Structural and dynamical changes in an α-subunit of a heterotrimeric G protein along the activation pathway

The G alpha subunits of heterotrimeric G proteins (G alpha beta gamma) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in switch sequences of Ga, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the G beta gamma subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of G alpha i1 through the G protein cycle, nitroxide side chains (1111) were introduced at sites in switch 11 and at a site in helix alpha 4, a putative effector binding region. In the inactive G alpha i1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with G beta gamma, motions of R1 are highly constrained, reflecting direct contact interactions at the G alpha i1-G/3 interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding alpha 2/beta 4 loop and smaller allosteric changes at the G alpha i1-G beta gamma interface distant from the receptor binding surface. Upon addition of GTP gamma S, the activated G alpha i1(GTP) subunit dissociates from the complex, and switch 11 is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation -dependent change in alpha 4, distant from the interaction surface, supports a role for this helix in effector binding.