Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism

Abstract

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of: 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating: a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.

FIGURE 1

(Left) Alignment between human α7 sequence and the chimerical AChBP-Torpedo marmorata subunits. The residues from α7 intracellular loop were removed. The residues of AChBP and Torpedo marmorata membrane domain are represented in red and blue, respectively. The positions of the Cys-loop (loop 7) and of α-helices are highlighted. (Right) Structure 0: model α7 receptor obtained after energy minimization of a preliminary structure obtained using comparative modeling. The extracellular and membrane domains are represented in red and blue, respectively.

FIGURE 2

Models 1O and 1C. View from the synaptic cleft. One subunit is represented in gray for clarity. The dashed circle allows for comparing the size of the pore in both structures, which illustrates the larger pore of the structure 1O.

FIGURE 3

Models 1C and 1O; lateral view.

FIGURE 4

Matrix representation of the relative movement in the protein between structures 1C and 1O. The distances between pairs of atoms are computed for the 1C and 1O structures and the difference is plotted as a matrix using the numbers of the residues in the model. The positions of the helices and β-sheets are represented in the upper-left half of the matrix.

FIGURE 5

Conformational change between structures 1C and 1O at the tertiary structure level. Only one subunit is represented. The internal β-sheet and M2 helix are represented in blue, the external β-sheet is represented in light blue and the rest of the protein is displayed in red.

FIGURE 6

(A) Models 1C and 1O including side chains. Only the membrane domain is represented in a space-filling representation. Slab mode is used in order to see the pore. (B) Radius of the pore at different levels for the structure 1C (red) 1O (green), and the structure derived from electron microscopy studies (dotted line). The axis of the pore is represented as a dashed line.

FIGURE 7

(A) Coupling zone between the extracellular and the membrane domains. The residues homologous to those identified on the GABA-A receptor (Kash et al., 2004, 2003) are represented in gray. (B) Three M2 segments on which the residues exposed to the solvent (262, 258, 255, 251, 248, 244, and 241) as identified by SCAM (Wilson and Karlin, 2001) and affinity labeling (Corringer et al., 2000) experiments are represented in red.