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J. Biol. Chem., Vol. 282, Issue 19, 14272-14282, May 11, 2007

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Critical Role of Electrostatic Interactions of Amino Acids at the Cytoplasmic Region of Helices 3 and 6 in Rhodopsin Conformational Properties and Activation^*

Eva Ramon¹, Arnau Cordomí², Laia Bosch², Eugeni Yu. Zernii^¶, Ivan I. Senin^¶, Joan Manyosa^||, Pavel P. Philippov^¶, Juan J. Pérez, and Pere Garriga³

From the Centre de Biotecnologia Molecular, Departament d'Enginyeria Química, Universitat Politècnica de Catalunya, 08222 Terrassa, Catalonia, Spain, Centre de Biotecnologia Molecular, Departament d'Enginyeria Química, Universitat Politècnica de Catalunya, 08028 Barcelona, Catalonia, Spain, the ^¶Department of Cell Signalling, A. N. Belozersky Institute of PhysicoChemical Biology, M.V. Lomonosov Moscow State University, 119992 Moscow, Russia, and ^||Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain

The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr²⁵¹ in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.

Received for publication, December 4, 2006 , and in revised form, February 5, 2007.

^* This work was supported in part by Spanish Ministry of Education Grants SAF2005-08148-C04-01 (to J. J. P.) and SAF2005-08148-C04-02 (to P. G.) and in part by NATO Scientific Programme, INTAS Grant 03-51-4548 (to I. I. S., J. M., and P. G.), FEBS fellowship program (to E. Yu. Z.), and Russian Foundation for Basic Research Grants 06-04-48018, 04-04-04001 (to P. P. P.), and 06-04-48761 (I. I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Center for Membrane Biology, Dept. of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, TX 77030.

² Recipient of doctoral fellowship from the Universitat Politècnica de Catalunya.

³ To whom correspondence should be addressed. Tel.: 34-937398998; Fax: 34-937398225; E-mail: pere.garriga{at}upc.edu.

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