Conformational Analysis of the Eight-Membered Ring of the Oxidized Cysteinyl-Cysteine Unit Implicated in Nicotinic Acetylcholine Receptor Ligand Recognition

• Published in: Journal of the American Chemical Society 2001-12, Vol.123 (50), p.12664-12669
• Main Authors: Creighton, Christopher J, Reynolds, Charles H, Lee, Daniel H. S, Leo, Gregory C, Reitz, Allen B
• Format: Article
• Language: English
• Subjects: Computer Simulation; Cysteine - chemistry; Ligands; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Peptide Fragments - chemistry; Peptides, Cyclic - chemistry; Protein Conformation; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - metabolism; Thermodynamics
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja016505m

Nicotinic acetylcholine receptors (nAChRs) are membrane-bound, pentameric ligand-gated ion channels associated with a variety of human disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia, and pain. Most known nAChRs contain an unusual eight-membered disulfide-containing cysteinyl-cysteine ring, ox-[Cys-Cys], as does the soluble acetylcholine binding protein (AChBP) found in the snail Lymnaea stagnalis. The cysteinyl-cysteine ring is located in a region implicated in ligand binding, and conformational changes involving this ring may be important for modulation of nAChR function. We have studied the preferred conformations of Ac-ox-[Cys-Cys]-NH2 by NMR in water and computationally by Monte Carlo simulations using the OPLS-AA force field and GB/SA water model. ox-[Cys-Cys] adopts four distinct low-energy conformers at slightly above 0 °C in water. Two populations are dependent on the peptide ω2 dihedral angle, with the trans amide favored over the cis amide by a ratio of ca. 60:40. Two ox-[Cys-Cys] conformers with a cis amide bond (C+ and C−) differ from each other primarily by variation of the χ3 dihedral angle, which defines the orientation of the helicity about the S−S bond (± 90°). Two trans amide conformers have the same S−S helicity (χ3 ≈ −90°), but are distinguished by a backbone rotation about φ2 and ψ1 (T− and T‘−). The ratio of T−/T‘−/C+/C− is 47:15:29:9. The orientation of the pendant moieties from the eight-membered ring is more compact for the major trans conformer (T−) than for the extended conformations adopted by T‘−, C+, and C−. These conformational preferences are also observed in tetrapeptide and undecapeptide fragments of the human α7 subtype of the nAChR that contains the ox-[Cys-Cys] unit. Conformer T− is nearly identical to the conformation seen in the X-ray structure of ox-[Cys187-Cys188] found in the unliganded AChBP, and is a Type VIII β-turn.