FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2004891458> ?p ?o ?g. }

• W2004891458 endingPage "20069" @default.
• W2004891458 startingPage "20059" @default.
• W2004891458 abstract "In this study, the x-ray crystal structures of the calcium-free and calcium-bound forms of phospholipase A2 (PLA2), produced extracellularly by Streptomyces violaceoruber, were determined by using the multiple isomorphous replacement and molecular replacement methods, respectively. The former and latter structures were refined to an R-factor of 18.8% at a 1.4-Å resolution and an R-factor of 15.0% at a 1.6-Å resolution, respectively. The overall structure of the prokaryotic PLA2 exhibits a novel folding topology that demonstrates that it is completely distinct from those of eukaryotic PLA2s, which have been already determined by x-ray and NMR analyses. Furthermore, the coordination geometry of the calcium(II) ion apparently deviated from that of eukaryotic PLA2s. Regardless of the evolutionary divergence, the catalytic mechanism including the calcium(II) ion on secreted PLA2 seems to be conserved between prokaryotic and eukaryotic cells. Demonstrating that the overall structure determined by x-ray analysis is almost the same as that determined by NMR analysis is useful to discuss the catalytic mechanism at the molecular level of the bacterial PLA2. In this study, the x-ray crystal structures of the calcium-free and calcium-bound forms of phospholipase A2 (PLA2), produced extracellularly by Streptomyces violaceoruber, were determined by using the multiple isomorphous replacement and molecular replacement methods, respectively. The former and latter structures were refined to an R-factor of 18.8% at a 1.4-Å resolution and an R-factor of 15.0% at a 1.6-Å resolution, respectively. The overall structure of the prokaryotic PLA2 exhibits a novel folding topology that demonstrates that it is completely distinct from those of eukaryotic PLA2s, which have been already determined by x-ray and NMR analyses. Furthermore, the coordination geometry of the calcium(II) ion apparently deviated from that of eukaryotic PLA2s. Regardless of the evolutionary divergence, the catalytic mechanism including the calcium(II) ion on secreted PLA2 seems to be conserved between prokaryotic and eukaryotic cells. Demonstrating that the overall structure determined by x-ray analysis is almost the same as that determined by NMR analysis is useful to discuss the catalytic mechanism at the molecular level of the bacterial PLA2. Phospholipase A2(PLA2 1The abbreviations used are: PLA2phospholipase A2Watwater molecule1The abbreviations used are: PLA2phospholipase A2Watwater molecule ; EC 3.1.1.4) is an enzyme that liberates fatty acid together with lysophospholipid by hydrolyzing the 2-ester bond of 1,2-diacyl-3-sn-phosphoglycerides. The enzyme, which has been found only by eukaryotic cells, can be broadly divided into two categories, namely secreted and cytosolic types (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). In recent years, a proposal for classification has been made after many types of PLA2 in each category were found. According to this classification, secreted PLA2s are grouped into classes I, II, III, V, IX, X, XI, and XII (2Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1207) Google Scholar, 3Gelb M.H. Valentin E. Ghomashchi F. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 39823-39826Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Classes IV, VI, VII, and VIII are included in cytosolic PLA2 (2Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1207) Google Scholar). We have discovered a bacterial PLA2 of the secreted type and propose to establish a new class, which is described in the accompanying paper (41Sugiyama M. Ohtani K. Izuhara M. Koike T. Suzuki K. Imamura S. Misaki H. J. Biol. Chem. 2002; 277: 20051-20058Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). phospholipase A2 water molecule phospholipase A2 water molecule All secreted PLA2s require calcium(II) ion