Critical role of electrostatic interactions of amino acids at the cytoplasmic region of helices 3 and 6 in rhodopsin conformational properties and activation.
The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.
Ramon
Eva
E
Centre de Biotecnologia Molecular, Departament d'Enginyeria Química, Universitat Politècnica de Catalunya, 08222 Terrassa, Catalonia, Spain.
Cordomí
Arnau
A
Bosch
Laia
L
Zernii
Eugeni Yu
EY
Senin
Ivan I
II
Manyosa
Joan
J
Philippov
Pavel P
PP
Pérez
Juan J
JJ
Garriga
Pere
P
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003198:Computer Simulation; D003593:Cytoplasm / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D013329:Structure-Activity Relationship
D012243:Rhodopsin