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• W1983257557 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 phosphate-bound state at 1.5 Å resolution. BVP adopts the characteristic cysteine-phosphatase α/β 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 118HCTHGXNRT126 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. 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 phosphate-bound state at 1.5 Å resolution. BVP adopts the characteristic cysteine-phosphatase α/β 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 118HCTHGXNRT126 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 5′ cap formation is initiated by hydrolysis of the γ-phosphate of 5′-triphosphate-terminated pre-mRNA. The resulting 5′-diphosphate end is then capped by transfer of GMP from GTP to form an inverted terminal dinucleotide structure, G(5′)ppp(5′)N. The first reaction is catalyzed by RNA 5′-triphosphatase and the second by GTP:RNA guanylyltransferase (reviewed in Refs. 1Shuman S. Prog. Nucleic Acids Res. Mol. Biol. 2000; 66: 1-40Crossref Google Scholar and 2Shuman S. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 301-312Crossref PubMed Scopus (36) Google Scholar). 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 (3Changela A. Ho C.K. Martins A. Shuman S. Mondragon A. EMBO J. 2001; 20: 2575-2586Crossref PubMed Scopus (79) Google Scholar), which includes phosphoprotein phosphatases (4Denu J.M. Dixon J.E. Curr. Opin. Chem. Biol. 1998; 2: 633-641Crossref PubMed Scopus (335) Google Scholar) and phosphoinositide phosphatases (5Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2578) Google Scholar). Cysteine phosphatase-type RNA triphosphatases act via a two-step mechanism entailing attack by a cysteine thiolate nucleophile of the enzyme on the γ-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 (3Changela A. Ho C.K. Martins A. Shuman S. Mondragon A. EMBO J. 2001; 20: 2575-2586Crossref PubMed Scopus (79) Google Scholar) 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 β sheet flanked by α 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 α/β fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by Mce1 and its baculovirus homologue BVP 1The abbreviations used are: BVP, baculovirus phosphatase; r.m.s.d., root mean square deviation; BSA, bovine serum albumin; DTT, dithiothreitol. 1The abbreviations used are: BVP, baculovirus phosphatase; r.m.s.d., root mean square deviation; BSA, bovine serum albumin; DTT, dithiothreitol. differs from that used by phosphoprotein phosphatases to hydrolyze phosphomonoesters. The key distinction is the absence of a carboxylate general acid catalyst in the RNA-specific triphosphatases. Residues conserved uniquely among the RNA phosphatase subfamily are important for cap formation in vivo and have been proposed to play a role in substrate recognition. However, the structure of the Mce1 triphosphatase apoenzyme provided no direct information regarding the nature of the atomic contacts involved. Baculovirus phosphatase (BVP) is a 168-amino acid protein encoded by Autographa californica nucleopolyhedrovirus (6Gross C.H. Shuman S. J. Virol. 1998; 72: 7057-7063Crossref PubMed Google Scholar, 7Takagi T. Taylor G.S. Kusakabe T. Charbonneau H. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9808-9812Crossref PubMed Scopus (44) Google Scholar). BVP displays primary structure similarity to the N-terminal RNA triphosphatase domain of Mce1. BVP differs from the cellular capping enzyme in several respects. First, it is a monofunctional triphosphatase that is not covalently linked to a guanylyltransferase domain. Second, unlike the cellular capping enzyme, which hydrolyzes only the γ-phosphate of a triphosphate-terminated substrate, BVP can also hydrolyze the β-phosphate of either diphosphate-terminated RNA or free NDPs (6Gross C.H. Shuman S. J. Virol. 1998; 72: 7057-7063Crossref PubMed Google Scholar, 7Takagi T. Taylor G.S. Kusakabe T. Charbonneau H. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9808-9812Crossref PubMed Scopus (44) Google Scholar). Monofunctional RNA phosphatases are not confined to baculovirus; the monofunctional human phosphatase PIR1 is a homologue of BVP that also displays RNA triphosphatase and diphosphatase activities (8Deshpande T. Takagi T. Hao L. Buratowski S. Charbonneau H. J. Biol. Chem. 1999; 274: 16590-16594Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Additionally, PIR1-like proteins are present in many metazoan proteomes, but their functions are not known (8Deshpande T. Takagi T. Hao L. Buratowski S. Charbonneau H. J. Biol. Chem. 1999; 274: 16590-16594Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). BVP activity also embraces the hydrolysis of inorganic tripolyphosphate and pyrophosphate (9Martins A. Shuman S. Virology. 2002; 304: 167-175Crossref PubMed Scopus (6) Google Scholar). Despite its broad specificity for hydrolysis of phosphoanhydrides in vitro, BVP can act as an RNA triphosphatase in the cap synthetic pathway in vivo in yeast cells (9Martins A. Shuman S. Virology. 2002; 304: 167-175Crossref PubMed Scopus (6) Google Scholar). The availability of biochemical and genetic readouts of BVP activity has prompted an extensive mutational analysis of BVP, whereby amino acids essential for triphosphatase activity in vitro and in vivo were identified initially by alanine scanning (10Martins A. Shuman S. J. Biol. Chem. 2000; 275: 35070-35076Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), after which structure-activity relationships at the essential residues were determined via conservative substitutions (11Martins A. Shuman S. Biochemistry. 2002; 41: 13403-13409Crossref PubMed Scopus (5) Google Scholar). What is lacking is a structural explanation for the mutational effects based on atomic interactions with substrates, intermediates, or products, as well as a structural framework for the unique specificity of BVP for phosphoanhydrides and the broad specificity of BVP for triphosphate and diphosphate ends versus the more stringent specificity of the bifunctional capping enzymes for triphosphate ends. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 Å resolution, which provides new insights into these issues. Purification and Crystallization of BVP—BVP was produced as an N-terminal His10-BVP fusion in Escherichia coli BL21(DE3)pLysS cells and purified by nickel-agarose chromatography as described previously (11Martins A. Shuman S. Biochemistry. 2002; 41: 13403-13409Crossref PubMed Scopus (5) Google Scholar). For crystallization, His-tagged BVP was purified further by gel filtration chromatography, dialyzed into 50 mm Tris-HCl (pH 8.0), 0.5 m NaCl, 1 mm EDTA, and 1 mm DTT, and concentrated to 3–4 mg/ml. Attempts to remove the His tag prior to crystallization resulted in protein precipitation, and hence the crystallization trials were done by using the His-tagged protein. Initial crystallization trials of His-tagged BVP at 22 °C in hanging drops equilibrated against 2.0 m sodium/potassium phosphate (pH 7.0) and 0.1 m sodium acetate (pH 4.5) yielded showers of small needle-like crystals. In order to improve crystal quality, the affinity tag was cleaved off during crystallization by including 0.1 m guanidine HCl and trypsin (1:5000 molar ratio of trypsin to BVP) in the original mother liquor prior to setting up the crystallization experiment. In this manner, strongly diffracting crystals of the untagged form of BVP appeared within 1 week and grew as large needles of ∼0.06 × 0.06 × 0.4 mm3. Data Collection and Structure Determination—Prior to data collection, crystals were transferred in a single step to crystallization solution supplemented with 25% glycerol for 1–2 min and then flash-cooled in liquid nitrogen. All data were collected at 100 K using a MAR-CCD detector at beamline 5ID-B at the Advanced Photon Source. Data were integrated using XDS (12Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3217) Google Scholar) and scaled with SCALA (13Project Collaborative Computational Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19733) Google Scholar). The crystals belong to space group P21 with unit cell dimensions of a = 40.9 Å, b = 74.1 Å, c = 105.2 Å, β = 92.4°, and there are three molecules (molecules A–C) in the asymmetric unit. The structure was solved by molecular replacement using the program AMoRe (13Project Collaborative Computational Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19733) Google Scholar). The crystal structure of the RNA triphosphatase domain of the mouse mRNA capping enzyme (Mce1) (3Changela A. Ho C.K. Martins A. Shuman S. Mondragon A. EMBO J. 2001; 20: 2575-2586Crossref PubMed Scopus (79) Google Scholar), with the nonconserved loop and C-terminal regions removed, was used as the search model. A clear solution for the positions of all three monomers was found with an R-factor of 47.5% and a correlation coefficient of 0.388. An initial model was built using an electron density map calculated to 2.5 Å resolution with improved phases obtained from prime-and-switch phasing in RESOLVE (14Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (432) Google Scholar). Manual model building was carried out in O (15Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar) and alternated with cycles of simulated annealing and positional and individual temperature factor refinement in CNS (16Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar). Two strong Fo – Fc difference density peaks for phosphate ions were found at the active site of each monomer. In molecule B, additional electron density adjacent to one of the phosphates (site 2) was assigned as a second phosphate with low occupancy. It is not clear whether these two phosphates are part of a diphosphate molecule or not, and they were treated as two separate ions. Once most of the model had been built, the resolution was extended using data to 1.5 Å resolution obtained from a crystal grown in the presence of 5 mm ATP. No ATP was observed in the electron density maps. Subsequent refinement was performed with REFMAC5 (17Murshudov G. Vagin A. Dodson E. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13818) Google Scholar). Molecules A and B are intact and contain residues 1–168, whereas in molecule C residues 1 and 34–41 are disordered. Additionally, in molecule B there is electron density for an N-terminal histidine residue encoded by the expression vector. Molecules A and B exhibit lower average temperature factors (average B-factor ∼12–14 Å2) than molecule C (average B-factor = 22 Å2). The final model has an Rfree = 19.4% and R-factor = 16.5% and includes 576 water molecules and 7 phosphate ions. 99.5% of all residues are found within the most favored or allowed regions in the Ramachandran plot with only 2 residues (Gln-88 in the A and C molecules) in disallowed regions. Data collection and refinement statistics are summarized in Table I.Table IData collection and refinement statisticsData collectionWavelength (Å)0.9479Cell dimensions (Å) (P21)a = 40.9 Å, b = 74.1 Å, c = 105.2 Å, β = 92.4°Resolution (Å)1.50Measured reflections424,098Unique reflections97,263Completeness (%)aNumbers in parentheses represent values in the highest resolution shell (1.50-1.54 Å).97.1 (90.4)Rsym (%)aNumbers in parentheses represent values in the highest resolution shell (1.50-1.54 Å).,bRsym = Σ|I — 〈I〉|/ΣI, where I indicates observed intensity, and 〈I〉 = average intensity obtained from multiple measurements.6.6 (25.0)Rmeas (%)aNumbers in parentheses represent values in the highest resolution shell (1.50-1.54 Å).,cRmeas as defined by Ref. 37.7.3 (30.2)Redundancy4.4 (2.8)I/σ(I)6.8 (2.7)RefinementResolution (Å)20.0-1.50No. reflectionsWorking set/test set92,343/4867R-factordR-factor = Σ∥Fo| — |Fc∥/Σ|Fo|, where |Fo| indicates observed structure factor amplitude, and |Fc| indicates calculated structure factor amplitude.16.5RfreeeRfree is the R-factor based on 5% of the data excluded from refinement.19.4No. of protein atoms4073No. of water molecules567No. of ions7R.m.s.d.Bond lengths (Å)0.01Bond angles (°)1.2Average B-factor (Å2):Main chain15.1Side chain17.4Solvent30.3a Numbers in parentheses represent values in the highest resolution shell (1.50-1.54 Å).b Rsym = Σ|I — 〈I〉|/ΣI, where I indicates observed intensity, and 〈I〉 = average intensity obtained from multiple measurements.c Rmeas as defined by Ref. 37Diederichs K. Karplus P.A. Nat. Struct. Biol. 1997; 4: 269-275Crossref PubMed Scopus (778) Google Scholar.d R-factor = Σ∥Fo| — |Fc∥/Σ|Fo|, where |Fo| indicates observed structure factor amplitude, and |Fc| indicates calculated structure factor amplitude.e Rfree is the R-factor based on 5% of the data excluded from refinement. Open table in a new tab Figures were generated using MOLSCRIPT (18Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), RASTER3D (19Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar), GRASP (20Nicholls A. Sharp K.A. Honig B.H. Proteins Struct. Funct. Genet. 1991; 11: 281-286Crossref PubMed Scopus (5314) Google Scholar), and SETOR and SETORPLOT (21Evans S.V. J. Mol. Graphics. 1993; 11: 134-138Crossref PubMed Scopus (1249) Google Scholar). Coordinates and structure factors for BVP have been deposited in the Protein Data Bank under the accession code 1YN9. Purification of Native BVP—In order to produce soluble, His tag-free BVP, the BVP gene was amplified by PCR from pET16b-BVP plasmid (6Gross C.H. Shuman S. J. Virol. 