Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

Abstract

Baculovirus RNA 5 -triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphatebound state at 1.5 Å resolution. BVP adopts the characteristic cysteine-phosphatase ␣/␤ fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif 118 HCTHGXNRT 126 in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages. mRNA 5Ј cap formation is initiated by hydrolysis of the ␥-phosphate of 5Ј-triphosphate-terminated pre-mRNA. The resulting 5Ј-diphosphate end is then capped by transfer of GMP from GTP to form an inverted terminal dinucleotide structure, G(5Ј)ppp(5Ј)N. The first reaction is catalyzed by RNA 5Ј-triphosphatase and the second by GTP:RNA guanylyltransferase (reviewed in Refs. 1 and 2). In metazoans and plants, the triphosphatase and guanylyltransferase activities reside in a single polypeptide composed of an N-terminal triphosphatase domain fused to a C-terminal guanylyltransferase domain. The metazoan and plant RNA triphosphatases belong to the cysteine phosphatase superfamily (3), which includes phosphoprotein phosphatases (4) and phosphoinositide phosphatases (5).Cysteine phosphatase-type RNA triphosphatases act via a twostep mechanism entailing attack by a cysteine thiolate nucleophile of the enzyme on the ␥-phosphate of triphosphate-terminated RNA to form a cysteinyl-phosphoenzyme intermediate, which is then hydrolyzed to liberate inorganic phosphate. The active site cysteine is located within a signature P-loop motif, HCTHGXNRT.We reported previously (3) the crystal structure of the RNA triphosphatase domain of mouse capping enzyme Mce1 at 1.65 Å resolution. Mce1 adopts a globular fold consisting of a five-stranded parallel ␤ sheet flanked by ␣ helices on both sides. The active site cysteine is located at the bottom of a deep, positively charged pocket formed by essential amino acids that line the pocket walls and surface rim. Structural, biochemical, and mutational results showed that despite sharing a HCXXXXXR(S/T) P-loop motif, a phosphoenzyme intermediate, and a core ␣/␤ fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by Mce1 and its baculovirus homologue BVP 1 differs from that used by phosphoprotein ...

Roles of LEF-4 and PTP/BVP RNA Triphosphatases in Processing of Baculovirus Late mRNAs

“…Here we refer to it by the hybrid name ptp/bvp. Crystallographic studies of PTP/BVP showed that its structure was similar to metazoan RNA capping enzymes, thus confirming its specificity for RNA substrates (5). Furthermore, in vivo analyses showed that PTP/ BVP could substitute for yeast RNA triphosphatase in the cap synthetic pathway in yeast cells (42).…”

Structure of Human PIR1, an Atypical Dual-Specificity Phosphatase

“…The phosphatase core consists of a central five-stranded β-sheet (β1−β5) (highlighted with a light blue background in Figure 2B) sandwiched between two clusters of five (h1–h5) and four (h6–h9) helices that make contacts with the solvent (Figure 2B). A search using the Dali server 40 identified baculovirus BVP (PDB entry 1YN9) 21 and the RNA 5′-phosphatase domain of the mouse mRNA capping enzyme (PDB entry 1I9S) 41 as the phosphatases that were most structurally similar to PIR1-core (rmsds of 1.4 and 1.5 Å, respectively). PIR1-C152S-core can also be superimposed on VH1 (PDB entry 3CM3) (Figure 2C) and VHR (PDB entry 1VHR) with rmsds of 2.5 and 2.2 Å, respectively, but presents four noticeable differences from the classical VH1-like DSP-core (colored red in Figure 2B,C).…”

Structural and functional characterization of a novel phosphatase from the Arabidopsis thaliana gene locus At1g05000

“…Cysteine phosphatase structures often contain phosphate(s) 12 or sulfate(s) 38 within a positively-charged pocket that includes the HCxxGxxR motif; sulfate presumably substitutes for the physiological phosphate. Two sulfate groups were observed in the catalytic site cavity of At1g05000, suggesting the possibility that a multiply phosphorylated physiological substrate may be recognized by the active site.…”

“…Two sulfate groups were observed in the catalytic site cavity of At1g05000, suggesting the possibility that a multiply phosphorylated physiological substrate may be recognized by the active site. Sulfates/phosphates in At1g05000 and in the baculovirus BVP 12 and human VHR 38 enzymes are rather distant (>3 Å) from the cysteine of the cysteine phosphatase active site motif, bringing their physiological relevance into doubt.…”