title: A dimer interface mutation in glyceraldehyde-3-phosphate dehydrogenase regulates its binding to AU-rich RNA.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3'-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD(+) binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250.
authors.forename: Michael R
authors.initials: MR
authors.lastname: White
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250.
authors.forename: Mohd M
authors.initials: MM
authors.lastname: Khan
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201.
authors.forename: Daniel
authors.initials: D
authors.lastname: Deredge
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and.
authors.forename: Christina R
authors.initials: CR
authors.lastname: Ross
authors.affiliation: Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210.
authors.forename: Royston
authors.initials: R
authors.lastname: Quintyn
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and.
authors.forename: Beth E
authors.initials: BE
authors.lastname: Zucconi
authors.affiliation: Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210.
authors.forename: Vicki H
authors.initials: VH
authors.lastname: Wysocki
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Wintrode
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Wilson
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, egarcin@umbc.edu.
authors.forename: Elsa D
authors.initials: ED
authors.lastname: Garcin
mesh_terms: D020413:3' Untranslated Regions; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016880:Anisotropy; D001665:Binding Sites; D018076:DNA, Complementary / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D006019:Glycolysis; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D012313:RNA / Q000737:chemistry*; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014965:X-Rays
chemical_list: D020413:3' Untranslated Regions; D018076:DNA, Complementary; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: AU-rich Elements; Conformational Change; Crystallography; Fluorescence Anisotropy; Fluorescence Resonance Energy Transfer (FRET); GAPDH; Hydrogen Exchange Mass Spectrometry; Oligomerization; RNA; Tumor Necrosis Factor (TNF)