The accessible surface of the nicotinic acetylcholine receptor. Identification by chemical modification and cross-linking with 14C-dimethyl suberimidate.
To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.
Watty
A
A
Institut für Biochemie, Fachbereich Chemie, Freie Universität Berlin, Germany.
Weise
C
C
Dreger
M
M
Franke
P
P
Hucho
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002250:Carbon Radioisotopes / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004120:Dimethyl Suberimidate / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000378:metabolism; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry*; D017421:Sequence Analysis; D014101:Torpedo / Q000502:physiology
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D009943:Organophosphorus Compounds; D011978:Receptors, Nicotinic; D004120:Dimethyl Suberimidate; D008239:Lysine