Agonist-mediated Conformational Changes in Acetylcholine-binding Protein Revealed by Simulation and Intrinsic Tryptophan Fluorescence

Fan Gao,Nina Bren,Thomas P Burghardt,Scott Hansen,Richard H Henchman,Palmer Taylor,J Andrew Mccammon,Steven M Sine

doi:10.1074/jbc.m412389200

Abstract

We delineated acetylcholine (ACh)-dependent conformational changes in a prototype of the nicotinic receptor ligand binding domain by molecular dynamics simulation and changes in intrinsic tryptophan (Trp) fluorescence. Prolonged molecular dynamics simulation of ACh-binding protein showed that binding of ACh establishes close register of Trps from adjacent subunits, Trp(143) and Trp(53), and draws the peripheral C-loop inward to occlude the entrance to the binding cavity. Close register of Trp(143) and Trp(53) was demonstrated by ACh-mediated quenching of intrinsic Trp fluorescence, elimination of quenching by mutation of one or both Trps to Phe, and decreased lifetime of Trp fluorescence by bound ACh. Occlusion of the binding cavity by the C-loop was demonstrated by restricted access of an extrinsic quencher of binding site Trp fluorescence by ACh. The collective findings showed that ACh initially establishes close register of conserved Trps from adjacent subunits and then draws the C-loop inward to occlude the entrance to the binding cavity.

Highlights

We delineated acetylcholine (ACh)-dependent conformational changes in a prototype of the nicotinic receptor ligand binding domain by molecular dynamics simulation and changes in intrinsic tryptophan (Trp) fluorescence
Prolonged molecular dynamics simulation of ACh-binding protein showed that binding of ACh establishes close register of Trps from adjacent subunits, Trp143 and Trp53, and draws the peripheral C-loop inward to occlude the entrance to the binding cavity
To delineate atomic-scale conformational changes associated with molecular recognition of ACh, we conducted molecular dynamics (MD) simulations of AChBP in the presence of explicit water molecules and counter ions, either with or without ACh bound to the five binding sites

Summary

EXPERIMENTAL PROCEDURES

Molecular Docking and Dynamics Simulation—We used the AMBER 7 program [13] to conduct molecular dynamics (MD) simulation of AChBP in the presence of explicit solvent molecules at room temperature, without ACh bound or with ACh bound to one, two, or all five binding sites. To prepare the protein structure for docking and MD simulation, water, ions, and HEPES were first removed from the crystal structure. Partial atomic charges were assigned to each atom of the protein using the restrained electrostatic potential charge model of the AMBER7 program. Partial atomic charges of ACh were obtained from electrostatic potential fitted charges from the HF/6 –31* quantum mechanics calculation. Time-resolved fluorescence intensity decay was measured on a timedomain analog lifetime instrument with flash lamp excitation (PTI). Tryptophans were excited at 298 Ϯ 2 nm, and emission was collected

RESULTS

WT ϩ ACh

DISCUSSION