High‐resolution crystal structures of the photoreceptor glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) with three and four‐bound NAD molecules

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD) 4 , and bGAPDH(NAD) 3 . The bGAPDH(NAD) 4 was solved at 1.52 Å , the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD) 4 and bGAPDH(-NAD) 3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54-56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies.Abbreviations: APS, advanced photon source; BGP, 1,3-diphosphoglycerate; bGAPDH, bovine GAPDH; BNL, Brookhaven National Laboratory; G3P, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; bGAPDH(NAD) 3 and bGAPDH(NAD) 4 , bovine GAPDH with NAD bound in three or four subunits; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; MS, mass spectroscopy; NAD, nicotinamide adenine dinucleotide; NO, nitric oxide; NSLS, National Synchrotron Light Source; PDE6, phosphodiesterase 6; PEG, polyethylene glycol; RMSD, root-mean-square-deviation(s); ROS, rod outer segment(s); SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography.Additional Supporting Information may be found in the online version of this article.Disclosure: The authors declare none.Accession numbers: The atomic coordinates have been deposited in the RCSB Protein Data Bank under accession number 4O63 for bGAPDH(NAD) 3 and 4O59 for bGAPDH(NAD) 4 .

The sweet side ofRNAregulation: glyceraldehyde‐3‐phosphate dehydrogenase as a noncanonicalRNA‐binding protein

“…The structures of GAPDH from archaea, bacteria, and eukaryotes have been determined by x‐ray crystallography, and show that the NAD + cofactor is bound, despite not being added during protein purification. In mammalian GAPDH structures, two, three, or even four NAD + molecules have been observed per homotetramer. In addition, we recently showed that GAPDH was present as a mixture of NAD + ‐bound and NAD + ‐free species in solution by nano‐ESI/MS/MS studies .…”

A cryoprotectant induces conformational change in glyceraldehyde-3-phosphate dehydrogenase

“…Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme that catalyses the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate using NAD + as a cofactor (Baker et al, 2014) and is known to be a moonlighting protein (Savreux-Lenglet et al, 2015). This enzyme plays multiple roles in the regulation of mRNA stability (Zhou et al, 2008), intracellular membrane trafficking (Sirover, 2012), iron uptake and transport (Zaid et al, 2009), DNA replication and repair (Zheng et al, 2003), and nuclear RNA transport (Dastoor & Dreyer, 2001).…”

Crystallization of Proteins from Crude Bovine Rod Outer Segments

“…Unit cell dimensions were a = 79.8 Å, b = 126.5 Å, c = 83.8 Å; α = 90°, β = 118°, γ =90°. Although bovine GAPDH existed as a homotetramer identical to known structures in this protein family, it had three NAD molecules bound instead of either two or four found in other mammalian GAPDHs, a feature that distinguishes it from others PDB entries, 1J0X (Cowan-Jacob, Kaufmann, Anselmo, Stark, & Grutter, 2003), 1U8F (Jenkins & Tanner, 2006), and 1ZNQ (Baker, Shi, Wang, & Palczewski, 2014). …”