title: Conserved Tyr223(5.58) plays different roles in the activation and G-protein interaction of rhodopsin.
abstract: Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via a protonated Schiff base. Coupling to the G-protein transducin (G(t)) occurs after light-induced 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 Tyr223(5.58) as part of the Y(5.58)X(7)K(R)(5.66) motif. Mutation to Ala, Phe, or Glu results in specific impairments of G(t)-activation measured by intrinsic G(t) fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal G(t)α 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 G(t). When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward G(t) remains substantial. From the FTIR features of Meta IIbH(+) we conclude that proton uptake to Glu134(3.49) 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.
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Berlin, Germany. matthias.elgeti@charite.de
authors.forename: Matthias
authors.initials: M
authors.lastname: Elgeti
authors.forename: Roman
authors.initials: R
authors.lastname: Kazmin
authors.forename: Martin
authors.initials: M
authors.lastname: Heck
authors.forename: Takefumi
authors.initials: T
authors.lastname: Morizumi
authors.forename: Eglof
authors.initials: E
authors.lastname: Ritter
authors.forename: Patrick
authors.initials: P
authors.lastname: Scheerer
authors.forename: Oliver P
authors.initials: OP
authors.lastname: Ernst
authors.forename: Friedrich
authors.initials: F
authors.lastname: Siebert
authors.forename: Klaus Peter
authors.initials: KP
authors.lastname: Hofmann
authors.forename: Franz J
authors.initials: FJ
authors.lastname: Bartl
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D012172:Retinaldehyde / Q000737:chemistry; D012243:Rhodopsin / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D015293:Transducin / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D014443:Tyrosine; D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde