Structural and Mutational Analyses of Drp35 from Staphylococcus aureus
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m607340200
ISSN1083-351X
AutoresYoshikazu Tanaka, Kazuya Morikawa, Yu Ohki, Min Yao, Kouhei Tsumoto, Nobuhisa Watanabe, Toshiko Ohta, Isao Tanaka,
Tópico(s)Trace Elements in Health
ResumoDrp35 is a protein induced by cell wall-affecting antibiotics or detergents; it possesses calcium-dependent lactonase activity. To determine the molecular basis of the lactonase activity, we first solved the crystal structures of Drp35 with and without Ca2+; these showed that the molecule has a six-bladed β-propeller structure with two calcium ions bound at the center of the β-propeller and surface region. Mutational analyses of evolutionarily conserved residues revealed that the central calcium-binding site is essential for the enzymatic activity of Drp35. Substitution of some other amino acid residues for the calcium-binding residues demonstrated the critical contributions of Glu48, Asp138, and Asp236 to the enzymatic activity. Differential scanning calorimetric analysis revealed that the loss of activity of E48Q and D236N, but not D138N, was attributed to their inability to hold the calcium ion. Further structural analysis of the D138N mutant indicates that it lacks a water molecule bound to the calcium ion rather than the calcium ion itself. Based on these observations and structural information, a possible catalytic mechanism in which the calcium ion and its binding residues play direct roles was proposed for the lactonase activity of Drp35. Drp35 is a protein induced by cell wall-affecting antibiotics or detergents; it possesses calcium-dependent lactonase activity. To determine the molecular basis of the lactonase activity, we first solved the crystal structures of Drp35 with and without Ca2+; these showed that the molecule has a six-bladed β-propeller structure with two calcium ions bound at the center of the β-propeller and surface region. Mutational analyses of evolutionarily conserved residues revealed that the central calcium-binding site is essential for the enzymatic activity of Drp35. Substitution of some other amino acid residues for the calcium-binding residues demonstrated the critical contributions of Glu48, Asp138, and Asp236 to the enzymatic activity. Differential scanning calorimetric analysis revealed that the loss of activity of E48Q and D236N, but not D138N, was attributed to their inability to hold the calcium ion. Further structural analysis of the D138N mutant indicates that it lacks a water molecule bound to the calcium ion rather than the calcium ion itself. Based on these observations and structural information, a possible catalytic mechanism in which the calcium ion and its binding residues play direct roles was proposed for the lactonase activity of Drp35. Staphylococcus aureus is a major cause of hospital- and community-acquired infections. S. aureus causes serious and fatal diseases, such as toxic shock syndrome or septicemia (1Dinges M.M. Orwin P.M. Schlievert P.M. Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1315) Google Scholar). Moreover, S. aureus has the remarkable and unfortunate feature that it can become readily resistant to antibiotics. Indeed, it has acquired resistance to almost all antibiotics so far, resulting in an increase in incidence of acute hospital-acquired infections (2Hiramatsu K. Aritaka N. Hanaki H. Kawasaki S. Hosoda Y. Hori S. Fukuchi Y. Kobayashi I. Lancet. 1997; 350: 1670-1673Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar). Extensive studies have focused on how S. aureus acquires resistance to antibiotics, and genome sequencing analysis confirmed the existence of many resistance genes acquired by horizontal transfer from other species (3Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1598) Google Scholar). In addition, S. aureus can cope with antibiotic stresses in an adaptive manner through regulation of the expression of many genes (4Kuroda M. Kuroda H. Oshima T. Takeuchi F. Mori H. Hiramatsu K. Mol. Microbiol. 