Black_2016_Biophys.J_111_722

Resistance to Inhibitors of Cholinesterase A (Ric-8A) is a 60-kDa cytosolic protein that has chaperone and guanine nucleotide exchange (GEF) activity toward heterotrimeric G protein alpha subunits of the i, q, and 12/13 classes, catalyzing the release of GDP from Galpha and subsequent binding of GTP. In the absence of GTP or GTP analogs, and subsequent to GDP release, Galpha forms a stable nucleotide-free complex with Ric-8A. In this study, time-resolved fluorescence anisotropy measurements were employed to detect local motions of Galphai1 labeled at selected sites with Alexa 488 (C5) fluorescent dye (Ax) in the GDP, GTPgammaS (collectively, GXP), and Ric-8A-bound states. Sites selected for Alexa 488 (C5) derivatization were in the alpha-helical domain (residue 106), the alpha-helical domain-Ras-like domain hinge (residue 63), Switch I (residue 180), Switch II (residue 209), Switch III (residue 238), the alpha4 helix (residue 305), and at the junction between the purine-binding subsite in the beta6-alpha5 loop and the C-terminal alpha helix (residue 330). In the GXP-bound states, the Alexa fluorophore reports local motions with correlation times ranging from 1.0 to 1.8 ns. The dynamics at Ax180 is slower in Galphai1*GDP than in Galphai1*GTPgammaS. The reverse is true at Ax209. The order parameters, S(2), for Alexa probes at switch residues are high (0.78-0.88) in Galphai1*GDP and lower (0.67-0.75) in Galphai1*GTPgammaS, although in crystal structures, switch segments are more ordered in the latter. Local motions at Ax63, Ax180, Ax209, and Ax330 are all markedly slower (2.3-2.8 ns) in Galphai1:Ric-8A than in Galphai1*GXP, and only modest (+/- 0.1) differences in S(2) are observed at most sites in Galphai1:Ric-8A relative to Galphai1*GXP. The slow dynamics suggests long-range correlated transitions within an ensemble of states and, particularly in the hinge and switch segments that make direct contact with Ric-8A. Induction of Galphai1 structural heterogeneity by Ric-8A provides a mechanism for nucleotide release.