Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist.
The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.
McKee
Timothy D
TD
Department of Biochemistry and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454, USA.
Lewis
Margaret R
MR
Kono
Masahiro
M
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D012243:Rhodopsin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D012243:Rhodopsin