2003; 49: 807-821Crossref PubMed Scopus (393) Google Scholar). Drp35 (a 35-kDa drug-responsive protein) is a cytoplasmic protein originally found to be markedly induced upon exposure of S. aureus to cell wall-affecting antibiotics (5Murakami H. Matsumaru H. Kanamori M. Hayashi H. Ohta T. Biochem. Biophys. Res. Commun. 1999; 264: 348-351Crossref PubMed Scopus (25) Google Scholar). Antibiotic susceptibility experiments using a drp35 defective strain and overexpressing strain of S. aureus revealed that Drp35 is correlated with bacitracin resistance, although it did not show significant changes in minimal inhibitory concentration for β-lactams, glycopeptides, or fosfomycin (6Morikawa K. Hidaka T. Murakami H. Hayashi H. Ohta T. FEMS Microbiol. Lett. 2005; 249: 185-190Crossref PubMed Scopus (8) Google Scholar). Drp35 can also be induced by a variety of detergents, including Nonidet P-40, Triton X-100, SDS, and CHAPS 2The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PON, paraoxonase family protein; MES, 4-morpholineethanesulfonic acid; DSC, differential scanning calorimetry; DFPase, diisopropylfluorophosphatase; r.m.s., root mean square. (6Morikawa K. Hidaka T. Murakami H. Hayashi H. Ohta T. FEMS Microbiol. Lett. 2005; 249: 185-190Crossref PubMed Scopus (8) Google Scholar). These findings suggest that a broad range of stresses that perturb membrane integrity are responsible for the induction of Drp35 and that Drp35 may be a factor responsible for such general stresses rather than specific anti-biotic stress. Interestingly, Drp35 possesses calcium-dependent lactonase activity, although it has not been clarified how this activity contributes to the ability of the S. aureus cell to cope with stress (6Morikawa K. Hidaka T. Murakami H. Hayashi H. Ohta T. FEMS Microbiol. Lett. 2005; 249: 185-190Crossref PubMed Scopus (8) Google Scholar). In eukaryotic cells, paraoxonase family proteins (PONs) act as lactonases, and these proteins also require calcium ions for their catalytic activity similarly to Drp35 (7Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (367) Google Scholar, 8Draganov D.I. La Du B.N. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (365) Google Scholar, 9Gonzalvo M.C. Gil F. Hernandez A.F. Rodrigo L. Villanueva E. Pla A. J. Biochem. Mol. Toxicol. 1998; 12: 61-69Crossref PubMed Scopus (45) Google Scholar). Based on these observations, it has been proposed that Drp35 is a bacterial counterpart of eukaryotic PONs (6Morikawa K. Hidaka T. Murakami H. Hayashi H. Ohta T. FEMS Microbiol. Lett. 2005; 249: 185-190Crossref PubMed Scopus (8) Google Scholar). PONs are promiscuous enzymes and can hydrolyze not only lactone but also paraoxon, phosphotriester, and esters and thereby inactivate various organophosphates, including insecticides or nerve agents, such as sarin and soman (7Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (367) Google Scholar, 8Draganov D.I. La Du B.N. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (365) Google Scholar). Several studies also proposed a relationship of PONs to diseases, such as antiatherosclerotic activity (8Draganov D.I. La Du B.N. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (365) Google Scholar, 10Lusis A.J. Nature. 2000; 407: 233-241Crossref PubMed Scopus (4697) Google Scholar, 11Ng C.J. Shih D.M. Hama S.Y. Villa N. Navab M. Reddy S.T. Free Radic. Biol. Med. 2005; 38: 153-163Crossref PubMed Scopus (251) Google Scholar, 12Rosenblat M. Gaidukov L. Khersonsky O. Vaya J. Oren R. Tawfik D.S. Aviram M. J. Biol. Chem. 2006; 281: 7657-7665Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The reaction mechanism of hydrolysis by PONs has been studied by structural analysis, directed evolution, and site-directed mutagenesis (13Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (539) Google Scholar, 14Aharoni A. Gaidukov L. Yagur S. Toker L. Silman I. Tawfik D.