Evidence for Structural Changes in Carboxyl-Terminal Peptides of Transducin α-Subunit upon Binding a Soluble Mimic of Light-Activated Rhodopsin

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Abstract
Although a high-resolution crystal structure for the ground state of rhodopsin is now available, portions of the cytoplasmic surface are not well resolved, and the structural basis for the interaction of the cytoplasmic loops with the retinal G-protein transducin (Gt) is still unknown. Previous efforts aimed at the design, construction, and functional characterization of soluble mimics for the light-activated state of rhodopsin have shown that grafting defined segments from the cytoplasmic region of bovine opsin onto a surface loop in a mutant form of thioredoxin (HPTRX) is sufficient to confer partial Gt activating potential [Abdulaev et al. (2000) J. Biol. Chem.275, 39354−39363]. To assess whether these designed mimics could provide a structural insight into the interaction between light-activated rhodopsin and Gt, the ability of an HPTRX fusion protein comprised of the second (CD) and third (EF) cytoplasmic loops (HPTRX/CDEF) to bind Gt α-subunit (Gtα) peptides was examined using nuclear magnetic resonance (NMR) spectroscopy. Transfer NOESY (TrNOESY) experiments show that an 11 amino acid peptide corresponding to the carboxyl terminus of Gtα (GtP), as well as a “high-affinity” peptide analogue, HAP1, binds to HPTRX/CDEF in the fast-exchange regime and undergoes similar, subtle structural changes at the extreme carboxyl terminus. Observed TrNOEs suggest that both peptides when bound to HPTRX/CDEF adopt a reverse turn that is consistent with the C-cap structure that has been previously reported for the interaction of GtP with the light-activated signaling state, metarhodopsin II (MII). In contrast, TrNOESY spectra provide no evidence for structuring of the amino terminus of either GtP or HAP1 when bound to HPTRX/CDEF, nor do the spectra show any measurable changes in the CD and EF loop resonances of HPTRX/CDEF, which are conformationally dynamic and significantly exchange broadened. Taken together, the NMR observations indicate that HPTRX/CDEF, previously identified as a functional mimic of MII, is also an approximate structural mimic for this light-activated state of rhodopsin.