An Activating Helix Switch at the Rhodopsin-Transducin Interface

Abstract

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R∗) and to catalyze GDP/GTP exchange in the α-subunit of intracellular G proteins (Gαβγ). Kinetic analysis of transducin (Gtαβγ) activation has shown that an intermediary R∗·Gtαβγ·GDP complex is formed which precedes GDP release and formation of the nucleotide-free R∗·G protein complex. Based on this reaction sequence we explore the dynamic interface between the proteins during formation of these complexes. We start from the R∗ conformation stabilized by a Gtα C-terminal peptide (GαCT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal α-helix of Gtα (α5) and shows how α5 can be docked to the open binding site of R∗. Two modes of interaction are found. One of them - termed stable or S-interaction - matches the position of the GαCT peptide in the crystal structure and reproduces the hydrogen bridge networks between the C-terminal reverse turn of GαCT and conserved E(D)RY and NPxxY(x)5,6F regions of the GPCR. The alternative fit - termed intermediary or I-interaction - is distinguished by a tilt and significant clockwise rotation of α5 relative to the S-interaction. It shows different α5 contacts with the NPxxY(x)5,6F region and the second cytoplasmic loop of R∗. From the two α5 interactions, we derive a ‘helix switch’ mechanism for the transition of R∗·Gtαβγ·GDP to the nucleotide-free R∗·G protein complex. It illustrates how α5 acts as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site. The results are discussed in light of the recent structure of the β2 adrenergic receptor-Gs protein complex. A detailed mechanism of complex formation and GDP release is derived.