1998; 72: 7057-7063Crossref PubMed Google Scholar) using a sense primer designed to introduce a BamHI restriction site immediately upstream of the start codon. The PCR product was digested with BamHI and inserted into the BamHI site of the vector pET28-His-Smt3 to generate the plasmid pET-His-Smt3-BVP, which encodes BVP fused to an N-terminal tag consisting of a His6 leader peptide followed by the 98-amino acid Saccharomyces cerevisiae Smt3 protein and a single serine. (Smt3 is the yeast orthologue of the small ubiquitin-like modifier SUMO.) The pET-His-Smt3-BVP plasmid was transformed into E. coli BL21(DE3)pLysS. A 1-liter bacterial culture amplified from a single transformant was grown at 37 °C in Luria-Bertani medium containing 50 μg/ml kanamycin and 30 μg/ml chloramphenicol until A600 reached ∼0.6. Expression was induced by adding isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 0.1 mm. The culture was then incubated at 30 °C for 4 h with continuous shaking. Cells were harvested by centrifugation, and the recombinant His6-Smt3-BVP protein was purified from the soluble bacterial extract by nickel-agarose chromatography as described previously (11Martins A. Shuman S. Biochemistry. 2002; 41: 13403-13409Crossref PubMed Scopus (5) Google Scholar). The 33-kDa His6-Smt3-BVP polypeptide was recovered in the 50 and 200 mm imidazole eluate fractions; the yield was ∼18 mg. The enzyme preparation was dialyzed against buffer B (50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 10% glycerol). The His6-Smt3 tag was removed by proteolytic cleavage with the S. cerevisiae Smt3-specific protease Ulp1 (22Mossessova E. Lima C.D. Mol. Cell. 2000; 5: 865-876Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar). Briefly, a reaction mixture (560 μl) containing 50 mm Tris-HCl (pH 8.0), 1 mm DTT, 350 mm NaCl, 9% glycerol, 1.5 mg of His6-Smt3-BVP, and 1.5 μg of His6Ulp1-NΔ403 was incubated on ice for 30 min. To separate the cleaved His-Smt3 tag from native BVP, the digest was applied to a 0.2-ml column of nickel-NTA resin (Qiagen) that had been equilibrated with buffer C (50 mm Tris-HCl (pH 8.0), 300 mm NaCl, 10% glycerol). The 19-kDa native BVP protein was recovered in the flow-through and wash fractions, whereas His-Smt3 was retained on the resin and was eluted with 0.2 m imidazole. BVP concentrations were determined by SDS-PAGE analysis of serial dilutions of the BVP preparations in parallel with serial dilutions of a BSA standard. The gels were stained with Coomassie Blue, and the staining intensities of the BVP and BSA polypeptides were quantified using a digital imaging and analysis system from Alpha Innotech Corp. Velocity Sedimentation—Aliquots (100 μg) of the His10-BVP and native BVP preparations were applied to 5.0 ml of 15–30% glycerol gradients containing 50 mm Tris-HCl (pH 8.0), 0.3 m NaCl, 1 mm EDTA, 1 mm DTT, 0.1% Triton X-100. The gradients were centrifuged in an SW50i rotor at 50,000 rpm for 18 h at 4 °C. Protein standards catalase (75 μg), BSA (75 μg), and cytochrome c (75 μg) were sedimented in a parallel gradient. Fractions (21 drops each) were collected from the bottoms of the tubes. Aliquots (10 μl) of even numbered gradient fractions were analyzed by SDS-PAGE. Aliquots of the fractions were assayed for triphosphatase activity as specified in the figure legend. Structure Determination of Baculovirus Phosphatase—Initial crystallization trials of His-tagged BVP yielded small crystals of the fusion protein that were unsuitable for diffraction studies. In order to improve crystal quality, attempts were made to remove the His10 tag from BVP in solution using various proteases, but all trials resulted in protein precipitation. However, strongly diffracting crystals could be grown by including small amounts of trypsin in the original mother liquor prior to setting up the crystallization experiment. SDS-PAGE analysis of crystals grown in the presence of trypsin suggested that most or all of the affinity tag had been cleaved off during crystallization, which was later confirmed by the crystal structure. Because of solubility problems and because we could obtain excellent crystals using the above described procedure, no attempts to crystallize BVP using a tag-free version of the protein were done. The structure