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.
Highlights
The glycolytic enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a non-canonical RNA-binding protein
We show that the mutation does not affect GAPDH oligomerization but promotes dynamic changes within protein regions clustered along the P axis of the GAPDH tetramer
Wild-type GAPDH Binds Tightly to the adenine-uridine rich elements (AREs) from TNF-␣ mRNA—We first tested GAPDH binding to an RNA oligonucleotide derived from TNF-␣ 3Ј-UTR
Summary
Results: A dimer interface mutation impairs the two-step binding of GAPDH to AU-rich elements from TNF-␣ mRNA and leads to RNA structural changes. 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. We propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces. Our results allow us to propose a new model for RNA binding to GAPDH
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