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• W2031659637 abstract "AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-Å resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded β-sheet flanked by three α-helices on either side, with the active site located above the carboxyl-terminal edge of the β-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases. AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-Å resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded β-sheet flanked by three α-helices on either side, with the active site located above the carboxyl-terminal edge of the β-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases. Francisella tularensis is a highly infectious intracellular bacterial pathogen and the cause of tularemia (1Oyston P.C. Sjostedt A. Titball R.W. Nat. Rev. Microbiol. 2004; 2: 967-978Crossref PubMed Scopus (411) Google Scholar). The organism can be isolated from numerous rodent hosts and arthropod vectors, readily grown in broth culture, and mechanically aerosolized. It is one of the most infectious pathogenic agents known, requiring fewer than ten organisms to establish infection (1Oyston P.C. Sjostedt A. Titball R.W. Nat. Rev. Microbiol. 2004; 2: 967-978Crossref PubMed Scopus (411) Google Scholar). Inhalation of aerosolized F. tularensis can result in pneumonic tularemia (2Dennis D.T. Inglesby T.V. Henderson D.A. Bartlett J.G. Ascher M.S. Eitzen E. Fine A.D. Friedlander A.M. Hauer J. Layton M. Lillibridge S.R. McDade J.E. Osterholm M.T. O'Toole T. Parker G. Perl T.M. Russell P.K. Tonat K. J. Am. Med. Assoc. 2001; 285: 2763-2773Crossref PubMed Scopus (1169) Google Scholar), which has a case fatality rate of up to 30% if untreated (1Oyston P.C. Sjostedt A. Titball R.W. Nat. Rev. Microbiol. 2004; 2: 967-978Crossref PubMed Scopus (411) Google Scholar). The U.S. Centers for Disease Control and Prevention considers F. tularensis to be a Category A bioterrorism agent, which has led to renewed interest in identifying genes and pathways that underlie virulence to facilitate development of new antimicrobial drugs and vaccines (1Oyston P.C. Sjostedt A. Titball R.W. Nat. Rev. Microbiol. 2004; 2: 967-978Crossref PubMed Scopus (411) Google Scholar, 3Sjostedt A. Curr. Opin. Microbiol. 2003; 6: 66-71Crossref PubMed Scopus (77) Google Scholar, 4Larsson P. Oyston P.C. Chain P. Chu M.C. Duffield M. Fuxelius H.H. Garcia E. Halltorp G. Johansson D. Isherwood K.E. Karp P.D. Larsson E. Liu Y. Michell S. Prior J. Prior R. Malfatti S. Sjostedt A. Svensson K. Thompson N. Vergez L. Wagg J.K. Wren B.W. Lindler L.E. Andersson S.G. Forsman M. Titball R.W. Nat. Genet. 2005; 37: 153-159Crossref PubMed Scopus (380) Google Scholar). Acid phosphatase A from F. tularensis (AcpA) 2The abbreviations used are: AcpA, acid phosphatase A from F. tularensis; ACP, acid phosphatase; pNPP, p-nitrophenylphosphate; pNPPC, p-nitrophenylphosphorylcholine; PLC, phospholipase C; PlcH, hemolytic PLC from P. aeruginosa; ASA, human arylsulfatase A; AlkP, alkaline phosphatase. is a highly expressed 57-kDa polyspecific periplasmic acid phosphatase (ACP) (5Reilly T.J. Baron G.S. Nano F.E. Kuhlenschmidt M.S. J. Biol. Chem. 1996; 271: 10973-10983Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). AcpA hydrolyzes a variety of substrates, including p-nitrophenylphosphate (pNPP), p-nitrophenylphosphorylcholine (pNPPC), peptides containing phosphotyrosine, inositol phosphates, AMP, ATP, fructose 1,6-bisphosphate, glucose and fructose 6-phosphates, NADP+, and ribose 5-phosphate (5Reilly T.J. Baron G.S. Nano F.E. Kuhlenschmidt M.S. J. Biol. Chem. 1996; 271: 10973-10983Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 6Reilly T.J. Felts R.L. Henzl M.T. Calcutt M.J. Tanner J.J. Protein Expr. Purif. 2006; 45: 132-141Crossref PubMed Scopus (23) Google Scholar). The enzyme is inhibited by the metal oxyanions orthovanadate, molybdate, and tungstate. Based on amino acid sequence analysis, AcpA is distinct from histidine ACPs (7Van Etten R.L. Davidson R. Stevis P.E. MacArthur H. Moore D.L. J. Biol. Chem. 1991; 266: 2313-2319Abstract Full Text PDF PubMed Google Scholar) and purple ACPs (8Vincent J.B. Crowder M.W. Phosphatases in Cell Metabolism and Signal Transduction: Structure, Function, and Mechanism of Action. R.G. Landes Company, Austin1995Google Scholar), as well as class A, B, and C bacterial nonspecific ACPs (9Rossolini G.M. Schippa S. Riccio M.L. Berlutti F. Macaskie L.E. Thaller M.C. Cell. Mol. Life Sci. 1998; 54: 833-850Crossref PubMed Scopus (134) Google Scholar). Purified AcpA inhibits the respiratory burst of stimulated neutrophils, which suggests that AcpA helps the pathogen elude the host oxidative defense system during the initial stages of macrophage infection (5Reilly T.J. Baron G.S. Nano F.E. Kuhlenschmidt M.S. J. Biol. Chem. 1996; 271: 10973-10983Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Furthermore, proteomics studies have shown that AcpA is expressed at a higher level in virulent F. tularensis strains compared with the nonvirulent vaccine strain (10Hernychova L. Stulik J. Halada P. Macela A. Kroca M. Johansson T. Malina M. Proteomics. 2001; 1: 508-515Crossref PubMed Google Scholar). Most recently, it has been shown that a mutant strain of F. tularensis subspecies novicida lacking a functional acpA gene is less virulent in mice than the wild-type strain due to a defect in phagosomal escape. 3J. S. Gunn, personal communication. Thus, AcpA appears to be important for survival of the microbe at two critical junctures of infection: colonization and intracellular survival. Amino acid sequence alignments show that AcpA belongs to a superfamily of bacterial enzymes that includes ACPs and phospholipases C (PLCs) from a variety of microbial pathogens, including Pseudomonas aeruginosa, Mycobacterium tuberculosis, Bordetella pertussis, and several Burkholderia species (11Stonehouse M.J. Cota-Gomez A. Parker S.K. Martin W.E. Hankin J.A. Murphy R.C. Chen W. Lim K.B. Hackett M. Vasil A.I. Vasil M.L. Mol. Microbiol. 2002; 46: 661-676Crossref PubMed Scopus (79) Google Scholar). AcpA is the only characterized enzyme from the ACP branch of the superfamily. PLCs from this superfamily are important virulence factors in P. aeruginosa (12Ostroff R.M. Wretlind B. Vasil M.L. Infect. Immun. 1989; 57: 1369-1373Crossref PubMed Google Scholar) and M. tuberculosis (13Raynaud C. Guilhot C. Rauzier J. Bordat Y. Pelicic V. Manganelli R. Smith I. Gicquel B. Jackson M. Mol. Microbiol. 2002; 45: 203-217Crossref PubMed Scopus (158) Google Scholar) infections, with the hemolytic PLC from P. aeruginosa (PlcH) being the best characterized example from the PLC branch of the superfamily (14Ostroff R.M. Vasil A.I. Vasil M.L. J. Bacteriol. 1990; 172: 5915-5923Crossref PubMed Scopus (128) Google Scholar). PlcH is particularly interesting, because it is a multifunctional enzyme that displays sphingomyelin synthase activity in addition to PLC activity (15Luberto C. Stonehouse M.J. Collins E.A. Marchesini N. El-Bawab S. Vasil A.I. Vasil M.L. Hannun Y.A. J. Biol. Chem. 2003; 278: 32733-32743Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). PLCs of the AcpA/PlcH superfamily share no sequence homology with the well studied zinc metallophospholipases Clostridium perfringens α-toxin and Bacillus cereus phosphatidylcholine-preferring PLC (16Titball R.W. Microbiol. Rev. 1993; 57: 347-366Crossref PubMed Google Scholar), which suggests that PLCs of the AcpA/PlcH superfamily have a novel, and as yet uncharacterized, catalytic mechanism. To gain insights into the structural basis of the catalytic activity of enzymes of the AcpA/PlcH superfamily, we have determined the crystal structure of AcpA bound to the competitive inhibitor orthovanadate. Crystallization and X-ray Diffraction Data Collection—Expression of recombinant F. tularensis AcpA in Escherichia coli, protein purification, and the growth of three different crystal forms were described previously (17Felts R.L. Reilly T.J. Tanner J.J. Biochim. Biophys. Acta. 2005; 1752: 107-110Crossref PubMed Scopus (9) Google Scholar). Structure determination utilized crystal form III, which was obtained by incubating the enzyme with the competitive inhibitor sodium orthovanadate (Na3VO4, 5 mm) prior to crystallization and using polyethylene glycol 1500 as the precipitating agent (17Felts R.L. Reilly T.J. Tanner J.J. Biochim. Biophys. Acta. 2005; 1752: 107-110Crossref PubMed Scopus (9) Google Scholar). These crystals have space group C2221 with unit cell dimensions a = 112 Å, b = 144 Å, c = 124 Å, two molecules per asymmetric unit, and 43% solvent content. The derivative used for phasing was produced by soaking an AcpA/orthovanadate crystal in 40 mm Sm(C2H3O2)3 for 10 min. Diffraction data extending to 2.4-Å resolution were collected from the Sm derivative at Advanced Photon Source beamline 19-ID using λ = 1.6531 Å, which corresponds to an energy between the L-I and L-II absorption edges of Sm. Data processing was done with HKL2000 (Table 1). Anomalous difference Patterson maps showed several strong features on the u = 0 Harker section.TABLE 1Data collection and refinement statisticsOrthovanadate (high energy)Orthovanadate (low energy)Sm derivativeWavelength (Å)1.12711.7401.6531Space groupC2221C2221C2221Unit cell dimensions (Å)a = 112.1, b = 144.4, c = 123.9a = 111.2, b = 142.6, c = 125.6a = 112.2, b = 144.2, c = 123.9Diffraction resolution (Å)50-1.75 (1.81-1.75)50-2.20 (2.28-2.20)50-2.40 (2.48-2.40)No. of observations397,516295,644572,236No. of unique reflections98,13751,19539,618Redundancy4.1 (4.0)5.8 (4.9)14.4 (13.4)Completeness (%)97.2 (94.9)99.9 (99.9)99.9 (99.9)Average 1/σ26.6 (2.2)16.8 (3.4)38.6 (23.8)Rsym (I)0.047 (0.357)0.098 (0.443)0.065 (0.118)No. of non-hydrogen atoms8,182No. of residues in chain A481No. of residues in chain B472No. of water molecules550Rcryst0.198 (0.244)RfreeaA 5% random test set.0.231 (0.291)Root mean square deviationbCompared to the Engh and Huber force field (50).Bond lengths (Å)0.012Bond angles (deg.)1.7Ramachandran plotcThe Ramachandran plot was generated with RAMPAGE (51).Favored (%)96.4Allowed (%)3.1Average B-factors (Å2)Protein25Orthovanadate27Active site metal ion16Water28PDB accession code2D1Ga A 5% random test set.b Compared to the Engh and Huber force field (50Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2545) Google Scholar).c The Ramachandran plot was generated with RAMPAGE (51Lovell S.C. Davis I.W. Arendall 3rd, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3859) Google Scholar). Open table in a new tab The data set used for phase extension and refinement calculations at 1.75-Å resolution was collected from an AcpA/orthovanadate crystal at Advanced Light Source beamline 8.3.1. A second data set, which was used for anomalous difference Fourier analysis of the active site metal center, was collected from another AcpA/orthovanadate crystal at beamline 8.3.1. This data set was collected at low energy (λ = 1.74 Å) to enhance the anomalous signal of the metal ion. Both data sets were processed with HKL2000 (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). See Table 1 for a summary of data processing statistics. Phasing and Refinement Calculations—The structure was solved using single wavelength anomalous diffraction phasing. SnB (19Weeks C.M. Miller R. J. Appl. Crystallogr. 1999; 32: 120-124Crossref Scopus (384) Google Scholar) was used to identify a 10-atom anomalous constellation for the Sm derivative, which was input to SHARP (20Bricogne G. Vonrhein C. Flensburg C. Schiltz M. Paciorek W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 2023-2030Crossref PubMed Scopus (554) Google Scholar) for single wavelength anomalous diffraction phase calculations and solvent flattening. The resulting SHARP phases had a figure of merit of 0.84 for reflections to 2.4-Å resolution. An electron density map calculated from the SHARP phases clearly showed features resembling protein secondary structural elements. A partial backbone tracing consisting of a few α-helices and β-strands was obtained with the automated model building program MAID (21Levitt D.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1013-1019Crossref PubMed Scopus (117) Google Scholar). The MAID tracing was used to determine the noncrystallographic symmetry transformation relating the two protein molecules in the asymmetric unit. In preparation for noncrystallographic symmetry averaging, the programs MAMA (22Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 941-944Crossref PubMed Scopus (156) Google Scholar) and CNS (23Brünger 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 (16967) Google Scholar) were used to create a mask that covered one of the molecules in the asymmetric unit. The SHARP phases were then improved and extended to 1.75-Å resolution with 2-fold noncrystallographic symmetry averaging and solvent flipping in CNS. The 1.75-Å resolution density-modified phases were input to ARP/wARP (24Morris R.J. Perrakis A. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 968-975Crossref PubMed Scopus (221) Google Scholar) for automated electron density map interpretation. The best model from ARP/wARP included the backbone for 97% of the expected residues in the asymmetric unit and 83% of the expected side chains. The model was improved with several rounds of model building in COOT (25Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23375) Google Scholar) followed by refinement with REFMAC5 (26Winn M.D. Murshudov G.N. Papiz M.Z. Methods Enzymol. 2003; 374: 300-321Crossref PubMed Scopus (681) Google Scholar). Refinement statistics are listed in Table 1. The asymmetric unit includes 953 amino acid residues belonging to two AcpA molecules (chains labeled A and B). The following sections of the polypeptide chains are disordered: A1-A4, A15-A18, A490-A498, B1-B5, B12-B18, B129-B137, and B490-B494. The root mean square difference between chains A and B is 0.27 Å for Cα atoms and 0.55 Å for all atoms, which indicates that the two chains have nearly identical conformations. Each AcpA molecule contains one orthovanadate ion (HVO42-) and one metal ion bound in the active site. The metal ion was modeled as Ca2+ for purposes of crystallographic refinement but appears with atom name X1 and residue name UNK in the coordinate file deposited in the Protein Data Bank (PDB (27Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27537) Google Scholar)) to indicate that the identity of the metal is unknown at this time. The solvent structure includes 550 ordered water molecules and 4 bound polyethylene glycol fragments. There is also a decavanadate ion (V10O286-) bound in a crystal contact region, where it interacts with the carboxyl-terminal histidine affinity tag of one of the AcpA molecules. Coordinates and structure factor amplitudes have been deposited in the PDB under accession code 2D1G. Site-directed Mutagenesis and Activity Assays—AcpA mutant Ser-175 → Ala was generated using the QuikChange mutagenesis kit (Stratagene), and the mutation was verified by DNA sequencing. The mutant enzyme was expressed and purified using methods employed for AcpA (17Felts R.L. Reilly T.J. Tanner J.J. Biochim. Biophys. Acta. 2005; 1752: 107-110Crossref PubMed Scopus (9) Google Scholar). SDS-PAGE analysis showed that Ser-175 → Ala had the expected molecular weight, and Western blots using rabbit anti-AcpA polyclonal and anti-His tag antibodies were positive. Enzymatic activities of AcpA and Ser-175 → Ala were measured using a discontinuous colorimetric assay with pNPP and pNPPC as substrates (6Reilly T.J. Felts R.L. Henzl M.T. Calcutt M.J. Tanner J.J. Protein Expr. Purif. 