for the expression of enzymatic activity. Interestingly, the kinetic study of the S. violaceoruber PLA2 with respect to the dissociation constant for calcium(II) ion suggested that the binding affinity of the ion for the prokaryotic PLA2 is 1 order of magnitude lower than that for the eukaryotic enzyme (41Sugiyama M. Ohtani K. Izuhara M. Koike T. Suzuki K. Imamura S. Misaki H. J. Biol. Chem. 2002; 277: 20051-20058Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In addition, the bacterial PLA2 prefers phosphatidylcholine as a substrate to phosphatidylethanolamine. The primary structure of the prokaryotic PLA2, except for residues Cys61–Tyr68, is distinct from those of eukaryotic secreted PLA2s that have already been analyzed for the tertiary structure. As a striking difference, eukaryotic PLA2s have 6–8 disulfide bonds, whereas the bacterial enzyme has only two disulfide bonds. Over 150 primary structures of PLA2 have been already determined, and the crystal (4Dijkstra B.W. Kalk K.H. Hol W.G.J. Drenth J. J. Mol. Biol. 1981; 147: 97-123Crossref PubMed Scopus (426) Google Scholar, 5Brunie S. Bolin J. Gewirth D. Sigler P.B. J. Biol. Chem. 1985; 260: 9742-9749Abstract Full Text PDF PubMed Google Scholar, 6Thunnissen M.M.G.M., AB, E. Kalk K.H. Drenth J. Dijkstra B.W. Kuipers O.P. Dijkman R. de Haas G.H. Verheij H.M. Nature. 1990; 374: 689-691Crossref Scopus (250) Google Scholar, 7Scott D.L. White S.P. Otwinowski Z. Yuan K. Gelb M.H. Sigler P.B. Science. 1990; 250: 1541-1546Crossref PubMed Scopus (675) Google Scholar, 8Scott D.L. Otwinowski Z. Gelb M.H. Sigler P.B. Science. 1990; 250: 1563-1566Crossref PubMed Scopus (283) Google Scholar, 9White S.P. Scott D.L. Otwinowski Z. Gelb M.H. Sigler P.B. Science. 1990; 250: 1560-1563Crossref PubMed Scopus (313) Google Scholar, 10Wery J.P. Schevitz R.W. Clawson D.K. Bobbitt J.L. Dow E.R. Gamboa G. Goodson T.Jr. Hermann R.B. Kramer R.M. McClure D.B. Mihelich E.D. Putnam J.E. Sharp J.D. Stark D.H. Teater C. Warrick M.W. Jones N.D. Nature. 1991; 352: 79-82Crossref PubMed Scopus (198) Google Scholar) and NMR structures (11van den Berg B. Tessari M. de Haas G.H. Verheij H.M. Boelens R. Kaptein R. EMBO J. 1995; 14: 4123-4131Crossref PubMed Scopus (47) Google Scholar, 12van den Berg B. Tessari M. Boelens R. Dijkman R. Kaptein R. de Haas G.H. Verheij H.M. J. Biomol. NMR. 1995; 5: 110-121Crossref PubMed Scopus (37) Google Scholar, 13van den Berg B. Tessari M. Boelens R. Dijkman R. de Haas G.H. Kaptein R. Verheij H.M. Nat. Struct. Biol. 1995; 2: 402-406Crossref PubMed Scopus (69) Google Scholar) of several PLA2s, such as bovine and porcine pancreatic PLA2s, snake and bee venom PLA2s, and human synovial fluid PLA2, have been solved. Here, we show the crystal structure of the calcium-free S. violaceoruber PLA2, which was determined by using the multiple isomorphous replacement methods and refined at a high resolution of 1.4 Å. Furthermore, we determined the crystal structure of the calcium-bound form by the molecular replacement method and refined it at a 1.6-Å resolution. This is the first report disclosing the x-ray crystal structure of prokaryotic PLA2. The x-ray crystal structures of these two forms of the S. violaceoruber PLA2 were compared with those by the NMR technique and also with the tertiary structures of eukaryotic PLA2s. The x-ray crystal structure of the calcium-bound S. violaceoruber PLA2 is concrete evidence that the catalytic hydrogen-bonding network is essentially flexible even in the crystal structure. We believe that this observation will aid in understanding the molecular mechanism for the catalysis of the secreted PLA2s. The S. violaceoruber PLA2was purified to homogeneity and lyophilized as described previously (41Sugiyama M. Ohtani K. Izuhara M. Koike T. Suzuki K. Imamura S. Misaki H. J. Biol. Chem. 2002; 277: 20051-20058Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Before crystallization, the lyophilized protein, dissolved in a 10 mm Tris-HCl buffer, pH 8.0, containing 20 mm CaCl2, was adjusted to 20 mg/ml as a final concentration. Initial crystals were obtained by the vapor diffusion method at 297 K using the hanging drop technique (14McPherson A. The Preparation and Analysis of Protein Crystals. 