Crystal structure of human diphosphoinositol phosphatase 1

Abstract

Phosphorylated variants of inositol play diverse and crucial roles in a wide range of cellular processes. The fully phosphorylated species, IP6 or phytic acid, was for long thought to be the endpoint of cellular inositol phosphorylation but some 15 years ago two laboratories independently discovered mono- and bis-diphosphorylated inositols PP-IP5 (IP7) and [PP]2-IP4 (IP8).1, 2 Since then, a large number of functions have been ascribed to these diphosphorylated inositols, for example, for telomere maintenance, DNA repair, protein phosphorylation, apoptosis, and even for effective exocytosis in pancreatic β-cells.3-5 The cellular turnover of inositol diphosphates is immense with a large part of the IP6 pool being converted to and from IP7 every hour.1, 3, 4 At any given time point, however, the cellular concentrations of IP7 and IP8 are very low (0.1–2 μM).3, 4 In mammalian cells, IP7 has for some time been known to be of the 5-[PP]-IP5 isoform.4 However, a second IP7 isoform was recently discovered and the new IP7 isoform was proposed to be either 1-[PP]-IP5 and 3-[PP]-IP5.6 Consequently, the IP8 isoform(s) present in mammalian cells are most likely either 1,5-[PP]2-IP4 and/or 3,5-[PP]2-IP4. The ambiguity of the IP7 and IP8 isoforms arises from the lack of stereo-selectivity in the techniques used so far. Early on, it was observed that cells incubated with fluoride display very high levels of inositol diphosphates and it was therefore presumed that the diphosphatase that breaks down inositol diphosphates to IP6 again is fluoride sensitive.1 Indeed, a fluoride sensitive diphosphoinositol diphosphatase (DIPP), was identified in 1998 by Shears and coworkers.7 From sequence similarity, it was found to belong to the Nudix family of hydrolases that commonly have nucleotide and nucleotide derivative substrates.7, 8 Mutagenesis in the Nudix motif, GX5EX7REUXEEXGU, of DIPP rendered the enzyme largely inactive.7, 9 Later, one could define a DIPP-family consisting of five highly homologous members that are all capable of selective inositol diphosphate degradation.10 Here, we describe the crystal structure of the first described DIPP, the human DIPP1, as determined by a combination of sulfur and phosphor SAD. Furthermore, we describe a cocrystal structure of DIPP1 with the product, IP6, and a magnesium fluoride cluster bound. On the basis of the cocrystal structure, we propose a catalytic mechanism for DIPPs. DNA encoding residues 1–148 of the DIPP1 gene (gi: 14043478) was cloned by ligation-independent cloning into a pET-28 based expression vector incorporating an N-terminal hexa-His-tag fusion (pNIC-Bsa4; gi:EF198106). After transformation and liquid culture growth using standard methods,11 recombinant expression of human DIPP1 in Escherichia coli strain BL21(DE3) was induced at 291 K by addition of 0.5 mM IPTG to Terrific Broth media. Induction was maintained for 18 h before harvesting. DIPP1 was purified using IMAC12 on a 1 mL HiTrap chelating HP column followed by gel filtration on a 120 mL Superdex 75 column. DIPP1 was essentially pure as judged by SDS-PAGE analysis and denaturing electrospray-ionization mass spectrometry verified protein integrity. All columns used were from GE-Healthcare, Uppsala, Sweden. Crystals of DIPP1 were grown by sitting-drop vapor diffusion at 277 K. DIPP1 (11 mg/mL) in 20 mM Hepes pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP, and 5mM myo-inositol hexakisphosphate (IP6) were mixed with an equal amount (100 nL) of reservoir solution (30% PEG 8000, 200 mM lithium sulfate, and 100 mM sodium acetate, pH 4.5) using a Phoenix crystallization robot (Art Robbins Instruments, Sunnyvale, CA). Crystals grew as 0.05 mm × 0.05 mm × 0.1 mm rods after 3 weeks. Crystals were flash-frozen in liquid-nitrogen directly from the crystallization solution. A single-wavelength anomalous dispersion dataset was collected with Cu-Kα radiation on an X8 PROTEUM system equipped with a four-circle Kappa goniostat and a PLATINUM-135 CCD (