Ligand-induced conformational changes in the acetylcholine-binding protein analyzed by hydrogen-deuterium exchange mass spectrometry.
Recent x-ray crystallographic studies of the acetylcholine-binding protein (AChBP) suggest that loop C, found at the circumference of the pentameric molecule, shows distinctive conformational changes upon antagonist and agonist occupation. We have employed hydrogen-deuterium exchange mass spectrometry to examine the influence of bound ligands on solvent exposure of AChBP. Quantitative measurements of deuterium incorporation are possible for approximately 56% of the Lymnaea AChBP sequence, covering primarily the outer surface of AChBP. In the apoprotein, two regions flanking the ligand occupation site at the subunit interface, loop C (residues 175-193) and loop F (residues 164-171), show greater extents of solvent exchange than other regions of the protein including the N- and C-terminal regions. Occupation by nicotinic agonists, epibatidine and lobeline, and nicotinic antagonists, methyllycaconitine, alpha-bungarotoxin, and alpha-cobratoxin, markedly restricts the exchange of loop C amide protons, influencing both the rates and degrees of exchange. Solvent exposure of loop C and its protection by ligand suggest that in the apoprotein, loop C exhibits rapid fluctuations in an open conformation. Bound agonists restrict solvent exposure through loop closure, whereas the larger antagonists restrict solvent exposure largely through occlusion of solvent. Loop F, found on the complementary subunit surface at the interface, also reveals ligand selective changes in amide proton exchange rates. Agonists do not affect solvent accessibility of loop F, whereas certain antagonists cause subtle accessibility changes. These results reveal dynamic states and fluctuating movements in the vicinity of the binding site for unligated AChBP that can be influenced selectively by ligands.
Department of Pharmacology and Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.
Jianxin
J
Shi
Julia R
JR
Koeppe
Elizabeth A
EA
Komives
Palmer
P
Taylor
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D008024:Ligands; D008195:Lymnaea / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012997:Solvents
C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins; D008024:Ligands; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D012997:Solvents; D006859:Hydrogen; D003903:Deuterium