title: Structural and dynamical changes in an alpha-subunit of a heterotrimeric G protein along the activation pathway.
abstract: The Galpha subunits of heterotrimeric G proteins (Galphabetagamma) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in "switch" sequences of Galpha, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gbetagamma subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Galphai1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix alpha4, a putative effector binding region. In the inactive Galphai1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gbetagamma, motions of R1 are highly constrained, reflecting direct contact interactions at the Galphai1-Gbeta 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 alpha2/beta4 loop and smaller allosteric changes at the Galphai1-Gbetagamma interface distant from the receptor binding surface. Upon addition of GTPgammaS, the activated Galphai1(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 alpha4, distant from the interaction surface, supports a role for this helix in effector binding.
authors.affiliation: Jules Stein Eye Institute, Departments of Ophthalmology and Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Ned
authors.initials: N
authors.lastname: Van Eps
authors.forename: William M
authors.initials: WM
authors.lastname: Oldham
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D006153:Guanosine Diphosphate