1st Ed. John Wiley & Sons Inc., New York1982: 96-97Google Scholar). Drops containing 5 μl of the protein solution and 5 μl of the reservoir solution were equilibrated against the reservoir solution. The reservoir solution contained 14% (w/v) polyethylene glycol 6000, 0.2m Li2SO4, and 0.1 msodium cacodylate at pH 6.0. However, the crystals were heavily twinned and dissolved at 292 K. Therefore, after the crystals were completely dissolved, the drops were placed again at 297 K. The resulting single crystals, which grew in 2 weeks to a size of 0.3 × 0.3 × 1.2 mm, were suitable for the diffraction analysis. These crystals belong to the monoclinic space group P21 with unit cell dimensions a = 29.4 Å, b = 57.8 Å, c = 31.7 Å, and β = 111.5° with one molecule per asymmetric unit. After the structure determination, it was revealed that no calcium ion bound to the protein. All x-ray data were collected at room temperature. Initially, the native data to 2.0-Å resolution was collected using an R-AXIS IIc imaging plate with graphite monochromated Cu Kα radiation from the rotating anode generator RU-200 run at 40 kV and 100 mA. Each oscillation frame was taken using a rotation of 3° for 20 min at a crystal-to-detector distance of 80 mm. The diffraction intensities were integrated and scaled using the Process program (15Higashi T. J. Appl. Crystallogr. 1990; 23: 253-257Crossref Google Scholar). A nearly complete data set (94.6%) to a 2.0-Å resolution with high redundancy was collected from a single crystal. This native data set was used for the phase estimation at lower resolution and the preliminary refinement of the constructed model. Higher resolution data to 1.4 Å were later collected using the same detector and the same x-ray source. The detector 2θ angle was set to 35°, and the distance between its center and the crystal was set to 85 mm. Each oscillation frame was taken using a rotation of 1.5° for 15 min. In the highest resolution bin between 1.45 and 1.40 Å, this data set was 37.4% complete with a signal-to-noise ratio (I/ς(I)) of 4.52. Details of the data collection statistics are summarized in the top of Table I.Table IData collection, phasing, and refinement statisticsData collectionCa2+-free-1Ca2+-free-2PtAuRhCa2+-boundReagentK2PtCl4NaAu(CN)4(NH4)3RhCl6Conditions (mm/day)10/4100/120/2Maximum resolution (Å)2.01.42.72.73.01.6Observed reflections46,97322,0159,1576,0252,52530,440Unique reflections6,59715,5582,3922,6081,67812,820Completeness (%)94.677.585.693.287.873.2Rmerge 1-aRmerge = Σ ‖I − 〈I〉‖/ΣI.(%)7.217.416.304.983.436.50Riso 1-bRiso = Σ ‖FPH −Fp‖/ΣFp, where FPH is the observed structure factor amplitude of derivative and FP is that of 2.0-Å native data (Ca2+-free-1).(%)10.94.15.3PhasingPt (isomorphous)Pt (anomalous)Au (isomorphous)Rh (isomorphous)Rcullis 1-cRcullis = Σ ∥FPH(obs) ± FP(obs) ‖ −FH(calc)‖/Σ‖FPH(obs) ± FP(obs)‖ for centric reflections, where Fp, FPH, and FH are structure factor amplitudes of the reflections for native, derivative, and heavy atoms, respectively.(%)46.441.950.0Rkraut 1-dRkraut = Σ‖FPH(obs) −Fp(cal)‖/ΣFPH(obs) for acentric reflections.(%)10.54.36.8Rano 1-eRano = Σ (‖FPH +(obs) −FPH +(cal) ‖ + ‖FPH −(obs) −FPH −(cal)‖)/Σ(FPH +(obs) + FPH −(obs)) for acentric reflections. Plus and minus signs indicate a Bijvoet pair of reflections.