of BVP was determined by molecular replacement using the structure of the RNA triphosphatase domain of mammalian capping enzyme (3Changela A. Ho C.K. Martins A. Shuman S. Mondragon A. EMBO J. 2001; 20: 2575-2586Crossref PubMed Scopus (79) Google Scholar) as a search model. The final model containing three BVP molecules (A–C) in the asymmetric unit was refined to 1.5 Å resolution with an Rfree = 19.4% and R-factor = 16.5% (Table I). All three monomers are nearly identical in conformation, with root mean square deviation (r.m.s.d.) values ranging between 0.3 Å and 0.4 Å for all C-α atoms. Molecules A and B are complete (residues 1–168 are visualized; Fig. 1A), whereas in molecule C the N-terminal methionine and surface loop residues 34–41 are disordered. Within the asymmetric unit, molecules B and C are each related to molecule A by local 2-fold symmetry. The occurrence of three protomers in the asymmetric unit of the crystal derived from BVP that was trypsinized in situ to remove the tag prompted us to compare the quaternary structures of His10-tagged BVP and native tag-free BVP. Zonal velocity sedimentation analysis of recombinant His10-tagged BVP revealed two distinct populations: a slow migrating species sedimenting between BSA (68 kDa) and cytochrome c (13 kDa) that we presumed was a BVP monomer, and a fast migrating species that sedimented on the light side of catalase (248 kDa) that could represent an octameric or higher order oligomeric complex (Fig. 2A). Both components possessed ATP phosphohydrolase activity (Fig. 2A). To obtain tag-free BVP, we first produced BVP in bacteria as a His6-Smt3 fusion protein, purified the fusion protein by nickel-agarose chromatography, removed the His10-Smt3 tag by digesting the protein with the Smt3-specific protease Ulp1, and then isolated the “native” BVP protein free of His6-Smt3 by passing the digest over a nickel-agarose column and recovering native BVP in the flow-through fraction. Native BVP sedimented as a single catalytically active monomeric component, with no detectable oligomeric forms (Fig. 2B). We surmised the following: (i) native BVP is monomeric; (ii) higher order oligomerization is induced by the His10 tag; and (iii) the arrangement of BVP molecules in the crystal structure does not reflect a biologically relevant oligomerization state. Although the structure presented here was derived from data obtained from a BVP crystal grown in the presence of ATP, no electron density was observed for an ATP molecule. Instead, strong density was observed in the Fo – Fc electron density map for two well ordered phosphate ions in the active site region of each monomer (Fig. 3A), which were likely derived from the 2 m sodium/potassium phosphate crystallization solution. Although the catalytic pockets in molecules A and B were blocked due to crystal packing interactions, the active site in molecule C was solvent-accessible (Fig. 4,B--D). Extensive cocrystallization trials and crystal soaks in the presence of excess ATP and other substrate analogues failed to replace the bound phosphates with alternative ligands.Fig. 4A, surface representation of the active site pocket with bound phosphate ions. A close-up view of the active site region in BVP is shown as a surface rendering colored by electrostatic potential. Blue and red regions correspond to positively and negatively charged areas, respectively. The phosphate at site 1 binds deep in the positively charged catalytic pocket, whereas the site 2 phosphate ion sits in a neighboring hollow. The approximate locations of the catalytic cysteine and other charged residues that are ideally positioned to interact with a triphosphate moiety are indicated. B–D, comparison of crystal packing interactions at each BVP monomer active site. Molecules A–C are colored red, green, and purple, respectively, and neighboring symmetry-related molecules and their residues are depicted in gray. The P-loop is highlighted in blue with catalytic residues Cys-119 and Arg-125 shown as ball-and-stick, and the bound phosphates are colored magenta.