Conserved Tyr2235.58 plays different roles in the activation and G-protein interaction of rhodopsin

Abstract

Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cisretinal via a protonated Schiff base. Coupling to the G-protein transducin (Gt) occurs after lightinduced cis/trans-retinal isomerization, which leads through photoproducts into a sequence of metarhodopsin (Meta) states: Meta I Meta IIa Meta IIb Meta IIbH+. The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting of conserved amino acids. Here we focus on Tyr2235.58 as part of the Y5.58X7K(R)5.66 motif. Mutation to Ala, Phe, or Glu results in specific impairments of Gt-activation measured by intrinsic Gt fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal Gtα peptide allows the assignment of these deficiencies to specific steps in the activation path. Effects of mutation occur already in Meta I but do not directly influence deprotonation of the Schiff base during formation of Meta IIa. Absence of the whole phenol ring (Y223A) allows the activating motion of TM6 in Meta IIb but impairs the coupling to Gt. When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward Gt remains substantial. From the FTIR features of Meta IIbH+ we conclude that proton uptake to Glu1343.49 is mandatory for Tyr223 5.58 to engage in the interaction with the key player Arg135 3.50 predicted by X-ray analysis. This polar interaction is partially recovered in Y223E, explaining its relatively high activity. Only the phenol side chain of tyrosine provides all characteristics for accumulation of the active state and G-protein activation. © 2011 American Chemical Society.