Rhodopsin-Transducin Interface: Studies with Conformationally Constrained Peptides

Abstract

To probe the interaction between transducin (G(t)) and photoactivated rhodopsin (R*), 14 analog peptides were designed and synthesized restricting the backbone of the R*-bound structure of the C-terminal 11 residues of G(t)alpha derived by transferred nuclear Overhauser effect (TrNOE) NMR. Most of the analogs were able to bind R*, supporting the TrNOE structure. Improved affinities of constrained peptides indicated that preorganization of the bound conformation is beneficial. Cys347 was found to be a recognition site; particularly, the free sulfhydryl of the side chain seems to be critical for R* binding. Leu349 was another invariable residue. Both Ile and tert-leucine (Tle) mutations for Leu349 significantly reduced the activity, indicating that the Leu side chain is in intimate contact with R*. The structure of R* was computer generated by moving helix 6 from its position in the crystal structure of ground-state rhodopsin (R) based on various experimental data. Seven feasible complexes were found when docking the TrNOE structure with R* and none with R. The analog peptides were modeled into the complexes, and their binding affinities were calculated. The predicted affinities were compared with the measured affinities to evaluate the modeled structures. Three models of the R*/G(t)alpha complex showed strong correlation to the experimental data.

Molecular Modeling of Meta II Rhodopsin

“…Subsequently, in agreement with the available literature data compiled and summarized in [24] we have attributed to and imposed on RD nine key distances defining expected rearrangement of all transmembrane helices, as referred to in the literature [23,24]. These distances (effective by constraints) are presented in Table 1.…”

“…Most of these distances were taken directly from the experimental studies reported in [24]. For details see Table 1.…”

The Hydrodynamic Properties of Dark- and Light-activated States of n-Dodecyl β-D-Maltoside-solubilized Bovine Rhodopsin Support the Dimeric Structure of Both Conformations

“…Additionally, oligomeric forms of T ␣ were the predominant species when highly specific photoactivated crosslinking reagents were employed (85). Compatible with these findings is the cooperativity reported for the interaction of Rho with T (72,73,86). Mixon et al (87) have described the threedimensional structure of G i ␣ 1 , an ␣ subunit isotype of G i .…”

“…The packing of Rho molecules as dimers provides a platform that can easily accommodate both T functional units (71,72) and is consistent with the kinetic studies of the Rhocatalyzed guanine nucleotide exchange (70), as well as with binding studies between Rho and T (73, 74), which demonstrated allosteric regulation of the interaction of T with Rho*. In addition, it has been speculated that one molecule of Rho in the dimer is needed for productive coupling with T, whereas the second one provides a partial scaffold to dock subunits of T (75).…”

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

“…More recently, crosslinking experiments confirmed the existence of rhodopsin dimers [47]. A dimeric or higher oligomeric functional form of rhodopsin is supported by the structural information available on G proteins and rhodopsin, and biophysical data that identify different interacting regions on the interface between these proteins [48]. In particular, the size of the G protein surface interacting with rhodopsin has been shown to be almost twice as large as the exposed cytoplasmic surface of a single rhodopsin molecule [49,50].…”