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Overview of the BVP Structure, a Cysteine Phosphatase Fold—BVP is a single domain protein consisting of a central, twisted, five-stranded β sheet surrounded by six α helices, two on one side and four on the other side (Fig. 1A). The conserved phosphate-binding P-loop motif connects the β5 strand and the α6 helix. The compact α/β domain structure adopted by BVP is nearly identical in topology to the RNA triphosphatase domain of Mce1 (Fig. 1B) and resembles the conserved catalytic core found in other cysteine phosphatases. Structural comparisons using the DALI server (23Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3556) Google Scholar) indicated that BVP shares the most similarity to Mce1, the phosphoinositide phosphatase PTEN (24Lee J.O. Yang H. Georgescu M.M. Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar), and several dual specificity protein phosphatases, including the kinase-associated phosphatase KAP (25Song H. Hanlon N. Brown N.R. Noble M.E. Johnson L.N. Barford D. Mol. Cell. 2001; 7: 615-626Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), human Cdc14B (26Gray C.H. Good V.M. Tonks N.K. Barford D. EMBO J. 2003; 22: 3524-3535Crossref PubMed Scopus (116) Google Scholar), and VHR (27Yuvaniyama J. Denu J.M. Dixon J.E. Saper M.A. Science. 1996; 272: 1328-1331Crossref PubMed Scopus (303) Google Scholar). The conformation of the P-loop residues 118HCTHGINRT126 and the overall active site environment are identical in all three BVP monomers. The Cys-119 nucleophile extended from the bottom of the substrate-binding pocket toward one of the bound phosphate ions (site 1) (Fig. 3B). Other conserved P-loop residues and residues from neighboring surface loops lent an overall positive surface potential, providing an attractive docking site for either a diphosphate or triphosphate substrate. Positively charged, shallow grooves extending from the catalytic pocket indicate additional regions possibly involved in substrate binding (Fig. 4A). In other cysteine phosphatases, the depth of the active site pocket was a critical factor in defining substrate specificity (24Lee J.O. Yang H. Georgescu M.M. Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar, 27Yuvaniyama J. Denu J.M. Dixon J.E. Saper M.A. Science. 1996; 272: 1328-1331Crossref PubMed Scopus (303) Google Scholar, 28Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (551) Google Scholar). It was postulated that BVP would use a shallower pocket to act on diphosphate-terminated substrates that cannot be accommodated by the deep pocket in Mce1 (3Changela A. Ho C.K. Martins A. Shuman S. Mondragon A. EMBO J. 2001; 20: 2575-2586Crossref PubMed Scopus (79) Google Scholar). However, the depth of the active site pocket in BVP was ∼8 Å, making it very similar to Mce1 and suggesting that other factors dictate substrate specificity in the RNA-specific cysteine phosphatase family (see below). Two Phosphate-binding Sites—One phosphate ion (site 1) binds deep in the catalytic pocket and is coordinated by conserved P-loop residues and main chain amide groups (Fig. 3B). The phosphate is directly above the catalytic Cys-119 at a sulfur to phosphorous distance of ∼3.8 Å, indicative of a noncovalent interaction. The phosphate in site 1 exemplifies the binding mode expected for the terminal phosphate of an RNA substrate prior to formation of the covalent cysteinyl-phosphoenzyme intermediate or a product complex generated after hydrolysis of the phosphoenzyme. The invariant P-loop residue, Arg-125, makes a bidentate contact with two of the phosphate oxygens at site 1, consistent with the role proposed for this residue in substrate binding and transition-state stabilization (11Martins A. Shuman S. Biochemistry. 2002; 41: 13403-13409Crossref PubMed Scopus (5) Google Scholar). The key contribution of the bidentate contact is underscored by previous findings that mutation of Arg-125 to alanine abolished BVP triphosphatase activity in vitro and in vivo, and conservative substitution by lysine did not rescue function (11Martins A. Shuman S. Biochemistry. 2002; 41: 13403-13409Crossref PubMed Scopus (5) Google Scholar). P-loop residues His-121 and Asn-124 make additional hydrogen bonds to the site 1 phosphate. The backbone amide and N-δ of" @default.
• W1983257557 created "2016-06-24" @default.
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• W1983257557 creator A5007952303 @default.
• W1983257557 creator A5014112082 @default.
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• W1983257557 date "2005-05-01" @default.
• W1983257557 modified "2023-09-27" @default.
• W1983257557 title "Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate" @default.
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