Dynamics of Glyceraldehyde-3-Phosphate Dehydrogenase Interfacial Regions Affect Binding to AU-Rich RNA

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The homotetrameric protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been shown to possess many functions aside from its role in glycolysis. Of particular interest is its role in post-transcriptional regulation. Despite lacking a canonical RNA binding motif, GAPDH has been shown to bind to many mRNAs and subsequently alter their translation. Most of these protein-nucleic acid interactions have been shown to occur by GAPDH binding to Adenine-Uridine Rich Elements (AREs) within the 3’ untranslated regions (UTRs) of specific mRNAs. While much evidence has been gathered in determining the means of RNA binding, the exact site and mechanism of binding still remain elusive. Variables that may be key to elucidating these two facets of RNA binding include the effects of posttranslational modifications, oligomerization, cofactor binding, and structural dynamics, of GAPDH. Herein, it is demonstrated for the first time that GAPDH binds to the core AREs of the tumor necrosis factor-α mRNA 3′UTR via a sequential two-step mechanism. As well, a single point mutation at the GAPDH dimer interface results in a reduction in binding affinity in the second step and an alteration in the bound RNA structure. In contrast to previous studies, it is shown here that this mutation does not affect protein oligomerization, but induces dynamic changes in protein regions localized along the P axis of the GAPDH tetramer. Based on our results, we propose a novel model for GAPDH binding to ARE-containing RNA that may be regulated by GAPDH post-translational modifications.