Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A

• Published in: Structure (London) 2019-07, Vol.27 (7), p.1137-1147.e5
• Main Authors: Zeng, Baisen, Mou, Tung-Chung, Doukov, Tzanko I., Steiner, Andrea, Yu, Wenxi, Papasergi-Scott, Makaia, Tall, Gregory G., Hagn, Franz, Sprang, Stephen R.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; BASIC BIOLOGICAL SCIENCES; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; GTP-Binding Protein alpha Subunits, Gi-Go - chemistry; GTP-Binding Protein alpha Subunits, Gi-Go - genetics; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; guanine nucleotide exchange factor; Guanine Nucleotide Exchange Factors - chemistry; Guanine Nucleotide Exchange Factors - genetics; Guanine Nucleotide Exchange Factors - metabolism; Guanosine Triphosphate; heteronuclear nuclear magnetic resonance; heterotrimeric G protein; Histidine - genetics; Histidine - metabolism; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Models, Molecular; molecular chaperone; Mutation; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Oligopeptides - genetics; Oligopeptides - metabolism; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; protein dynamics; Protein Interaction Domains and Motifs; Protein Multimerization; protein structure; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; small-angle X-ray scattering; X-ray crystallography
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2019.04.013

Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1–452, and is phosphorylated at Ser435 and Thr440. Residues 1–429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be a putative Gα-binding surface. Normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1–4, 5–6, and 7–9. 2D 1H-15N-TROSY spectra of [2H,15N]-labeled Gαi1 in the presence of R452 reveals chemical shift perturbations of the C terminus and Gαi1 residues involved in nucleotide binding. [Display omitted] •The G protein α subunit binding domain of Ric-8A is an Armadillo/HEAT repeat fold•Hydrogen-deuterium exchange data suggest a Gα binding surface on Ric-8A•Ric-8A binds elements of Gα also recognized by G protein-coupled receptors•The Ric-8A fold harbors a putative binding site for its C-terminal phosphoserine Zeng et al. use X-ray crystallography and small-angle scattering, in conjunction with NMR spectroscopy, to reveal the structure of and dynamics of the G protein chaperone and activator Ric-8A and probe its interaction with the G protein alpha subunit i1.