhead legends “ALA29”, “HIS37”, and “TRP41”.

FIGURE 10

Profile of the position-dependent diffusion coefficient of excess proton (dashed) and proton with the presence of AMT (dotted) and sodium cation (solid) for the proposed open state +3.

FIGURE 11

Permeation free energy profiles for sodium cation along the channel axis for the proposed open state +3 by applying all the AMBER force field (solid), the AMBER protein force field combined with the CHARMM ion force field (dotted), the AMBER force field with enhanced greater protein backbone flexibility (dashed), and the AMBER force field with a thicker water layer (dot-dashed). The reduced model was used in all simulations here.