2006; 45: 132-141Crossref PubMed Scopus (23) Google Scholar). Overall Structure of AcpA—The structure of AcpA comprises three domains and has approximate dimensions of 60 Å × 48 Å × 66 Å. The core domain is a highly twisted, 8-stranded β-sheet flanked by three α-helices on either side (Fig. 1A). The strand order of the β-sheet is 12, 2, 11, 10, 1, 9, 3, then 8, with all but strand 11 in parallel (Fig. 2). There are two smaller domains located above the carboxyl-terminal edge of the 8-stranded β-sheet. One of these small domains consists of residues 47-147 and features four short α-helices (labeled A-D) connected by rather long loops (Fig. 1, blue domain). This domain has a disulfide bond linking Cys-102 and Cys-138 (Figs. 1A and 2). As discussed below, this domain forms part of the dimer interface. The other small domain (residues 258-283) consists of a pair of 2-stranded anti-parallel β-sheets (β4-β7), which resembles a flap (Fig. 1, orange domain), and there is a disulfide bond that links Cys-269 of this domain to Cys-216 of the β-sheet core domain.FIGURE 2Secondary structure topology diagram of AcpA. α-Helices are shown as rectangles labeled A-J, and β-strands are shown as arrows numbered 1-12. Cys residues are represented in yellow with disulfides bridges shown as dashed lines. The green boxes denote active site residues, with red numbering for residues coordinating to the bound metal and blue numbering for residues interacting with the vanadate inhibitor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Purified AcpA forms an apparent dimer according to analytical ultracentrifugation and gel-filtration chromatography data (6Reilly T.J. Felts R.L. Henzl M.T. Calcutt M.J. Tanner J.J. Protein Expr. Purif. 2006; 45: 132-141Crossref PubMed Scopus (23) Google Scholar). The two proteins chosen for the asymmetric unit (Fig. 3A) form the largest intermolecular surface between any two proteins in the crystal lattice, based on analysis with PISA (28Krissinel E. Henrick K. Berthold M.R. Glen R. Diederichs K. Kohlbacher O. Fischer I. Computational Life Sciences: First International Symposium. Springer, Konstanz, Germany2005: 163-174Google Scholar). This interface buries 2398 Å2 of surface area, whereas the next largest interface buries only 932 Å2 of surface area. Furthermore, this interface had the highest possible PISA complexation significance score (1.0), compared with 0 for all other possible interfaces. It is concluded that the pair of protein molecules in the asymmetric unit represents the AcpA dimer in solution. The small helical domain (residues 47-147) and the β12 face of the β-sheet core domain form the dimer interface (Fig. 3A). Secondary structural elements involved in dimerization include αB and its adjacent loops (residues 73-87), the loop following αC (residues 116-119), a 10-residue section of the loop connecting β10 and β11 (394-404), β12 and its adjacent loops (residues 425-433), and residues in a loop near the carboxyl terminus (residues 459-466). Together, these residues form a flat surface (Fig. 3B) that spans 40 Å in one direction and 30 Å in the other. The dimer interface is highly hydrophilic, and hydrogen bonding appears to play a major role in dimer stability. There are 14 direct inter-subunit hydrogen bonds (Table 2) but no ion pairs. Hydrogen-bonding side chains in the interface include Asn-74, Thr-79, Gln-81, Asn-116, Gln-401, Asp-404, and Tyr-428. Note that the intersubunit hydrogen bonds display 2-fold symmetry (Table 2). In addition, there are 16 interfacial water molecules that mediate 20 intersubunit hydrogen bonds (Fig. 3C). As with the inter-subunit hydrogen bonds, the 16 bridging water molecules obey the 2-fold symmetry of the dimer (Fig. 3C). Although hydrogen bonding is prominent in the interface, a few nonpolar residues contribute significant surface area to the interface. For example, Leu-82 packs against Leu-119, whereas Leu-433 from one subunit packs against Leu-433 of the opposite subunit at the centroid of the dimer.TABLE 2Intersubunit hydrogen bondsHydrogen