(%)13.3Figure of merit1-fFigure of merit is a measure of phase precision in analysis. Herein, these values are based on the single isomorphous replacement or single anomalous scattering phases.0.3840.3100.4310.324Phasing power1-gPhasing power is the calculated heavy atom contribution divided by the lack of closure error in the analysis.2.451.962.271.47RefinementCa2+-freeCa2+-boundResolution range (Å)10.0–1.45.0–1.6R(%)18.815.0Rfree (%)23.1Solvent atoms127 waters88 waters and 1 calcium ion1-a Rmerge = Σ ‖I − 〈I〉‖/ΣI.1-b Riso = Σ ‖FPH −Fp‖/ΣFp, where FPH is the observed structure factor amplitude of derivative and FP is that of 2.0-Å native data (Ca2+-free-1).1-c Rcullis = Σ ∥FPH(obs) ± FP(obs) ‖ −FH(calc)‖/Σ‖FPH(obs) ± FP(obs)‖ for centric reflections, where Fp, FPH, and FH are structure factor amplitudes of the reflections for native, derivative, and heavy atoms, respectively.1-d Rkraut = Σ‖FPH(obs) −Fp(cal)‖/ΣFPH(obs) for acentric reflections.1-e Rano = Σ (‖FPH +(obs) −FPH +(cal) ‖ + ‖FPH −(obs) −FPH −(cal)‖)/Σ(FPH +(obs) + FPH −(obs)) for acentric reflections. Plus and minus signs indicate a Bijvoet pair of reflections.1-f Figure of merit is a measure of phase precision in analysis. Herein, these values are based on the single isomorphous replacement or single anomalous scattering phases.1-g Phasing power is the calculated heavy atom contribution divided by the lack of closure error in the analysis. Open table in a new tab The phase problem was solved to 2.7-Å resolution using the PHASES program (16Furey W. Swaminathan S. Methods Enzymol. 1990; 276: 472-494Google Scholar). Heavy atom derivatives were prepared by soaking the crystal in the reservoir solution containing each heavy atom reagent. Three derivatives (Pt, Au, and Rh) were used for the phasing (middle of Table I). The position of the primary Pt site was easily found in a difference Patterson map. The resulting single isomorphous replacement phases were used to solve other sites of the derivatives. The heavy atom parameters were refined, and a phase set with a figure of merit of 0.74 was obtained. Anomalous scattering information of the Pt derivative was utilized in this process, and anisotropic temperature factors were used for each heavy atom. A density modification with an extension to a 2.0-Å resolution was performed by the solvent-flattening and histogram-matching method using the SQUASH program (17Zhang K.Y.J. Main P. Acta Crystallogr. A. 1990; 46: 377-381Crossref Scopus (114) Google Scholar). The molecular boundary was determined in the electron density map applying a value of the solvent content of 20%, which is smaller than the value calculated on the basis of one molecule per asymmetric unit (33%). The resulting Fourier map enabled us to trace the main chain easily, and the difference map calculated from the native anomalous differences and the obtained phases confirmed the location of two disulfide bonds and one sulfur atom of the Met residue. An initial model was built into this modified map using the program Xfit in the XtalView software package (18McRee D.E. J. Mol. Graphics. 1992; 10: 44-46Crossref Google Scholar). This model gave an R-factor of 39.8% for the reflections from 10.0- to 3.0-Å resolution. Refinement of the model was performed by a combination of simulated annealing (19Brünger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar) and conventional restrained refinement methods (20Konnert J.H. Hendrickson W.A. Acta Crystallogr. Sect. A. 1980; 36: 344-350Crossref Scopus (298) Google Scholar) using the X-PLOR program (21Brünger A.T. X-PLOR Version 3.1 Manual. Yale University Press, New Haven, CT1993Google Scholar). A subset of 10% of the reflections was used to monitor the free R-factor (Rfree) (22Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3853) Google Scholar). The refinement began with data from 10.0- to 3.0-Å resolution, and the upper limit was raised to a 2.0-Å resolution. Each refinement cycle included the refinement of positional parameters and individual isotropic temperature factors, the revision of the model using the omit map, and the addition of the solvent molecules. When the R-factor for the reflections from 10.0- to 2.0-Å resolution fell below 20%, the native data was changed to the 1.4-Å resolution data. Further rebuilding, the addition of the solvent molecules, and the X-PLOR refinement yielded the current model (bottom of Table I). The root mean square deviations from the ideal geometry (23Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2539) Google Scholar) are 0.008 Å in bond length, 1.9° in bond angle, and 1.13° in improper angle. For crystallization, the protein solution was prepared as described previously. Initial crystals were obtained by the vapor diffusion at 297 K using the hanging drop method (14McPherson A. The Preparation and Analysis of Protein Crystals. 