New Concepts Rhodopsin Activation Follows Precoupling with Transducin: Inferences from Computational Analysis †,‡ Francesca Fanelli* and Daniele Dell’Orco Department of Chemistry, UniVersity of Modena and Reggio Emilia, and Dulbecco Telethon Institute, Via Campi 183 41100 Modena, Italy ReceiVed August 2, 2005; ReVised Manuscript ReceiVed September 15, 2005 ABSTRACT: The electrostatic and shape complementarities between the crystal structures of dark rhodopsin and heterotrimeric transducin (Gt) have been evaluated by exhaustively sampling the roto-translational space of one protein with respect to the other. Structural complementarity, reliability, and consistency with in vitro evidence all converge in the same rhodopsin-Gt complex, showing that the functionally important R135 of the E/DRY motif is almost accessible to the C-terminus of Gt R already in the dark state. The main inference from this study is that activation of rhodopsin and Gt may be concurrent processes, consisting of conformational changes in a supramolecular complex formed prior to the light-induced activation of the photoreceptor. Dark Rhodopsin Has the Determinants for Recognizing Heterotrimeric Transducin: EVidence from in Vitro Experiments Of the different states that define the reaction path of rhodopsin activation, the dark or ground state is the only one known at the atomic detail (1-5). At least four intermediates intervene between dark rhodopsin and Meta II (MII), 1 the signaling state capable of activating heterot- rimeric G protein (i.e., transducin (Gt)) (6). The 5.5 Å electron density map of Meta I (MI), the photostationary state that precedes MII, has been recently released, providing evidence that rhodopsin remains in a conformation similar to that of the ground state until late in the photobleaching process and that the gross conformational changes occur during the MI to MII transition (7). It is widely thought that rhodopsin binds Gt in the MII state (6). The patchwork of the most relevant information from in vitro experiments on rhodopsin-transducin recogni- tion suggests that the R4/6 loop and the C-terminus of Gt R recognize a solvent-accessible cleft on MII, formed by amino acids from the cytosolic extensions of H3, H5, and H6 (the letter “H” stands for “helix”), from the N-terminus of the second intracellular loop (I2), and from the N-terminus of H8 (reviewed in refs 8-10). Recent experiments have demonstrated that the role of H6 movements during MII formation is to provide a binding site on the cytoplasmic face of rhodopsin for the Gt R C-terminus (11). This move- ment appears to open a cleft and expose a hydrophobic patch, † This study was supported by a Telethon-Italy Grant No. S00068TELA (To F.F.). ‡ The coordinates of the selected complexes involving Gt_mut1, Gt_mut2, Gt_mut3, Gt_chim1, and Gt_chim2 will be released upon request. * Corresponding author. Phone: +39 059 2055114. Fax: +39 059 373543. E-mail: fanelli@unimo.it. 1 Abbreviations: Gt, transducin; MII, Meta II; MI, Meta I; I2, second intracellular loop; PWR, plasmon-waveguide resonance; GPCRs, G protein-coupled receptors; PDB, Protein Data Bank; CR-RMSD, CR-atom root mean square deviation; I3, third intracellular loop. © Copyright 2005 by the American Chemical Society Volume 44, Number 45 NoVember 15, 2005 10.1021/bi051537y CCC: $30.25 © 2005 American Chemical Society Published on Web 10/22/2005