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

Abstract

Chemical signaling throughout the organism is initiated by molecular recognition of small biomolecules by receptor binding sites. Recognition is accompanied by conformational changes of the binding site, through induced fit, selection of pre-existing conformers, or a combination of the two, and these are amplified over distance and time to produce the biological response. In the case of rapidly gated synaptic receptors, the amplification propagates within the receptor macromolecule itself and occurs within as little as tens of microseconds (1). Structural counterparts of the initial recognition step likely evolved to provide this close spatial and temporal coupling. To understand the initial recognition step in rapidly gated synaptic receptors, we examined conformational changes due to agonist occupancy in AChBP, 1 a prototype of the nicotinic receptor ligand binding domain (2).The nicotinic receptor belongs to the Cys loop superfamily of rapidly gated synaptic receptors that transduce agonist binding into opening of an intrinsic ion pore. Although a complete atomic structure has not been determined for any member of the superfamily, atomic resolution data recently emerged for AChBP (3, 4), a close homolog of the ligand binding domain of the ␣7 neuronal nicotinic receptor. AChBP has proven to be a valuable model system for clarifying aspects of ligand recognition in nicotinic receptors, including structural modeling (5-7), dynamics of receptor ligand binding domains (8), computational docking of ligands (9, 10), and direct monitoring of ligand occupancy by changes in intrinsic Trp fluorescence (11, 12).Here we delineated ACh-dependent conformational changes in AChBP by prolonged MD simulation and compared these with conformational changes detected by steady-state and time-resolved measurements of intrinsic Trp fluorescence. MD simulation revealed asymmetric relaxation of the AChBP homopentamer without ACh bound, but maintenance of the symmetrical structure with ACh bound. Differences between the ACh-free and ACh-bound structures revealed conformational changes linked to agonist occupancy. Conformational changes predicted by simulation were verified by measurements of intrinsic Trp fluorescence, showing that binding of ACh establishes close register of two Trp side chains from opposing subunits and draws the peripheral C-loop inward to occlude the entrance of the binding cavity. EXPERIMENTAL PROCEDURESMolecular 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. Atomic coordinates of AChBP (3) were downloaded from the Protein Data Bank (Protein Data Bank code 1I9B). 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 then assigned to each atom of the protein using the restrained electrostatic poten...

A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor

“…The C loop (residues 180-197 in each subunit) encloses the pocket in which the ligand would bind. This finding is consistent with previous studies (50,51) that indicate that the C loop is mobile when no ligand is present and that contact of the C loop with a bound ligand, as in the liganded AChBP structure (23), helps stabilize the binding pocket and the C loop in a more closed state. Therefore an open͞mobile C loop, in at least two subunits, seems to correlate with a closed state of the TM channel.…”

“…The lack of motion in the Cys loop is somewhat contrary to previous observations seen in LBD-only simulations (32), and this difference likely represents the influence of the associated TMD. Movements of the C loop, similar to those seen here, have been observed (50,51), and the opposite behavior, with the C loop being held over the binding site by a ligand, also has been noted (50).…”

“…The fact that in this simulation, and previous simulations of the LBD (32,50), only two nonadjacent subunits seem to undergo substantial displacements is of both functional and perhaps evolutionary importance. It seems characteristic of these pentameric receptors that only two subunits are necessary to convey the activation͞gating action of the channel, because the maximum Hill coefficient observed is two (83).…”

Spontaneous conformational change and toxin binding in α7 acetylcholine receptor: Insight into channel activation and inhibition

“…McCammon and coworkers (17) also carried out targeted MD simulations, in which the C loop is pulled from the ''up'' to the ''down'' position (side view in Fig. S1B); the latter position is observed when an agonist is bound (20). This downward motion of the C loop triggers the motion of the tip of the F loop, the C terminus of ␤10, and the ␤1-␤2 loop in the opposite direction.…”

Enhanced meta‐analysis of acetylcholine binding protein structures reveals conformational signatures of agonism in nicotinic receptors

“…17 The method we discuss here extends this analysis by (a) using multiple samples to calibrate state definitions and (b) demonstrating the ability to predict the states of a ligandbound AChBP structures.…”