1st Ed. John Wiley & Sons Inc., New York1982: 96-97Google Scholar). Drops containing 5 μl of the protein solution and 5 μl of the reservoir solution were equilibrated against 1.0 ml of reservoir solution. The typical reservoir solution contained 50–60% (v/v) 2-methyl-2,4-pentanediol and 0.1 m Tris-HCl at pH 8.5. However, the obtained crystals were heavily twinned. To obtain single appropriate crystals, repeated seeding was performed using the crushed crystals as seeds. Single crystals suitable for the diffraction analysis (0.1 × 0.5 × 2.0 mm) grew in 1 week. They belong to the space group P21 with unit cell dimensions a = 38.3 Å, b = 54.3 Å,c = 30.6 Å, and β = 90.2° with one molecule per asymmetric unit. These preliminary crystallographic data have been published (24Matoba Y. Kumagai T. Sugiyama M. J. Inorg. Biochem. 2000; 82: 221-223Crossref PubMed Scopus (2) Google Scholar). X-ray analysis was performed at room temperature using an R-AXIS IIc imaging plate with mirror monochromated Cu Kα radiation from the RU-300 rotating anode generator run at 40 kV and 100 mA. The crystal was mounted with the crystallographic a* axis parallel to the crystal rotation axis. The crystal-to-detector distance was set to 60 mm. Each frame of 2.5° crystal oscillation was taken for 12 min. Diffraction spots in a rotation range of 142.5° were recorded on a total of 57 frames. The diffraction intensities to a 1.6-Å resolution were integrated and scaled by the Process program (15Higashi T. J. Appl. Crystallogr. 1990; 23: 253-257Crossref Google Scholar). In the highest resolution bin between 1.7- and 1.6-Å resolution, this data set is 48.0% complete with a signal/noise ratio (I/ς(I)) of 2.41 and an Rmerge of 28.3%. Details of the data collection statistics are shown in Table I (top). The crystal structure of the calcium-bound S. violaceoruberPLA2 was solved by the molecular replacement procedure using the programs in X-PLOR (21Brünger A.T. X-PLOR Version 3.1 Manual. Yale University Press, New Haven, CT1993Google Scholar). The calcium-free form structure was used as search probe in a cross-rotation function against the data in a resolution range of 10.0- to 4.0-Å. A single peak at Euler angles θ1 = 183.3°, θ2 = 72.5°, and θ3 = 30.8° was 8.0 ς above the mean. A translational search was carried out in two dimensions (x and z). A top peak (x = 0.214, z= 0.425 in fractions of the unit cell) was observed at 7.2 ς above the mean. Refinement was first performed using the X-PLOR program (21Brünger A.T. X-PLOR Version 3.1 Manual. Yale University Press, New Haven, CT1993Google Scholar). Atomic coordinates, obtained by the molecular replacement method, were refined against the data between 10.0 and 4.0 Å for 20 cycles with the entire molecule as a rigid body, resulting in a crystallographic R-factor of 35.8%. Atomic refinement of the model was performed by using a combination of simulated annealing (19Brünger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar) and conventional restrained refinement methods (20Konnert J.H. Hendrickson W.A. Acta Crystallogr. Sect. A. 1980; 36: 344-350Crossref Scopus (298) Google Scholar). A subset of 10% of the reflections was used to monitor the Rfree(22Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3853) Google Scholar). The refinement began with data from 10.0- to 3.0-Å resolution, and the upper limit was raised to a 1.6-Å resolution. Each refinement cycle included the refinement of positional parameters and individual isotropic temperature factors. After each refinement step, 2 Fo − Fc and Fo − Fc electron density maps were computed. The molecular modeling program Xfit in the XtalView program suit (18McRee D.E. J. Mol. Graphics. 