Electrostatic steering at acetylcholine binding sites.
The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.
Meltzer
Robert H
RH
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Thompson
Errol
E
Soman
Kizhake V
KV
Song
Xing-Zhi
XZ
Ebalunode
Jerry O
JO
Wensel
Theodore G
TG
Briggs
James M
JM
Pedersen
Steen E
SE
D000109:Acetylcholine / Q000737:chemistry*; D000110:Acetylcholinesterase / Q000737:chemistry*; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry*; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer; D008564:Membrane Potentials; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000737:chemistry*; D055672:Static Electricity
D011978:Receptors, Nicotinic; D000110:Acetylcholinesterase; D000109:Acetylcholine