Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge.
The intramolecular interactions that stabilize the inactive conformation of rhodopsin are of primary importance in elucidating the mechanism of activation of this and other G protein-coupled receptors. In the present study, site-directed spin labeling is used to explore the role of a buried salt bridge between the protonated Schiff base at K296 in TM7 and its counterion at E113 in TM3. Spin-label sensors are placed at positions in the cytoplasmic surface of rhodopsin to monitor changes in the structure of the helix bundle caused by point mutations that disrupt the salt bridge. The single point mutations E113Q, G90D, and A292E, which were previously reported to cause constitutive activation of the apoprotein opsin, are found to cause profound movements of both TM3 and TM6 in the dark state, the latter of which is similar to that caused by light activation. The mutant M257Y, which constitutively activates opsin but does not disrupt the salt bridge, is shown to cause related but distinguishable structural changes. The double mutants E113Q/M257Y and G90D/M257Y produce strong activation of the receptor in the dark state. In the E113Q/M257Y mutant investigated with spin labeling, the movement of TM6 and other changes are exaggerated relative to either E113Q or M257Y alone. Collectively, the results provide structural evidence that the salt bridge is a key constraint maintaining the resting state of the receptor, and that the disruption of the salt bridge is the cause, rather than a consequence, of the TM6 motion that occurs upon activation.
Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.
Jong-Myoung
JM
Kim
Christian
C
Altenbach
Masahiro
M
Kono
Daniel D
DD
Oprian
Wayne L
WL
Hubbell
H Gobind
HG
Khorana
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013113:Spin Labels; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism
D013113:Spin Labels; D012243:Rhodopsin; D015293:Transducin