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 482-487Crossref PubMed Scopus (258) Google Scholar, 15Khersonsky O. Tawfik D.S. J. Biol. Chem. 2006; 281: 7649-7656Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). However, their physiological role has not been determined despite many studies, including molecular characterization, of these proteins. In analogy with the promiscuous function of PONs, Drp35 may play some important role in detoxification of compounds that affect the cytoplasmic membrane, and therefore, elucidating the precise function of Drp35 may provide some insight to overcome staphylococcal pathogenesis. However, Drp35 has not been characterized in detail at the molecular level, including determination of its physiological role. In the present study, we investigated its lactonase activity from a structural view-point. First, we report the crystal structures of Drp35 with and without calcium ions. In the presence of Ca2+, Drp35 has a six-bladed β-propeller structure with a calcium ion at its center. Comparison of lactonase activity with mutant proteins indicated the significance of residues coordinating to the calcium ion. Structural analysis of the D138N mutant and functional analyses using several mutants suggested that a water molecule coordinated to the calcium ion in the wild type may be essential for catalysis. Based on these findings, we discuss the mechanism of lactonase activity of Drp35. Materials—All enzymes used in genetic engineering were obtained from Takara Shuzo (Kyoto, Japan) and Toyobo (Osaka, Japan). Isopropyl β-d-thiogalactopyranoside was obtained from Wako Fine Chemicals Inc. (Osaka, Japan). All other reagents were of biochemical research grade. Construction of Expression Vector for Drp35 and Mutant Proteins—The gene encoding Drp35 was amplified using KOD-Plus DNA polymerase (Toyobo), with S. aureus Mu50 genomic DNA as a template. The NcoI recognition sequence, CCATGG, in the drp35 gene was replaced with CCGTGG, which does not change the amino acid sequence, by the two-stage PCR method using the following primers: Drp35-S (5′-NNNNCCATGGCCATGTCACAACAAGATTTACCTACATTATTTTATAGC-3′), Drp35-AS (5′-CCGCTCGAGTTGAAACTGAAAACTTTGATGACCTTTTGC-3′), Drp35-mut-S (5′-ACAGCTGAACCGTGGCTTGAAATT-3′), and Drp35-mut-AS (5′-AATTTCAAGCCACGGTTCAGCTGT-3′) (restriction sites for digestion and ligation are underlined, and mutated nucleotides are indicated in italic type). The PCR products were inserted into the NcoI and BamHI sites of the pET28b vector (EMD Biosciences, San Diego, CA). All expression vectors for mutant proteins were prepared with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using synthesized primers and the Drp35 expression vector described above as the template. The correctness of the DNA sequences was confirmed using an ABI 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). Expression and Purification of Drp35 and Mutants—Transformed Escherichia coli strain B834 (DE3), harboring Drp35 expression vector and pT-RIL (Stratagene, Madison, WI), was grown at 37 °C in LB medium supplemented with 50 μgml-1 kanamycin and 34 μgml-1 chloramphenicol until the early stationary phase. To induce expression of the desired protein, isopropyl β-d-thiogalactopyranoside was added to a final concentration of 0.5 mm, and the culture was continued for 18 h at 25 °C. The selenomethionine derivative of Drp35 was obtained by the same method as described above except using M9 medium supplemented with 1 mm selenomethionine instead of LB medium. Cells were harvested by centrifugation at 5000 × g for 10 min at 4 °C and then disrupted using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan) in 50 mm Tris-HCl (pH 8.0), 300 mm NaCl. The cell debris was removed by centrifugation at 40,000 × g for 30 min at 4 °C, and the supernatant was loaded onto a HisTrap column (GE Healthcare Biosciences AB, Uppsala, Sweden) preequilibrated with 50 mm Tris-HCl (pH 8.0), 300 mm NaCl. The column was washed with 50 mm Tris-HCl (pH 8.0), 300 mm NaCl, and then the adsorbed protein was eluted with 50 ml of a 0-0.5 m gradient of imidazole in 50 mm Tris-HCl (pH 8.0), 300 mm NaCl. Fractions containing Drp35 were dialyzed against 50 mm Tris-HCl (pH 8.0), 300 mm NaCl, 1 