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Conformational dynamics of the acetylcholine binding protein, a Nicotinic receptor surrogate

Nicotinic acetylcholine receptors (nAChRs) are ligand- gated ion channels that mediate rapid neurotransmission in the central and peripheral nervous systems. The acetylcholine binding protein (AChBP) is a soluble structural and functional surrogate of the extracellular, ligand-binding domain of the nAChR that allows for studies not amenable to study of the nAChR as an integral membrane protein. In particular, AChBP provides a system in which to study solution dynamics and conformational changes related to ligand binding. To deduce ligand binding sites and infer conformational changes, cysteine mutants of AChBP were generated and labeled with the solvent- sensitive fluorophore, acrylodan. The fluorescence emission spectra from acrylodan-labeled AChBP mutants were examined in the absence and presence of nicotinic ligands. Binding of small molecules and large peptide toxins caused acrylodan conjugated to Q178C to move into environments of opposite polarities. From this, I proposed a hinge position at Q178 that allows for flexibility of the C-loop, such that it can expand or contract as a rigid body to accommodate bound ligand. Distinctive changes in acrylodan emission were also observed in the F-loop, indicating that this region of the protein likely plays a role in ligand binding, a finding not evident from existing structures. Complementary hydrodynamic and fluorescence anisotropy studies of AChBP free in solution and in complex with an 8 kD three-fingered alpha-neurotoxin indicated that the bound alpha-toxin has minimal effects on the translational and rotational diffusion properties of AChBP. Anisotropy experiments showed that, when bound, the alpha-toxin is highly dynamic. Its central finger, that contains beta- sheet structure and interacts with the agonist binding site, is the most rigid portion. The segmental flexibility of AChBP was studied by measuring decay of fluorescence anisotropy from fluorescein-labeled AChBP mutants. The results revealed that AChBP exhibits wide regional variation in alpha-carbon backbone flexibility, with the C -loop being overall most rigid in the picosecond- nanosecond time domain. In studying the effect of ligand binding on conformational flexibility, we found that the F -loop conformational change associated with ligand binding is pharmacologically correlated. Solution-based structural studies of conformation related to ligand binding should facilitate structure-guided drug design and increase understanding of nAChR function