Highly conserved tyrosine stabilizes the active state of rhodopsin

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-11, Vol.107 (46), p.19861-19866
• Main Authors: Goncalves, Joseph A., South, Kieron, Ahuja, Shivani, Zaitseva, Ekaterina, Opefi, Chikwado A., Eilers, Markus, Vogel, Reiner, Reeves, Philip J., Smith, Steven O., Henderson, Richard
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Amino Acid Substitution - genetics; Amino acids; Animals; Biochemistry; Biological Sciences; Cattle; Chemical equilibrium; Conserved Sequence - genetics; Crystal structure; Crystallography, X-Ray; Fluorescence; HEK293 Cells; Humans; Hydrolysis; Ion Channel Gating; Line spectra; Magnetic Resonance Spectroscopy; Membranes; Molecular biology; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation; Mutation - genetics; NMR; Nuclear magnetic resonance; Opsins; Pigments; Protein Conformation; Protein Stability; Protein Structure, Secondary; Proteins; Receptors; Rhodopsin - chemistry; Rhodopsin - metabolism; Signal Transduction; Spectroscopy; Structure-Activity Relationship; Tyrosine - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1009405107

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 Arg135 3.50 and Met257 6.40 forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu134 3.49 -Arg135 3.50 -Glu247 6.30 ionic lock. We also show that Tyr223 5.58 and Tyr306 7.53 form molecular contacts with Met257 6.40 . 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 Tyr223 5.58 and Ala132 3.47 in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr223 5.58 to phenylalanine or mutation of Ala132 3.47 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 Tyr306 7.53 to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.