Structure of Human PIR1, an Atypical Dual-Specificity Phosphatase

Abstract

PIR1 is an atypical dual-specificity phosphatase (DSP) that dephosphorylates RNA with a higher specificity than phosphoproteins. Here we report the atomic structure of a catalytically inactive mutant (C152S) of the human PIR1 phosphatase core (PIR1-core, residues 29–205), refined at 1.20 Å resolution. PIR1-core shares structural similarities with DSPs related to Vaccinia virus VH1 and with RNA 5′-phosphatases such as the baculovirus RNA triphosphatase and the human mRNA capping enzyme. The PIR1 active site cleft is wider and deeper than that of VH1 and contains two bound ions: a phosphate trapped above the catalytic cysteine C152 exemplifies the binding mode expected for the γ-phosphate of RNA, and ∼6 Å away, a chloride ion coordinates the general base R158. Two residues in the PIR1 phosphate-binding loop (P-loop), a histidine (H154) downstream of C152 and an asparagine (N157) preceding R158, make close contacts with the active site phosphate, and their nonaliphatic side chains are essential for phosphatase activity in vitro. These residues are conserved in all RNA 5′-phosphatases that, analogous to PIR1, lack a “general acid” residue. Thus, a deep active site crevice, two active site ions, and conserved P-loop residues stabilizing the γ-phosphate of RNA are defining features of atypical DSPs that specialize in dephosphorylating 5′-RNA.

Genetically modified proteins: functional improvement and chimeragenesis

“…An important signaling enzyme, dualDownloaded by [University of Exeter] at 18:01 28 July 2015 specificity phosphatase (DSP), whose misregulation is linked to cancer, diabetes, inflammation, and Alzheimer's disease, possesses broad substrate specificity and dephosphorylates phosphoprotein monoesters and the β-phosphate of RNA (Table 1). 8 A conservative mutation of L192 to arginine and noncatalytic C-terminal extension (residues 206−330) affected on enzymatic activity resulting in the appearence of diphosphatase activity in the monospecific ancestral protein phosphatase. 8 Two α-galactosidases AgaA and AgaB from thermophilic bacterium Geobacillus stearothermophilus have 97% identity and differ from each other by only 22 amino acid residues (Table 1).…”

“…8 A conservative mutation of L192 to arginine and noncatalytic C-terminal extension (residues 206−330) affected on enzymatic activity resulting in the appearence of diphosphatase activity in the monospecific ancestral protein phosphatase. 8 Two α-galactosidases AgaA and AgaB from thermophilic bacterium Geobacillus stearothermophilus have 97% identity and differ from each other by only 22 amino acid residues (Table 1). 4 This has drastically reflected on their different catalytic efficiency and themperature optimum.…”

Dimeric Quaternary Structure of Human Laforin

“…Given the open conformation of the laforin acidic loop (Fig. 1C), we reasoned this loop may either close upon binding to a phospho-substrate to bring Asp-235 closer to the active site or laforin phosphatase activity could be independent of a general acid, as reported for PIR1 (17) and PTEN (38).…”

“…In vitro glucan phosphatase assay was carried out using 100 g of amylopectin (Sigma) and 0.032 mg ml Ϫ1 laforin, as described (13,16). Phosphate released upon hydrolysis was measured at 620 nm using the Malachite green reagent and quantified using a phosphate standard curve (17). Phosphatase kinetics with 3-O-methylfluorescein phosphate (OMFP) was measured using laforin at 0.0084 mg ml Ϫ1 as described (17,18).…”

“…Phosphate released upon hydrolysis was measured at 620 nm using the Malachite green reagent and quantified using a phosphate standard curve (17). Phosphatase kinetics with 3-O-methylfluorescein phosphate (OMFP) was measured using laforin at 0.0084 mg ml Ϫ1 as described (17,18).…”