Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop.
Previous X-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy, and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that after binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. In this study, we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with acetylcholine (ACh) bound. Substitution of [15N]cysteine for the native cysteines 123, 136, 187, and 188 provided intrinsic monitors of the chemical environments of the Cys- and C-loops, respectively. Two-dimensional transverse relaxation-optimized spectroscopy 15N-1H HSQC spectroscopy of apo-AChBP revealed seven well resolved cross-peaks for the group of cysteines. The spectrum of AChBP with Ser substituted for Cys 187 and 188 shows only two main cross-peaks, corresponding to Cys 123 and 136 from the Cys-loop, enabling resonance assignments. After binding of ACh, the five cross-peaks associated with cysteines from the C-loop condense into two predominant cross-peaks not observed in the spectrum from the apo protein, indicating a restricted range of conformations and change in chemical environment of the C-loop. The results show that isotopic cysteine can be incorporated into specified positions of AChBP expressed from a eukaryotic source, that the C-loop assumes multiple conformations without ACh, but that its conformation becomes restricted with ACh bound. The collective findings suggest a structural mechanism for agonist recognition in AChBP and related Cys-loop receptors.
Gao
Fan
F
Receptor Biology Laboratory, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester MN 55905, USA.
Mer
Georges
G
Tonelli
Marco
M
Hansen
Scott B
SB
Burghardt
Thomas P
TP
Taylor
Palmer
P
Sine
Steven M
SM
D000109:Acetylcholine / Q000378:metabolism*; D019943:Amino Acid Substitution; D002460:Cell Line; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011950:Receptors, Cholinergic / Q000737:chemistry; D012997:Solvents
D011950:Receptors, Cholinergic; D012997:Solvents; D000109:Acetylcholine