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

Baisen Zeng; Tung-Chung Mou; Tzanko I Doukov; Andrea Steiner; Wenxi Yu; Makaia Papasergi-Scott; Gregory G Tall; Franz Hagn; Stephen R Sprang

doi:10.1016/j.str.2019.04.013

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

Structure. 2019 Jul 2;27(7):1137-1147.e5. doi: 10.1016/j.str.2019.04.013. Epub 2019 May 30.

Authors

Baisen Zeng¹, Tung-Chung Mou², Tzanko I Doukov³, Andrea Steiner⁴, Wenxi Yu⁵, Makaia Papasergi-Scott⁶, Gregory G Tall⁵, Franz Hagn⁴, Stephen R Sprang⁷

Affiliations

¹ Graduate Program in Biochemistry and Biophysics, University of Montana, Missoula, MT 59812, USA.
² Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA; Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA.
³ Macromolecular Crystallography Group, Stanford Synchrotron Radiation Light Source, SLAC National Accelerator Laboratory, Stanford University, Stanford, CA 94309, USA.
⁴ Bavarian NMR Center at the Department of Chemistry and Institute for Advanced Study, Technical University of Munich, Ernst-Otto-Fischer-Strasse 2, 85748 Garching, Germany; Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany.
⁵ Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
⁶ Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.
⁷ Graduate Program in Biochemistry and Biophysics, University of Montana, Missoula, MT 59812, USA; Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA; Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA. Electronic address: [email protected].

Abstract

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.

Keywords: X-ray crystallography; guanine nucleotide exchange factor; heteronuclear nuclear magnetic resonance; heterotrimeric G protein; molecular chaperone; protein dynamics; protein structure; small-angle X-ray scattering.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Amino Acid Sequence
Animals
Binding Sites
Cloning, Molecular
Crystallography, X-Ray
Escherichia coli / genetics
Escherichia coli / 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
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Guanine Nucleotide Exchange Factors / chemistry*
Guanine Nucleotide Exchange Factors / genetics
Guanine Nucleotide Exchange Factors / metabolism
Guanosine Triphosphate
Histidine / genetics
Histidine / metabolism
Models, Molecular
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 Interaction Domains and Motifs
Protein Multimerization
Rats
Recombinant Fusion Proteins / chemistry*
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism

Substances

Guanine Nucleotide Exchange Factors
His-His-His-His-His-His
Nuclear Proteins
Oligopeptides
Recombinant Fusion Proteins
Ric8a protein, rat
Histidine
Guanosine Triphosphate
GTP-Binding Protein alpha Subunits, Gi-Go

Abstract

Publication types

MeSH terms

Substances

Grants and funding