mm EDTA and then further purified on a HiLoad 26/60 Superdex 200-pg column (GE Healthcare Biosciences AB) equilibrated with 50 mm Tris-HCl (pH 8.0), 300 mm NaCl, 1 mm EDTA. Fractions containing the desired protein were collected and used for further experiments. The Ca2+-bound form of Drp35 and Drp35-D138N mutant were prepared by the same methods as described above except using the following buffers: for cell disruption and equilibration for HisTrap column, 50 mm Tris-HCl (pH 7.5), 300 mm NaCl, 1 mm CaCl2; for elution from HisTrap column, 50 mm Tris-HCl (pH 7.5), 300 mm NaCl, 1 mm CaCl2, 500 mm imidazole; for size exclusion chromatography, 50 mm Tris-HCl (pH 7.5), 300 mm NaCl, 1 mm CaCl2. For the enzyme assay, Drp35 and mutants were purified by the same method as described above, except for the use of a Hiprep 26/10 desalting column (GE Healthcare Biosciences AB) preequilibrated with 50 mm Tris-HCl (pH 7.5), 300 mm NaCl, 1 mm CaCl2 instead of dialysis and size exclusion chromatography. Crystallization of Drp35, Drp35-Ca2+ Complex, and Drp35-D138N Mutant—Purified Drp35 with or without CaCl2 was dialyzed against 20 mm Tris-HCl (pH 7.5) and 20 mm Tris (pH 8.0), with or without 5 mm CaCl2, respectively, and then concentrated up to 20 mg ml-1. Initial crystallization conditions were screened by the sparse matrix method at 20 °C, using a Crystal Screen kit, Crystal Screen 2 kit (Hampton Research, Laguna Hills, CA), Wizard I, and Wizard II (Emerald Biostructures, Bainbridge Island, WA). Crystals of the apo form of Drp35 most suitable for further analyses were grown by the hanging drop vapor diffusion method from 100 mm Tris buffer, pH 8.8, 23% polyethylene glycol 4000, 0.1 m lithium sulfate. Crystals of Drp35 complexed with Ca2+ were grown from 100 mm HEPES (pH 7.6), 1050 mm succinic acid (pH 7.0), 1% polyethylene glycol monomethyl ether 2000. Crystals of the Drp35-D138N mutant were grown from a buffer containing 100 mm HEPES (pH 7.2), 1000 mm succinic acid (pH 7.0), 1% polyethylene glycol monomethyl ether 2000. X-ray Diffraction—X-ray diffraction of selenomethionine-substituted Drp35 was performed on beamline NW12 at Photon Factory (Tsukuba, Japan) under cryogenic conditions (100 K). For single-wavelength anomalous diffraction (SAD) phasing, a wavelength of 0.97908 Aå was chosen on the basis of the fluorescence spectrum of the selenium K absorption edge. The diffraction data of the Drp35-Ca2+ complex and Drp35-D138N mutant were collected on beamline BL44B2 at SPring-8 (Harima, Japan) and on beamline NW12 at Photon Factory (Tsukuba, Japan), respectively. All of the diffraction data were indexed, integrated, scaled, and merged using the HKL2000 program package (16Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). The data collection and processing statistics are summarized in Table 1.TABLE 1X-ray data collection and refinement statisticsSelenomethionine-substituted structure (SAD)Calcium complexD138N mutantData collectionSpace groupP21P21P21Cell dimensionsa = 86.09, b = 146.06, c = 151.95, β = 93.39a = 76.53, b = 181.98, c = 81.80, β = 115.41a = 76.55, b = 182.43, c = 81.45, β = 115.58BeamlinePhoton Factory NW12SPring-8 BL44B2Photon Factory NW12Resolution (Aå)aThe values in parentheses refer to data in the highest resolution shell.50-2.40 (2.49-2.40)50-1.72 (1.78-1.72)50-2.10 (2.18-2.10)Wavelength (Aå)0.979080.900000.90000Rsym (%)aThe values in parentheses refer to data in the highest resolution shell.bRsym = ∑h∑i|Ih,i - 〈Ih 〉|/∑h∑i|Ih,i|, where 〈Ih 〉 is the mean intensity of a set of equivalent reflections.10.3 (46.6)6.3 (39.5)10.5 (34.9)Completeness (%)aThe values in parentheses refer to data in the highest resolution shell.100 (100)99.2 (95.0)100 (100)Observed reflections1,561,878806,546446,630Unique reflections145,982211,193116,543MultiplicityaThe values in parentheses refer to data in the highest resolution shell.10.3 (10.2)3.8 (3.4)3.8 (3.7)Refinement and model qualityResolution range (Aå)20-2.420-1.7220-2.10No. of reflections138,199211,106116,380R factorcR factor = ∑|Fo - Fc|/∑Fo, where Fo