Homology modeling and molecular dynamics simulations of transmembrane domain structure of human neuronal nicotinic acetylcholine receptor

Abstract

A three-dimensional model of the transmembrane domain of a neuronal-type nicotinic acetylcholine receptor (nAChR), (alpha4)2(beta2)3, was constructed from a homology structure of the muscle-type nAChR recently determined by cryo-electron microscopy. The neuronal channel model was embedded in a fully hydrated DMPC lipid bilayer, and molecular-dynamics simulations were performed for 5 ns. A comparative analysis of the neuronal- versus muscle-type nAChR models revealed many conserved pore-lining residues, but an important difference was found near the periplasmic mouth of the pore. A flickering salt-bridge of alpha4-E266 with its adjacent beta2-K260 was observed in the neuronal-type channel during the course of the molecular-dynamics simulations. The narrowest region, with a pore radius of approximately 2 A formed by the salt-bridges, does not seem to be the restriction site for a continuous water passage. Instead, two hydrophobic rings, formed by alpha4-V259, alpha4-L263, and the homologous residues in the beta2-subunits, act as the gates for water flow, even though the region has a slightly larger pore radius. The model offers new insight into the water transport across the (alpha4)2(beta2)3 nAChR channel, and may lead to a better understanding of the structures, dynamics, and functions of this family of ion channels.

FIGURE 1

The sequence of the human α4 (top) and β2 (bottom) are aligned with the Torpedo californica α1 and the β1, respectively. The numbering corresponds to the sequences of α4 and β2. The sequence break points between the TM3 and the TM4 domains in the structure of 1OED are indicated by the vertical arrows (↑).

FIGURE 2

(A) A top view and (B) side view of the TM domain model of (α4)₂(β2)₃ nAChR embedded in a fully hydrated DMPC lipid bilayer after 3.61-ns simulations.

FIGURE 3

(A) Structural drift revealed as RMSDs of all Cα atoms in the helical regions of each subunit. The grayscale on the top left of the figure reflects the strength of applied harmonic restraint forces to the backbone of the protein. Stepwise jumps of RMSDs correlate to a gradual reduction of harmonic restraints. A complete relief of restraints at 2.11 ns led to smooth increases of RMSD, which reached a plateau ∼3 ns. (B) Averaged helical tilt angles relative to the bilayer norm (z axis) as a function of MD simulation time. The course of applied harmonic restraints was identical to that in A.

FIGURE 4

Structural flexibility shown as RMSFs of all Cα atoms averaged over all subunits from the last 3-ns of unrestrained simulations.

FIGURE 5

Interactions (≤3.5 Å) of tryptophan (in TM4) or tyrosine (in TM1, TM3, and TM4) with DMPC lipid headgroups. The number of atoms involved in the interactions is shown as a function of position along the bilayer normal (Z) and simulation time.

FIGURE 6

Salt-bridges between α4 and β2. The distances from the carboxylate oxygen of α4-Glu²⁶⁶ to an adjacent side-chain amine of β2-Lys²⁶⁰ (see inset) are plotted as a function of simulation time.

FIGURE 7

Snapshots of the pore profiles and the water passage through the pore. For clarity, only part of the protein backbone and water in or close to the pore are shown. (The pore-lining residues are color-coded: yellow, α4-Thr²⁴⁸ and β2-Thr²⁴²; green, α4-Ser²⁵² and β2-Ser²⁴⁶; purple, α4-Leu²⁵⁵ and β2-Leu²⁴⁹; cyan, α4-Val²⁵⁹ and β2-Val²⁵³; orange, α4-Leu²⁶³ and β2-Leu²⁵⁷; and blue, α4-Glu²⁶⁶ and β2-Lys²⁶⁰.) The pore profile was generated using (the) HOLE. (The color codes for the pore radius: red, <2.0 Å; green, between 2.0 Å and 3.0 Å; and blue, >3.0 Å.) (A) A water vacuum is formed after 546 ps of simulation. (B) A water vacuum is reduced to between residues α4-Val²⁵⁹ and α4-Leu²⁶³ after 1110 ps of simulation. (C) A continuous water passage is formed after 4110 ps of simulation. A single file of water flows through the hydrophobic rings at α4-Val²⁵⁹ and α4-Leu²⁶³.