Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.
To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the photoactivatable, hydrophobic probe 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine([125I]TID). The pattern of [125I]TID incorporation into the M3 and M4 hydrophobic segments of each subunit was the same both in the presence and absence of the agonist carbamoylcholine and in the presence of an excess of nonradioactive TID, consistent with nonspecific photoincorporation from the lipid-protein interface. [125I]TID reacted with five residues in alpha-M4 [Blanton, M.P., & Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three residues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [125I]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-305 as well as with Ile-288 preceding M3. Residues at corresponding positions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma-M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well as M4 segments of each subunit are organized as transmembrane alpha-helices each with substantial contact with lipid. In addition, in alpha-M1 [125I]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, and Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a "face" of an alpha-helix or a beta-sheet.
Blanton
M P
MP
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115.
Cohen
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001389:Azirines / Q000378:metabolism; D001665:Binding Sites; D002217:Carbachol / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004557:Electric Organ / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D050356:Lipid Metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo
D001389:Azirines; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D002217:Carbachol