Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin.
Disruption of an interhelical salt bridge between the retinal protonated Schiff base linked to H7 and Glu113 on H3 is one of the decisive steps during activation of rhodopsin. Using previously established stabilization strategies, we engineered a stabilized E113Q counterion mutant that converted rhodopsin to a UV-absorbing photoreceptor with deprotonated Schiff base and allowed reconstitution into native-like lipid membranes. Fourier-transform infrared difference spectroscopy reveals a deprotonated Schiff base in the photoproducts of the mutant up to the active state Meta II, the absence of the classical pH-dependent Meta I/Meta II conformational equilibrium in favor of Meta II, and an anticipation of active state features under conditions that stabilize inactive photoproduct states in wildtype rhodopsin. Glu181 on extracellular loop 2, is found to be unable to maintain a counterion function to the Schiff base on the activation pathway of rhodopsin in the absence of the primary counterion, Glu113. The Schiff base becomes protonated in the transition to Meta III. This protonation is, however, not associated with a deactivation of the receptor, in contrast to wildtype rhodopsin. Glu181 is suggested to be the counterion in the Meta III state of the mutant and appears to be capable of stabilizing a protonated Schiff base in Meta III, but not of constraining the receptor in an inactive conformation.
Structural Studies Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Jörg
J
Standfuss
Ekaterina
E
Zaitseva
Mohana
M
Mahalingam
Reiner
R
Vogel
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics*; D005973:Glutamine / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D011498:Protein Precursors / Q000737:chemistry; D017433:Protein Structure, Secondary; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*; D012545:Schiff Bases; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship
D004220:Disulfides; D050505:Mutant Proteins; D011498:Protein Precursors; D011522:Protons; D012545:Schiff Bases; D005973:Glutamine; D018698:Glutamic Acid; D012243:Rhodopsin