Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, catalyses the conversion of D-glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. While mammalian and yeast GAPDHs are multifunctional proteins that have additional functions beyond those involved in glycolysis, including reactions related to nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis, Escherichia coli GAPDH (ecGAPDH) has only been reported to function in glycolysis. The S-loop of GAPDH is required for interaction with its cofactor and with other proteins. In this study, the three-dimensional crystal structure of GAPDH treated with trehalose is reported at 2.0 Å resolution. Trehalose was used as a cryoprotectant for the GAPDH crystals. The structure of trehalose-bound ecGAPDH was compared with the structures of both NAD+-free and NAD+-bound ecGAPDH. At the S-loop, the bound trehalose in the GAPDH structure induces a 2.4° rotation compared with the NAD+-free ecGAPDH structure and a 3.1° rotation compared with the NAD+-bound ecGAPDH structure.
Highlights
Many crystals of biological macromolecules are sensitive to X-rays near room temperature and frequently suffer from radiation damage, especially when X-ray experiments are carried out on highly intense synchrotron beamlines (Hope, 1990)
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from E. coli without any genetic manipulation
2 mg of purified Escherichia coli GAPDH (ecGAPDH) was obtained from a 1 l culture
Summary
Many crystals of biological macromolecules are sensitive to X-rays near room temperature and frequently suffer from radiation damage, especially when X-ray experiments are carried out on highly intense synchrotron beamlines (Hope, 1990). Hanging-drop vapour diffusion 24-well plates 4 20 20 mM Tris pH 7.5, 0.1 M NaCl 0.1 M MES pH 5.5–6.5, 2.8 M ammonium sulfate 2 ml: ml 1 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). The GAPDH crystals were cryoprotected with a solution containing 15% of the disaccharide trehalose
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