bond partnersDistanceChain AChain BÅAsn-74 (Nδ2)Asp-404 (Oδ1)3.1Asn-74 (N)Asp-404 (Oδ2)3.0Asp-404 (Oδ1)Asn-74 (Nδ2)2.8Asp-404 (Oδ2)Asn-74 (N)3.1Thr-79 (Oγ1)Gly-117 (O)2.6Gly-117 (O)Thr-79 (Oγ1)2.7Gln-81 (Nϵ2)Asn-116 (Oδ1)3.3Asn-116 (Oδ1)Gln-81 (Nϵ2)3.1Gln-401 (Nϵ2)Tyr-428 (OH)3.2Gln-401 (Nϵ2)Val-429 (O)3.0Gln-401 (Oϵ1)His-431 (N)2.8Tyr-428 (OH)Gln-401 (Nϵ2)3.2Val-429 (O)Gln-401 (Nϵ2)3.1His-431 (N)Gln-401 (Oϵ1)2.9 Open table in a new tab Active Site Architecture and Implications for Catalytic Mechanism—The location of the active site was clearly indicated by a strong electron density feature corresponding to the bound orthovanadate inhibitor (Fig. 4A). The active site is located above the carboxyl-terminal edge of the 8-stranded β-sheet near β1 and αF (Fig. 1A). The inhibitor binds in one end of a 12-Å long trough, which is located in a broad, shallow depression formed by residues from all three domains (Fig. 1B). Note that four water molecules are bound in the trough (Fig. 1B). The shape of the trough suggests that it may be involved in binding the leaving group of the substrate. This idea was tested by modeling pNPP in the active site. We found that the nitrophenyl group of pNPP fits edgewise into the trough (water removed) without causing steric clash. Surprisingly, there is a metal ion bound in the active site, based on the observation of a very strong electron density feature that could not be assigned to the protein, inhibitor, or solvent (Fig. 4A). Four lines of evidence suggest that this feature represents a metal ion. First, it is surrounded by an octahedral array of six oxygen ligands (Fig. 4, A and B): Glu-43, Asn-44, Ser-175, Asp-386, Asp-387, and the vanadate inhibitor. Three of the six coordinating ligands are carboxyl groups, which is suggestive of a bound metal ion with a charge of at least +2. Second, the proposed metal site corresponded to the highest peak in an anomalous difference Fourier map calculated from diffraction data collected at low energy (λ = 1.74 Å). The anomalous difference density feature was quite prominent when viewed at the 3.5 σ contour level (Fig. 4A, red cage) and remained visible even at 10 σ. Third, the proposed metal ion site was the second strongest binding site of the Sm derivative used for single wavelength anomalous diffraction phasing. We note that lanthanides readily replace metal ions of protein active/binding sites, including Mg2+, Ca2+, and first row transition metal ions (29Turro C. Fu P.K.-L. Bradley P.M. Sigel A. Sigel H. Metal Ions in Biological Systems. 40. Marcel Dekker, New York2003: 323-353Google Scholar). Fourth, as described in the next section, structural homologs of AcpA, such as arylsulfatase A (ASA) and alkaline phosphatase (AlkP), have metal ions bound in the active site at locations that are structurally analogous to the proposed AcpA metal site. Electron density maps were analyzed to gain insights into the elemental identity of the bound metal ion. The anomalous difference Fourier peak corresponding to the metal ion was much stronger than that of the vanadate (Fig. 4A, red cage), which implies that the metal ion is a stronger anomalous scatterer than the V atom of the inhibitor. This result is consistent with the active site metal ion being a first row transition metal. Also, several simulated annealing refinements were performed against the 1.75-Å data set with different metal ions modeled in the active site. The resulting difference electron density maps (σA-weighted mFo-DFc) suggested that the metal ion has at least the number of electrons of Ca2+ and is more likely a first row transition metal ion. Therefore, the metal ion was conservatively modeled as Ca2+ in the current structure pending further biochemical and analytical studies of the metal content of AcpA. The orthovanadate inhibitor exhibits distorted trigonal bipyramidal geometry and is bound by six side chains and the metal ion (Fig. 4, B and C). The