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

The Gα subunits of heterotrimeric G proteins (Gαβγ) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in “switch” sequences of Gα, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gβγ subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Gαi1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix α4, a putative effector binding region. In the inactive Gαi1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gβγ, motions of R1 are highly constrained, reflecting direct contact interactions at the Gαi1–Gβ 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 α2/β4 loop and smaller allosteric changes at the Gαi1–Gβγ interface distant from the receptor binding surface. Upon addition of GTPγS, the activated Gαi1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in α4, distant from the interaction surface, supports a role for this helix in effector binding.