Research on the Molecular Mechanism of Adenosine 3', 5'-Bisphosphate Inhibiting DXO
Yun J S, et al. Biochemical and biophysical research communications, 2018, 504(1), 89-95.
The decapping exoribonuclease DXO plays a role in pre-mRNA capping quality control. In order to understand the inhibitory mechanism of DXO, the crystal structure of DXO complex with adenosine-3',5'-bisphosphate (pAp) and Mg2+ was determined at 1.8 Å resolution. The effect of pAp on DXO 5'-3' ectoribonuclease activity was also examined. Structural and biochemical studies indicate that pAp inhibits the activity of DXO by occupying the active site and acting as a competitive inhibitor. Protein expression, purification, and crystallization · In brief, E234A mutant protein and the wild-type enzyme were purified by Ni-NTA and gel filtration chromatography using the same protocol. · Free enzyme crystals of DXO were obtained with the sitting-drop vapor diffusion method at 20 °C, using a reservoir solution containing 20% (w/v) PEG 3350. · The pAp-Mg2+ complex was obtained by soaking the free enzyme crystals with 100 mM pAp and 10 mM MgCl2 for 60 min in the presence of 15% ethylene glycol. · Crystals were flash-frozen in liquid nitrogen for diffraction analysis and data collection at 100 K.
Crystal Structure of Adenosine 3',5'-Bisphosphate in Complex with Yeast PAPS Reductase
Yu Z, et al. Biochemistry, 2008, 47(48), 12777-12786.
In yeast, PAPS reductase reduces PAPS to sulfite and PAP. The crystal structure of eukaryotic PAPS reductase from yeast, whose product is bound to adenosine 3',5'-bisphosphate (PAP), is reported. The structure reveals the complete C-terminal tail bound to the active site pocket and provides additional clues to the catalytic mechanism. Crystallization of NatiVe and SeMet PAPS Reductase · Purified PAPS reductase was extensively dialyzed against Tris-HCl buffer and concentrated to 10 mg/mL for crystallization using Amicon spin concentrators. · Native protein was crystallized with the presence of 5 mM TCEP and 1 mM PAPS by hanging drop vapor diffusion using 2 µL drops of protein mixed with an equal volume of reservoir buffer. · After 3 weeks, the crystals grew to full size of about 0.25 mm × 0.2 mm × 0.15 mm. Diffraction of crystals improved significantly by soaking with DTT followed by PAP. · Crystals were transferred and soaked in a buffer containing 10 mM DTT for 30 min and then soaked in a buffer containing 2 mM PAP for 10 min. · The SeMetmodified protein crystals were grown by hanging drop against 30% PEG 6000 and 0.1 M HEPES, pH 7.0, and grew to about one-third the size of native crystals.
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Yun J S, et al. Biochemical and biophysical research communications, 2018, 504(1), 89-95.
Yu Z, et al. Biochemistry, 2008, 47(48), 12777-12786.