inhibitor axial oxygen atom interacts with His-106 and His-350, whereas the equatorial oxygen atoms bind to Asn-44, His-287, His-288, Asp-208, His-350, and the metal ion. Asp-208 appears to share a proton with the inhibitor. The location of Ser-175 relative to the inhibitor and metal ion suggests that it plays the role of nucleophile that attacks the substrate P atom. The hydroxyl oxygen atom of Ser-175 is 1.8 Å from the metal ion and 2.2 Å from the inhibitor V atom (Fig. 4C). Ser-175 appears to be in an ideal location for backside nucleophilic attack at the substrate P atom. Thus, the active site structure strongly suggests that Ser-175 is the enzyme nucleophile, and the role of the metal ion is to activate Ser-175 for nucleophilic attack (Fig. 5A, step 2). This hypothesis implies formation of a covalent Ser-175-phosphoryl intermediate during catalysis (Fig. 5A, step 2). We engineered the Ser-175 → Ala mutant to test the importance of this residue for catalysis. The mutant exhibited no detectable activity using either pNPP or pNPPC as the substrate even at enzyme concentrations >0.01 mm and substrate concentrations up to 20 mm. Thus, Ser-175 plays an essential role in catalysis, which is consistent with our hypothesis that it is the enzyme nucleophile. Hydrolysis of the Ser-175-phosphoryl intermediate (Fig. 5A, step 3) presumably requires a general base to activate a water molecule. Residues that bind the inhibitor and that are located on the solvent side of the active site are possible candidates for this role. Asp-208 is, perhaps, the most likely candidate, because aspartic acid residues serve as the general base in other phosphatases, such as protein tyrosine phosphatase (30Lohse D.L. Denu J.M. Santoro N. Dixon J.E. Biochemistry. 1997; 36: 4568-4575Crossref PubMed Scopus (158) Google Scholar), and the carboxyl of Asp-208 forms a hydrogen bond (2.8 Å) with a water molecule (Wat-285) in our structure. Comparison to Other Protein Structures—To understand the relationship of AcpA to other phosphatases, we searched the PDB for structural homologs of AcpA using the program DALI (31Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3563) Google Scholar). Surprisingly, the closest homolog was not a phosphatase but was human arylsulfatase A (ASA, PDB code 1AUK, DALI Z-score = 16), followed by phosphoglycerate mutase (PDB code 1EJJ, Z = 15), phosphonoacetate hydrolase (PDB code 1EI6, Z = 10), and E. coli alkaline phosphatase (AlkP, PDB code 1B8J, Z = 9). All four enzymes belong to the AlkP superfamily, which has been described in detail (32Galperin M.Y. Jedrzejas M.J. Proteins. 2001; 45: 318-324Crossref PubMed Scopus (111) Google Scholar). AcpA shares a common β-sheet core domain and active site location with AlkP super-family members. The shared secondary structural elements consist of the middle six strands of the central β-sheet along with the six flanking α-helices (Fig. 6A). We note that DALI did not identify a single ACP with structural similarity to AcpA. Structural homology of AcpA to AlkP enzymes, especially ASA and AlkP, extends to details of the active site. ASA uses a formylglycine (FGly-69) as the nucleophile and binds a single metal ion in the active site (Ca2+ or Mg2+) (33Lukatela G. Krauss N. Theis K. Selmer T. Gieselmann V. von Figura K. Saenger W. Biochemistry. 1998; 37: 3654-3664Crossref PubMed Scopus (270) Google Scholar). The array of met-al-binding ligands in ASA is remarkably similar to that of AcpA (Fig. 6B). In both enzymes, the metal ion has octahedral coordination with three carboxyl groups, an asparagine side chain, the nucleophilic oxygen atom, and the inhibitor/phosphoryl. Recognition of the substrate phosphoryl is also similar in the two enzymes. For example, His-287 an" @default.
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• W2031659637 title "Structure of Francisella tularensis AcpA" @default.
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