Identification of core amino acids stabilizing rhodopsin.
Rhodopsin is the only G protein-coupled receptor (GPCR) whose 3D structure is known; therefore, it serves as a prototype for studies of the GPCR family of proteins. Rhodopsin dysfunction has been linked to misfolding, caused by chemical modifications that affect the naturally occurring disulfide bond between C110 and C187. Here, we identify the structural elements that stabilize rhodopsin by computational analysis of the rhodopsin structure and comparison with data from previous in vitro mutational studies. We simulate the thermal unfolding of rhodopsin by breaking the native-state hydrogen bonds sequentially in the order of their relative strength, using the recently developed Floppy Inclusion and Rigid Substructure Topography (FIRST) method [Jacobs, D. J., Rader, A. J., Kuhn, L. A. & Thorpe, M. F. (2001) Proteins 44, 150-165]. Residues most stable under thermal denaturation are part of a core, which is assumed to be important for the formation and stability of folded rhodopsin. This core includes the C110-C187 disulfide bond at the center of residues forming the interface between the transmembrane and the extracellular domains near the retinal binding pocket. Fast mode analysis of rhodopsin using the Gaussian network model also identifies the disulfide bond and the retinal ligand binding pocket to be the most rigid region in rhodopsin. Experiments confirm that 90% of the amino acids predicted by the FIRST method to be part of the core cause misfolding upon mutation. The observed high degree of conservation (78.9%) of this disulfide bond across all GPCR classes suggests that it is critical for the stability and function of GPCRs.
Rader
A J
AJ
Center for Computational Biology and Bioinformatics, Department of Molecular Biology and Biochemistry, School of Medicine, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15261, USA.
Anderson
Gülsüm
G
Isin
Basak
B
Khorana
H Gobind
HG
Bahar
Ivet
I
Klein-Seetharaman
Judith
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry*; D012984:Software
D000596:Amino Acids; D004220:Disulfides; D012243:Rhodopsin