Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

Abstract

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the ␣-subunit of intracellular G proteins (G␣␤␥). Kinetic analysis of transducin (Gt␣␤␥) activation shows that an intermediary R*⅐Gt␣␤␥⅐GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*⅐G 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 G t␣ C-terminal peptide (G␣CT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal ␣-helix of Gt␣ (␣5) and shows how ␣5 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 G␣CT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of G␣CT 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 (42°) and rotation (90°) of ␣5 relative to the S-interaction and shows different ␣5 contacts with the NPxxY(x)5,6F region and the second cytoplasmic loop of R*. From the 2 ␣5 interactions, we derive a ''helix switch'' mechanism for the transition of R*⅐Gt␣␤␥⅐GDP to the nucleotide-free R*⅐G protein complex that illustrates how ␣5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.G protein coupled receptors (GPCRs) use the free energy of agonist binding to transmit physical or chemical signals into the cell. Bound agonists stabilize the 7 transmembrane (7TM) helix bundle of the receptor in an active conformation (R*). R* in turn interacts with intracellular heterotrimeric G proteins (G␣␤␥, G) to catalyze the exchange of GDP for GTP in the G␣ subunit and thus activate downstream effectors. According to classical receptor theory, R* is also stabilized by G protein binding (1). We used a synthetic peptide derived from the C terminus of the ␣-subunit of transducin (G␣CT), the key binding site for the receptor (2, 3), to stabilize and crystallize the R* conformation of opsin (4, 5). Opsin is the ligand-free form of the photoreceptor rhodopsin, and transducin (G t ␣␤␥, G t ) is its cognate G protein. X-ray structure analysis of the R*⅐G␣CT complex revealed that the cytoplasmic side of the TM5/TM6 helix pair (corresponding to the cytoplasmic loop C3 of the 7TM bundle) forms a mitt-like structure in which G␣CT is held. The contacts with R* induce in G␣CT an ␣-helical conformation with a C-terminal reverse turn (C-cap) (4, 6, 7), which is recognized by the receptor on the basis of its geometry (4).In GPCR mediated signal transduction, the signal is eventually established in the GTP-bound form of the G protein, which is the form that activates downstream effectors. The key step in whi...

Recognition in the Face of Diversity: Interactions of Heterotrimeric G proteins and G Protein-coupled Receptor (GPCR) Kinases with Activated GPCRs

“…IL2 of the receptor also collides with the ␣N-␤1 and ␣2-␤4 loops on the G␣ subunit. These collisions would be alleviated, at least in part, by rotation of the bulk of the heterotrimer with respect to the ␣C helix by ϳ60°(clockwise in this view), similar to that proposed previously (70). Collisions could also be avoided if the ␣C helix were kinked upon receptor binding, but current data suggest that an intact ␣C helix is required for functional receptor interactions (42,43).…”

Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit

“…The G αi C-terminal helix was fused with the highaffinity G α C-terminal peptide bound to opsin (for details, see SI Text and Figs. S5 and S6), which provided a convenient starting point for the model (19). The myristoylated N-terminal amphipathic helix was placed parallel to the membrane surface and the heterotrimer oriented such that both the myristoyl group and the nearby farnesylated C terminus of the Gγ-subunit can be inserted into the membrane; together these hydrophobic interactions cooperatively drive membrane binding of the intact heterotrimer (20).…”

Oligomerization of GPCRs involved in endocrine regulation

“…Correspondingly, activation is characterized by a spatial rearrangement of the TMHs relative to one another (Scheerer et al 2008, Schertler 2008. This structural rearrangement is supported by amino acids acting as 'micro-switches' (Ahuja & Smith 2009, Hofmann et al 2009, Nygaard et al 2009). …”