and Fc represent observed and calculated structure factor amplitudes, respectively.0.2000.1690.168Rfree factordRfree factor was calculated for R factor, with a random 10% subset from all reflections.0.2220.1970.202Total protein atoms30,09615,07615,082Total ligand atoms08430Total water atoms12952141876Solvent content (%)42.046.746.7Average B factor (Aå2)39.418.826.3r.m.s. deviation from idealityBond lengths (Aå)0.0130.0050.005Bond angles (degrees)1.71.41.4Improper angles (degrees)0.90.790.79Dihedral angles (degrees)26.326.025.8Ramachandran plotResidues in most favored regions (%)81.284.285.2Residues in additional allowed regions (%)18.015.314.4Residues in generously allowed regions (%)0.80.50.4a The values in parentheses refer to data in the highest resolution shell.b Rsym = ∑h∑i|Ih,i - 〈Ih 〉|/∑h∑i|Ih,i|, where 〈Ih 〉 is the mean intensity of a set of equivalent reflections.c R factor = ∑|Fo - Fc|/∑Fo, where Fo and Fc represent observed and calculated structure factor amplitudes, respectively.d Rfree factor was calculated for R factor, with a random 10% subset from all reflections. Open table in a new tab Structure Solution and Refinement—The initial phasing was achieved by the SAD method with the program SHELX (17Sheldrick G.M. Hauptman H.A. Weeks C.M. Miller R. Uson I. International Tables for Crystallography. 2001; F (Arnold, E., ed) Vol. , pp. , Kluwer Academic Publishers, Dordrecht, The Netherlands: 333-351Crossref Google Scholar). The subsequent phase improvement was performed with the programs SOLVE/RESOLVE (18Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar, 19Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1872-1877Crossref PubMed Scopus (64) Google Scholar, 20Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1634) Google Scholar, 21Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 38-44Crossref PubMed Scopus (593) Google Scholar, 22Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 45-49Crossref PubMed Scopus (113) Google Scholar) and DM (23Schuller D.J. Acta Crystallogr Sect. D. Biol. Crystallogr. 1996; 52: 425-434Crossref PubMed Scopus (21) Google Scholar, 24Wang B.C. Methods Enzymol. 1985; 115: 90-112Crossref PubMed Scopus (943) Google Scholar, 25Cowtan K. Joint CCP4 and ESFEACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar). SHELX identified the positions of all 60 selenium sites. The program SOLVE/RESOLVE was used for heavy atom refinement and initial phasing. The initial electron density map was obtained after phase improvement by the program DM with the operator of noncrystallographic symmetry obtained by RESOLVE. A total of 2756 of 3996 residues were built automatically by RESOLVE, and 3792 residues were rebuilt manually using the program LSQKAB (26Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2358) Google Scholar) as part of the CCP4 suite (27(1994) Acta Crystallogr. Sect. D Biol. Crystallogr 50, 760-763Google Scholar) and the graphics program O (28Jones T.A. Zou J.Y. Cowan S.W. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar), based on the initial electron density map. The additional model building, positional energy minimization, and individual B factor refinements were carried out automatically with the program LAFIRE (29Yao M. Zhou Y. Tanaka I. Acta Crystallogr Sect. D Biol. Crystallogr. 2006; 62: 189-196Crossref PubMed Scopus (74) Google Scholar). To monitor the refinement, a random 10% subset from all reflections was set aside for calculation of the free R factor (Rfree). After automatic refinement and model fitting by LAFIRE, several cycles of refinement with the program CNS (30Brunger 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) and manual model fitting were carried out, and then the water molecules were located automatically. Due to the abundance of residues in an asymmetric unit against observed reflections, the difference between R and Rfree factor remarkably increased without the noncrystallographic symmetry restriction even in the final step of refinement. Therefore noncrystallographic symmetry restriction was