Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation.
In the G protein-coupled receptor rhodopsin, the conserved NPxxY(x)(5,6)F motif connects the transmembrane helix VII and the cytoplasmic helix 8. The less geometrically constrained retinal analogue 9-demethyl-retinal prevents efficient transformation of rhodopsin to signaling metarhodopsin (Meta) II after retinal photoisomerization. Here, we demonstrate that Ala replacement mutations within the NPxxY(x)(5,6)F domain, which eliminate an interaction between aromatic residues Y306 and F313, allow formation of Meta II despite the presence of 9-demethyl-retinal. Also a disulfide bond linking residues 306 and 313 in the 9-demethyl-retinal-reconstituted mutant Y306C/F313C/C316S prevented Meta II formation, whereas the reduced form of the mutant readily transformed to Meta II after illumination. These observations suggest that the interaction between residues 306 and 313 is disrupted during the Meta I/Meta II transition. However, this enhancement in Meta II formation is not reflected in the G protein activation, which is dramatically reduced for these mutants, suggesting that changes in the Y306-F313 interaction also lead to a proper realigning of helix 8 after photoisomerization. The E134Q mutation, located in the second conserved motif, D(E)RY, rescues activity in 9-demethyl-retinal-reconstituted mutants to different degrees, depending on the position of the Ala replacement in the NPxxY(x)(5,6)F motif, thus revealing distinct roles for the NP and Y(x)(5,6)F portions. Our studies underscore the importance of the NPxxY(x)(5,6)F and D(E)RY motifs in providing structural constraints in rhodopsin that rearrange in response to photoisomerization during formation of the G protein-activating Meta II. The dual control of the structural rearrangements secures reliable transformation of quiescent rhodopsin to activating Meta II.
Fritze
Olaf
O
Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität, Schumann Strasse 20-21, D-10098 Berlin, Germany.
Filipek
Sławomir
S
Kuksa
Vladimir
V
Palczewski
Krzysztof
K
Hofmann
Klaus Peter
KP
Ernst
Oliver P
OP
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D006801:Humans; D006860:Hydrogen Bonding; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays
C110176:9-demethylretinal; D004220:Disulfides; D012243:Rhodopsin; D012172:Retinaldehyde