1992; 10: 44-46Crossref Google Scholar) was used on the Silicon Graphics workstations for visualizing and rebuilding the model. Several cycles of the X-PLOR refinement resulted in decreasing both the R-factor and Rfree to 20.1 and 26.5%, respectively, for the reflections from 10.0 to 1.6-Å resolution with F > 2 ς. After the convergence on X-PLOR, further refinement was performed using the SHELXL-97 program (25Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1877) Google Scholar) against all of the reflections from 5.0- to 1.6-Å resolution with F > 0. Refinement statistics are given at the bottom of Table I. The root mean square deviations from the ideal geometry (23Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2539) Google Scholar) are 0.008 Å in bond length, 1.9° in bond angle, and 1.13° in improper angle. As shown in Fig. 1, a and b, the final 2 Fo − Fc maps of the hydrophobic core region in the calcium-free form and the calcium-binding site in the calcium-bound form structures exhibit very high quality. Almost all aromatic amino acid residues as well as Pro residues have a low density hole through the center of the rings, as expected from the high resolution maps. Mean coordinate errors were estimated by a Luzatti plot (26Luzzati V. Acta Crystallogr. 1952; 5: 802-810Crossref Google Scholar) to be 0.15 and 0.16 Å for the calcium-free and calcium-bound PLA2s, respectively (data not shown). Although all side chain atoms in the calcium-free form are included in the final model, those of Gln7, Arg28, Gln47, Phe53, Lys103, and Trp112 have poorly defined electron density. In the calcium-bound form, together with the side chain atoms of these 6 residues, those of Asn29, Glu102, and Lys119 were poorly defined. The C-terminal main- and side-chain atoms in the calcium-bound form structure are flexible. Mainly, the difference of distribution of the flexible residues between calcium-free and calcium-bound forms seems to be caused by the differences of their crystal-packing effects, as described below. In both cases, the Ramachandran plot (27Ramachandran G.N. Ramakrishnan C. Sasisekharan V. J. Mol. Biol. 1963; 7: 95-99Crossref PubMed Scopus (2658) Google Scholar, 28Kleywegt G.J. Jones T.A. Structure. 1996; 4: 1395-1400Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar) shows that only Leu44 is observed in the forbidden regions. The unusual backbone conformation of Leu44 is stabilized by a hydrogen bond formed between its backbone amide nitrogen and the Thr42 hydroxyl oxygen. On the geometry of class I/II PLA2s, an anti-parallel β-sheet composed of two strands is present together with α-helices accounting for 50% of the overall structure (4Dijkstra B.W. Kalk K.H. Hol W.G.J. Drenth J. J. Mol. Biol. 1981; 147: 97-123Crossref PubMed Scopus (426) Google Scholar, 5Brunie S. Bolin J. Gewirth D. Sigler P.B. J. Biol. Chem. 1985; 260: 9742-9749Abstract Full Text PDF PubMed Google Scholar, 6Thunnissen M.M.G.M., AB, E. Kalk K.H. Drenth J. Dijkstra B.W. Kuipers O.P. Dijkman R. de Haas G.H. Verheij H.M. Nature. 1990; 374: 689-691Crossref Scopus (250) Google Scholar, 7Scott D.L. White S.P. Otwinowski Z. Yuan K. Gelb M.H. Sigler P.B. Science. 1990; 250: 1541-1546Crossref PubMed Scopus (675) Google Scholar, 9White S.P. Scott D.L. Otwinowski Z. Gelb M.H. Sigler P.B. Science. 1990; 250: 1560-1563Crossref PubMed Scopus (313) Google Scholar, 10Wery J.P. Schevitz R.W. Clawson D.K. Bobbitt J.L. Dow E.R. Gamboa G. Goodson T.Jr. Hermann R.B. Kramer R.M. McClure D.B. Mihelich E.D. Putnam J.E. Sharp J.D. Stark D.H. Teater C. Warrick M.W. Jones N.D. Nature. 1991; 352: 79-82Crossref PubMed Scopus (198) Google Scholar). Class III bee venom PLA2 consists of three kinds of long α-helices and a few β-strands (8Scott D.L. Otwinowski Z. Gelb M.H. Sigler P.B. Science. 