Nanosecond Dynamics of Gαi1 Bound to Nucleotides or Ric-8A, a Gα Chaperone with GEF Activity

Authors

Labe A. Black, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana; Department of Chemistry and Biochemistry, University of Montana, Missoula, Montana.
Celestine J. Thomas, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana; Department of Chemistry and Biochemistry, University of Montana, Missoula, Montana.
Gwendolyn N. Nix, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana; Division of Biological Sciences, University of Montana, Missoula, Montana.
Michelle C. Terwilliger, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana; Department of Chemistry and Biochemistry, University of Montana, Missoula, Montana.
Stephen R. Sprang, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana; Division of Biological Sciences, University of Montana, Missoula, Montana. Electronic address: stephen.sprang@umontana.edu.
J B. Ross, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana; Department of Chemistry and Biochemistry, University of Montana, Missoula, Montana. Electronic address: sandy.ross@umontana.edu.

Document Type

Article

Publication Title

Biophysical Journal

Publication Date

8-23-2016

Volume

111

Issue

4

Disciplines

Biology | Life Sciences

Abstract

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 α subunits of the i, q, and 12/13 classes, catalyzing the release of GDP from Gα and subsequent binding of GTP. In the absence of GTP or GTP analogs, and subsequent to GDP release, Gα forms a stable nucleotide-free complex with Ric-8A. In this study, time-resolved fluorescence anisotropy measurements were employed to detect local motions of Gαi1 labeled at selected sites with Alexa 488 (C5) fluorescent dye (Ax) in the GDP, GTPγS (collectively, GXP), and Ric-8A-bound states. Sites selected for Alexa 488 (C5) derivatization were in the α-helical domain (residue 106), the α-helical domain-Ras-like domain hinge (residue 63), Switch I (residue 180), Switch II (residue 209), Switch III (residue 238), the α4 helix (residue 305), and at the junction between the purine-binding subsite in the β6-α5 loop and the C-terminal α 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 Gαi1•GDP than in Gαi1•GTPγS. The reverse is true at Ax209. The order parameters, S(2), for Alexa probes at switch residues are high (0.78-0.88) in Gαi1•GDP and lower (0.67-0.75) in Gαi1•GTPγS, 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 Gαi1:Ric-8A than in Gαi1•GXP, and only modest (± 0.1) differences in S(2) are observed at most sites in Gαi1:Ric-8A relative to Gαi1•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 Gαi1 structural heterogeneity by Ric-8A provides a mechanism for nucleotide release.

DOI

10.1016/j.bpj.2016.07.021

Rights

© 2016 Biophysical Society

Recommended Citation

Black, Labe A.; Thomas, Celestine J.; Nix, Gwendolyn N.; Terwilliger, Michelle C.; Sprang, Stephen R.; and Ross, J B., "Nanosecond Dynamics of Gαi1 Bound to Nucleotides or Ric-8A, a Gα Chaperone with GEF Activity" (2016). Biological Sciences Faculty Publications. 483.
https://scholarworks.umt.edu/biosci_pubs/483