Structural and functional role of helices I and II in rhodopsin. A novel interplay evidenced by mutations at Gly-51 and Gly-89 in the transmembrane domain.
The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.
Bosch
Laia
L
Centre de Biotecnologia Molecular, Departament d'Enginyeria Química, Universitat Politècnica de Catalunya, Colom 1, 08222 Terrassa, Catalonia, Spain.
Ramon
Eva
E
Del Valle
Luis J
LJ
Garriga
Pere
P
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D012243:Rhodopsin; D005998:Glycine