STRUCTURAL STUDIES OF ACETYLCHOLINE BINDING SITE

乙酰胆碱受体（AChR）是一个大的膜结合体

The acetylcholine receptor (AChR) is a large membrane bound protein complex of 300 kDalton size, forming a sodium ion channel at the junction between nerve and muscle cells. Its structure is only known to 9 Angstroms resolution. Thus no details of the structure at atomic resolution are available. alpha-Bungarotoxin (BGTX), a potent neurotoxin from the venom of the banded krait Bungarus Multicinctus, is a very potent inhibitor of AChR, blocking the acetylcholine binding site. We have completed the refinement of the NMR solution structure of BGTX. The smallest peptide fragment of AChR that binds strongly to BGTX is a 12-residue fragment corresponding to residues 185-196 of the alpha-subunit of AChR. We have been able to observe and assign most of the 1H resonances in the complex of BGTX and the 12-residue fragment of AChR, and have calculated solution structures consistent with the NMR constraints, which we have published. Details of the interaction between this peptide and BGTX include a hydrophobic pocket is created between val-188, tyr-190 of the 12mer peptide, and valines 39 and 40 of BGTX. We have also been working on the study of the conformations of a five residue fragment of AChR containing the two sequential cysteines (192 and 193) which are disulfide-linked to each other in vivo. These cysteines have been demonstrated to be essential for channel opening, and are within 8 Angstroms of bo und acetylcholine. We have been able to identify three conformations of this peptide, two with the amide bond between cys-192 and cys-193 in the cis conformation, and one with this amide bond in the trans conformation. The barrier in going from the lowest energy cis conformation to the other cis conformation is approximately 15 kcal/mole, and the barrier in going from the cis forms to the trans form is approximately 20 kcal/mole. These barriers correlate well with the barriers in going from the resting state to the desensitized state of the AChR, so that these conformational processes of the eight-membered ring formed between the sequential disulfides 192 and 193 may be responsible for the desensitization of the receptor in vivo. We have completed theoretical calculations using AMBER, and we are in the process of writing up these results for publication. The Computer Graphics Labroatory is essential in analyzing the results of our modeling of the conformations of this important eight-membered ring containing peptide.