Electrostatic Steering at Acetylcholine Binding Sites

Abstract

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.

Acetylcholine Promotes Binding of α‐Conotoxin MII at α3β2 Nicotinic Acetylcholine Receptors

“…In contrast to the closed C-loop model, the open model consistently docks ACh in the aromatic pocket deep in the binding loop region, in agreement with the binding poses reported in literature. [9e, 28] This result further supports the conclusion that our open C-loop model has a more accessible binding pocket than the closed homology model. A common binding motif observed in the docking calculations was the cation-π pharmacophore interactions deep within the binding pocket between the quaternary ammonium group of ACh and the aromatic residues.…”

“…These findings, described below, are consistent with previously reported ACh binding motifs. [9e, 28] It is noted that although ACh binding at non-α subunit interfaces was observed in this study, this does not necessarily indicate that the binding results in channel gating. The role of ACh in the gating mechanism of nAChRs is beyond the scope of the docking studies presented in this paper and is currently being investigated in a separate study.…”

Lack of Dependence of the Sizes of the Mesoscopic Protein Clusters on Electrostatics

“…In light of the important role that Coulomb forces play for the clusters of classes I and II, here we probe their effects on the properties of the mesoscopic clusters as a test of the colloid clustering scenario. From a fundamental perspective, Coulomb forces determine protein three-dimensional structure (38,39), substrate binding (40,41), enzyme activation (42,43), signal transduction (44), etc. Importantly, Coulomb forces govern two major classes of protein aggregation: amyloid fibrillation (45)(46)(47) and crystallization (48,49).…”

Monte Carlo-based linear Poisson-Boltzmann approach makes accurate salt-dependent solvation free energy predictions possible

“…[1][2][3][4][5][6][7] Thus, it is important to have an accurate and efficient treatment of ionic strength effects on the ever in-creasing number of three dimensional structures that are made available by the various structural genomic and proteomic initiatives. These biomolecules can assume a broad range of absolute net charges from zero to a very large value O͑100͒e.…”