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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.

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From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250.
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and.
Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210.
From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250,


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.


AU-rich Elements; Conformational Change; Crystallography; Fluorescence Anisotropy; Fluorescence Resonance Energy Transfer (FRET); GAPDH; Hydrogen Exchange Mass Spectrometry; Oligomerization; RNA; Tumor Necrosis Factor (TNF)

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