applied to only the regions in which atoms could be well imposed throughout the refinement. The average of r.m.s. deviation of 12 molecules is 0.144 Aå. Finally, 3849 residues and 1295 water molecules could be placed in the structure of Se-Drp35 with crystallographic R values and Rfree values of 20.0 and 22.2%, respectively. The structure of Drp35-Ca2+ complex and D138N mutant were determined by the molecular replacement method using the program MOLREP (31Vagin A. Teplyako A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar) using the structure of Se-Drp35 as a search probe. To monitor the refinement, a random 10% subset from all reflections was set aside for calculation of the Rfree factor. The positional and individual B factor refinements were carried out automatically with the program LAFIRE. After automatic refinement and model fitting by LAFIRE, several cycles of refinement with the program CNS and manual model fitting were carried out. Finally, the water molecules were picked automatically by the program LAFIRE, and then calcium ions and ligand molecules were placed manually. The crystallographic R values and Rfree values for the Drp35-Ca2+ complex and Drp35-D138N mutant converged to 16.9% (19.7%) and 16.8% (20.2%), respectively. The stereochemical qualities of the final refined models were analyzed using the program PROCHECK (32Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The refinement statistics are summarized in Table 1. Although crystals of Drp35-Ca2+ complex and Drp35-D138N mutant belong to space group P21 in analogy with Se-Drp35, none of the relationship was observed in cell parameters. The packing of noncrystallographically related molecules also does not have a simple correlation. Enzyme Assay—Lactonase activity was determined as described previously using dihydrocoumarin as the substrate (6Morikawa K. Hidaka T. Murakami H. Hayashi H. Ohta T. FEMS Microbiol. Lett. 2005; 249: 185-190Crossref PubMed Scopus (8) Google Scholar). The kinetic parameters for Drp35 were determined in the range of 4.1-9.0 from Lineweaver-Burk plots. Buffers used were acetate (pH 4.1-5.5), MES (pH 5.6-6.8), and Tris-HCl (pH 7.4-9.0). kcat/Km values for each pH value ((kcat/Km)H) were fitted to a bell-shaped model using the equation, (kcat/Km)H = (kcat/Km)max/((10-pH/10-pKa1) + (10-pKa2/10-pH) + 1), where (kcat/Km)max is the pH-independent value, and pKa1 and pKa2 are the apparent pKa values for the acidic and basic groups, respectively. Differential Scanning Calorimetry (DSC)—All DSC measurements were carried out with a VP-CAPILLARY DSC SYSTEM (MicroCal, Northampton, MA). Proteins were dialyzed against 50 mm acetate, pH 5.6, and 1 mm EDTA or against 50 mm acetate, pH 5.6, and 1 mm CaCl2. The dialysis buffer was used as a reference solution for the DSC scan. Protein samples of 0.60-0.94 mg ml-1 were heated from 10 to 85 °C at a scanning rate of 1 K min-1. Crystal Structure of Se-Drp35 and Drp35-Ca2+ Complex—The crystal structure of selenomethionine-substituted Drp35 was determined at a resolution of 2.4 Aå by the SAD method (Table 1). This structural information enabled a molecular replacement method to determine the structure of the Drp35-Ca2+ complex at a resolution of 1.72 Aå. The subunit structures of the Drp35-Ca2+ complex are well superposed with those of Se-Drp35 with an averaged r.m.s. deviation of 0.30 Aå. Each monomer consists of five helices (α1-α5) and 25 β-strands (β1-β25). These β-strands form six β-sheets, five of which are composed of four β-strands and one of which is composed of five β-strands (Fig. 1A). These β-sheets are located in a circular arrangement, resulting in a six-bladed β-propeller structure. As is often the case with many kinds of β-propeller protein, a "molecular clasp" tethering the N- and C-terminal regions within a β-sheet was also observed in two β-sheets (blades 5 and 6) in Drp35 (33Jawad Z. Paoli M. Structure. 