Self-assembly of a simple membrane protein: coarse-grained molecular dynamics simulations of the influenza M2 channel

Abstract

The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of alpha-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 x 5 micros) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2x rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of approximately 16 degrees. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel.

FIGURE 1

CG (A) and atomistic (B) systems, with the M2 tetramer, one DPPC lipid molecule, and the phosphate particles/atoms of the remaining DPPC molecules represented. Colors for the atoms: cyan, carbon; red, oxygen; blue, nitrogen; and bronze, phosphorous. Colors for CG particles: cyan, apolar; red, polar; blue, positively charged; and bronze, negatively charged. The CG (C) and atomistic (D) single M2 helices are shown using the same color schemes, although the atomistic backbone has been colored gray for clarity.

FIGURE 2

Snapshots of the system during the bilayer-forming simulation, showing the helix partitioning into the bilayer as it forms (water particles omitted for clarity). The helix partitions into the arranging lipids that form a stable bilayer after ∼23 ns. This bilayer remains intact (and the helix continues to span the bilayer) for the remainder of the simulation.

FIGURE 3

Distances between the centers of mass of the helices as they aggregate together into a tetramer (A), and snapshots of the positions of the helices (B) as tetramerization takes place. Helix 1, blue; helix 2, red; helix 3, gray; and helix 4, orange. This indicates that helices 1 and 4 form a dimer after ∼120 ns, which becomes a trimer (with helix 2) ∼100 ns later. A four-helix aggregate is formed after ∼530 ns, which then rearranges into a left-handed helical bundle.

FIGURE 4

Ensemble represents conformations 1–4 rotated by 90°, 180°, or 270° so that they can all be superimposed upon one another.

FIGURE 5

(A) Helix distances show that helices 1 and 2, and helices 3 and 4 are opposite each other in the arrangement of the helical bundle. It can be seen that at several points the two diagonal distances “switch” so that the closer helices move apart, whereas the distant helices approach one other. (B) Comparison of 2×-symmetrical (“representative”) and 4×-symmetrical (“transitional”) M2 helix bundle structures.

FIGURE 6

Comparison of the CG (A) and atomistic (B) models that was based on the CG structure. The trace of the CG backbone particles and the atomistic α-carbons are represented (helix 1, blue; helix 2, red; helix 3, gray; and helix 4, orange). The side-chain particles for the CG residues H37 and W41, and the heavy atoms of the equivalent atomistic residues are shown in more detail. (C) The x-ray structure (66) and (D) the NMR structure (67) of the M2 TM helix bundle are shown fitted to the converged CG model. E and F show (respectively) the Cα traces of the x-ray and NMR structures (red) fitted onto our atomistic model (blue) with the H37 and W41 side chains also represented. The nearest helix has been removed from E and F for clarity.

FIGURE 7

RMSD (top) and RMSF (bottom) for the two atomistic simulations containing the histidine-neutral (solid) system and the histidine-4⁺ (shaded) system.

FIGURE 8

Pore profile of the atomistic channel, taken from a snapshot during the histidine-neutral simulation. The side chains of the key gating residues (H37) are shown. The color scheme for the pore surface is as follows: red, radius < 1.15 Å (no water can pass); green, 1.15 Å < radius < 2.30 Å (single-file water); and blue, radius > 2.30 Å (multiple waters). Helix 1, blue; helix 2, red; helix 3, gray; and helix 4, orange.