Highly conserved tyrosine stabilizes the active state of rhodopsin

Abstract Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg1353.50 and Met2576.40 forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu1343.49-Arg1353.50-Glu2476.30 ionic lock. We also show that Tyr2235.58 and Tyr3067.53 form molecular contacts with Met2576.40. Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr2235.58 and Ala1323.47 in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr2235.58 to phenylalanine or mutation of Ala1323.47 to leucine decreases the lifetime of the Meta II intermediate. Furthermore, the Y223F mutation is coupled to structural changes in EL2. In contrast, mutation of Tyr3067.53 to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.