2002; 10: 447-454Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Interestingly, in all molecules of Se-Drp35, Drp35-Ca2+ complex, and Drp35-D138N mutant (see below), cis-peptide conformation was observed in His232-Glu233, located in the connecting loop between blades 1 and 6 (Fig. 1A). A structural alignment of Drp35 against all proteins in the Protein Data Bank by secondary structure matching (SSM) showed that the structure of Drp35 was similar to that of diisopropylfluorophosphatase (DFPase) from Loligo vulgaris and serum paraoxonase 1 (PON1) (r.m.s. deviation of 1.87 Aå for 264 Cα atoms and 2.50 Aå for 237 Cα atoms, respectively), although there was not significant amino acid sequence similarity (BLAST E-scores 4.3 and 1.6 for DFPase and PON1, respectively) (13Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (539) Google Scholar, 34Scharff E.I. Koepke J. Fritzsch G. Lucke C. Ruterjans H. Structure. 2001; 9: 493-502Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). These two eukaryotic proteins are classified as phosphotriesterases (EC 3.1.8) and possess a common biological activity (i.e. calcium-dependent hydrolysis activity) (9Gonzalvo M.C. Gil F. Hernandez A.F. Rodrigo L. Villanueva E. Pla A. J. Biochem. Mol. Toxicol. 1998; 12: 61-69Crossref PubMed Scopus (45) Google Scholar, 35Hartleib J. Geschwindner S. Scharff E.I. Ruterjans H. Biochem. J. 2001; 353: 579-589Crossref PubMed Scopus (28) Google Scholar). Similarly, Drp35 can hydrolyze lactones in a calcium-dependent manner and is a functional counterpart of PON (i.e. Drp35 is related structurally and functionally to DFPase and PON). The cis-peptide conformation found in Drp35 is not observed in the corresponding region in DFPase and PON1. Another apparent structural difference of Drp35 from PON is the absence of a canopy composed of α-helices that is required for binding of PON to the lipid layer of high density lipoprotein (13Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (539) Google Scholar). This is consistent with the observation that Drp35 is a soluble cytosolic protein and is not detected in the membrane fraction (6Morikawa K. Hidaka T. Murakami H. Hayashi H. Ohta T. FEMS Microbiol. Lett. 2005; 249: 185-190Crossref PubMed Scopus (8) Google Scholar). The reaction mechanisms for DFPase and PON have been reported. In both enzymes, a pocket at the center of the β-propeller acts as a substrate entrance tunnel in which substrates are captured and reacted. Drp35 also has a pocket at the identical position (Fig. 1C), suggesting that it would hold the active site (see below). In the structure of Drp35-Ca2+ complex, two calcium ions (Ca1 and Ca2) were bound to one molecule of Drp35 (Fig. 1A). The averaged distance between Ca1 and Ca2 is 17.0 Aå. Ca1 was bound at the bottom of the pocket located at the center of the β-propeller, where eight oxygen atoms derived from the side chains of Glu48, Asp138, Asn185, Asp236, Ser237, and three water molecules coordinated to Ca1 (Fig. 1, B and C). One of the water molecules was bound not only to Ca1 but also to Oδ in Asp138. The average distances between Ca1 and each ligated oxygen atom were as follows: 2.48 Aå for Oɛ in Glu48, 2.52 Aå for Oδ in Asp138, 2.37 Aå for Oδ in Asn185, 2.36 Aå for Oδ in Asp236, 2.41 Aå for Oγ in Ser237, and 2.56, 2.42, and 2.38 Aå for three water molecules. The other calcium, Ca2, is bound on the surface, in which the carbonyl oxygens from Thr133, Ser110, Asp130, Tyr135, and Gly112 and Oγ from Thr133 were bound to Ca2. The average distances between Ca2 and each ligated oxygen atom were as follows: 2.53 Aå for oxygen in Thr133, 2.30 Aå for oxygen in Ser110, 2.52 Aå for oxygen in the main chain of Asp130, 2.50 Aå for oxygen in Tyr135, 2.47 Aå for oxygen in Gly112, and 2.51 Aå for Oγ in Thr133. Although all coordinating atoms were oxygens as in Ca1, none of the carboxylate groups, which are the most frequently observed groups as ligands in Ca2+-protein complexes (36Pidcock E. Moore G.R. J. Biol. Inorg. Chem. 2001; 6: 479-489Crossref PubMed Scopus (183) Google Scholar), participated in the Ca2 binding. In crystal structures of DFPase and PON1 reported previously, a catalytically important calciu
Referência(s)