Year of Award
Doctor of Philosophy (PhD)
Biochemistry & Biophysics
Department or School/College
Department of Chemistry and Biochemistry
Bruce Bowler, Klára Briknarová, Michael Kavanaugh, J. Stephen Lodmell
G-Protein, Ric-8A, SAXS, X-ray Crystallography
University of Montana
Heterotrimeric G-proteins (Gαβγ) regulate many cellular processes in the G-protein signaling pathways. The α-subunit (Gα) in the heterotrimer is activated by G-protein-coupled receptor (GPCR) as the guanine-nucleotide exchange factor (GEF), which catalyzes the GDP-release and GTP-binding reactions at Gα nucleotide-binding site, at the cell membrane. Intracellular GEFs for Gα subunits have been identified; among them, the mammalian isoform A of resistance to inhibitors of cholinesterase-8 (Ric-8A) catalyzes nucleotide exchange and functions as a folding chaperone for inhibitory Gα (Gαi1). In a nucleotide-free complex with Gαi1, Ric-8A likely assumes the GEF and chaperone roles by inducing a molten globule-like state. Tall et al. recently discovered that Casein Kinase II phosphorylates Ric-8A at two conserved sites (S435 and T440), which upon phosphorylation, elevate both the GEF and chaperone activities.
To understand the molecular mechanism under which Ric-8A interacts with Gαi1, we conducted hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) and identified a putative protein-protein interactive site (residues 454-470) on Ric-8A. Site-directed mutagenesis generated single alanine mutants of Ric-8A along the putative Gαi1-binding sequence and tryptophan fluorescence GEF assays identified five residues (V455, T456, R458, P466, and G469) as binding “hotspot”. We also solved a 2.2Å resolution, X-ray crystal structure of a 452-residue long fragment (R452) of the full-length Ric-8A. The crystal structure depicts a phosphorylated Ric-8A 1-452 molecule (pR452). Mapping sequence conservation scores and HDX protection profile on the pR452 crystal structure provides insights about the Ric-8A, Gαi1 interaction. Low-resolution, solution structures of both R452 and pR452 were also determined using Small Angle X-ray Scattering (SAXS). Phosphorylation of R452 at S435 and T440 likely induces subtle conformational changes on the molecule. Steady-State GTPase assay results indicated that not only does R452 retain measurable GEF activity towards Gαi1, phosphorylation of R452 also elevates the GEF activity at high Ric-8A concentrations.
With information from the biochemical assessments and Ric-8A protein structures, we conclude that (A) Gαi1 likely binds to Ric-8A residues 454 to 470 and other under-characterized sites on Ric-8A because (B) R452 retains important structural elements for the GEF activity towards Gαi1 and (C) phosphorylation of Ric-8A induces elevated Ric-8A GEF activity which is accompanied by conformational changes.
Zeng, Baisen, "A STRUCTURAL AND BIOCHEMICAL STUDY ON RIC-8A, AN INTRACELLULAR GUANINE NUCLEOTIDE EXCHANGE FACTOR AND FOLDING CHAPERONE FOR THE INHIBITORY G-PROTEIN ALPHA SUBUNIT-1" (2019). Graduate Student Theses, Dissertations, & Professional Papers. 11298.
© Copyright 2019 Baisen Zeng