Binding Interface of Gapdh to the Au Rich Elements from TNF-Alpha mRNA Revealed by Hydrogen Deuterium Exchange Coupled with Mass Spectrometry

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous protein primarily known for its glycolytic function. More recent studies have revealed GAPDH as a multifaceted, multifunctional protein involved in various cellular processes including iron homeostasis, heme transport, apoptosis, tumerogenesis, oxidative stress response, chromatin structure among many others. As part of its multifunctional purpose, GAPDH has been shown to bind to various cellular RNAs and has been classified as a non-canonical RNA binding protein. In particular, it has been shown to bind AU-rich element of mRNA transcripts and consequently affecting translation. However, the detailed structural characterization of the RNA bound complex has remained elusive. The RNA binding site remains unknown thereby restricting potential therapeutic strategies. Our previous studies established that GAPDH binds the AU rich element of TNF-α mRNA transcript via a two-step model. In addition, we have shown that a T229K mutant of GAPDH displayed altered RNA and NAD+ binding properties, potentially signaling to a linkage between RNA binding and the cofactor. Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) revealed that the T229K mutant induced conformational changes into distal grooves along the P-axis of the tetrameric protein. In this study, we have mapped the binding interfaces of three GAPDH constructs displaying varying NAD+ binding properties with the AU rich element from TNF-α mRNA using HDX-MS. A largely common pattern of localized protection from exchange pointed to the grooves along the P-axis as the RNA binding interfaces, as hypothesized. Additionally, some variations in HDX protection point to potential conformational basis for the linkage with NAD+ binding.