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

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Abstract
Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the α-subunit of intracellular G proteins (Gαβγ). Kinetic analysis of transducin (G t αβγ) activation shows that an intermediary R*·G t αβγ·GDP complex is formed that 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 G t α 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 G t α (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-bonding networks between the C-terminal reverse turn of GαCT and conserved E(D)RY and NPxxY(x) 5,6 F regions of the GPCR. The alternative fit – termed intermediary or I-interaction – is distinguished by a tilt (42°) and rotation (90°) of α5 relative to the S-interaction and shows different α5 contacts with the NPxxY(x) 5,6 F region and the second cytoplasmic loop of R*. From the 2 α5 interactions, we derive a “helix switch” mechanism for the transition of R*·G t αβγ·GDP to the nucleotide-free R*·G protein complex that illustrates how α5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.

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