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J Biol Chem. 2015 Jan 16;290(3):1770-85. doi: 10.1074/jbc.M114.618165. Epub 2014 Dec 1.

A dimer interface mutation in glyceraldehyde-3-phosphate dehydrogenase regulates its binding to AU-rich RNA.

Author information

1
From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250.
2
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201.
3
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and.
4
Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210.
5
From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, egarcin@umbc.edu.

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.

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)

PMID:
25451934
PMCID:
PMC4340419
DOI:
10.1074/jbc.M114.618165
[Indexed for MEDLINE]
Free PMC Article

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