1990; 250: 1563-1566Crossref PubMed Scopus (283) Google Scholar). However, the S. violaceoruber PLA2 only has an α-helical secondary structure consisting of five α-helices and two helical segments (Fig. 2 and Fig. 3 a). The current crystal structure model of the bacterial PLA2 is almost the same as the NMR structure (41Sugiyama M. Ohtani K. Izuhara M. Koike T. Suzuki K. Imamura S. Misaki H. J. Biol. Chem. 2002; 277: 20051-20058Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To clarify the tertiary structure of bacterial PLA2, we conveniently divided the structure into the N- and C-terminal domains; the former domain consists of two α-helices (α1 and α2) and two helical segments. The latter domain consists of three long α-helices (α3, α4, and α5) and forms a three-helix bundle. The three α-helices are tightly packed against each other with an unusual slightly right-handed twist. The angles among the three α-helices are 11° (α3–α4), 18° (α4–α5), and 19° (α3–α5), respectively. Both domains are linked to a long loop consisting of residues Glu37 to Phe57. The α3-helix contains a homologous segment consisting of the residues Cys61 to Tyr68, and its orientation is almost anti-parallel to the α4-helix. The nearly anti-parallel helices are also formed by α4- and α5-helices. These three α-helices are approximately perpendicular to the α2-helix.Figure 3a, amino acid sequence of the S. violaceoruber PLA2 and structurally homologous segments of typical eukaryotic PLA2s. i), S. violaceoruber; ii), N. naja atraPLA2 (class I/II); iii), bee venom PLA2 (class III). The catalytic and calcium-binding sites are indicated in red and blue, respectively. The secondary structures are defined by the MOLSCRIPT program (39Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).b, stereoview of the superposition of the α-carbon backbone of the calcium-free S. violaceoruberPLA2 (in red) on those of PLA2s that are contained in N. naja atra (in blue) and bee (in green) venoms. The structures of N. naja atra and bee venom PLA2s are described in Refs. 7Scott D.L. White S.P. Otwinowski Z. Yuan K. Gelb M.H. Sigler P.B. Science. 1990; 250: 1541-1546Crossref PubMed Scopus (675) Google Scholar and 8Scott D.L. Otwinowski Z. Gelb M.H. Sigler P.B. Science. 1990; 250: 1563-1566Crossref PubMed Scopus (283) Google Scholar, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The α-carbon backbone of the S. violaceoruberPLA2 is superimposed on that of PLA2 from Naja naja atra, the cobra snake (7Scott D.L. White S.P. Otwinowski Z. Yuan K. Gelb M.H. Sigler P.B. Science. 1990; 250: 1541-1546Crossref PubMed Scopus (675) Google Scholar), by the least squares fit of the catalytic residues (Fig. 3 b). The tertiary structural similarity of backbone is limited to two long anti-parallel α-helices containing the residues composing the catalytic site. A two-stranded β-sheet, commonly found in the class I/II PLA2s, is absent in the bacterial PLA2. Although the C-terminal α5-helix in the S. violaceoruber PLA2 corresponds to the N-terminal α-helix in the class I/II PLA2s, its orientation is opposite. Similarly, the α-carbon backbone of the S. violaceoruber PLA2 is superimposed on that of PLA2 from bee venom (8Scott D.L. Otwinowski Z. Gelb M.H. Sigler P.B. Science. 1990; 250: 1563-1566Crossref PubMed Scopus (283) Google Scholar) (Fig. 3 b). The orientation of three long α-helices in the C-terminal domain of the bacterial PLA2 is similar to that of PLA2 from bee venom, whereas the overall structure of bacterial PLA2is obviously different from that of the bee venom PLA2. The overall structure of the S. violaceoruber PLA2can be expressed as a shape in which the N-terminal domain of the bacterial PLA2 is topologically added to a core region consisting of three α-helices of the bee venom PLA2. Despite the difference of the crystal lattice, the crystal structure of the calcium-free form is identical with that of the calcium-bound form. Fig. 4 a shows the superposition of the two forms using the main-chain atoms. The root mean square positional differences between the two structures are 0.93 and 1.10 Å for main-chain atoms and for all protein atoms, respectively. Marked structural deviations occur at the Cys45—Ala48 residues in the long loop (Fig. 4 b) and at the Lys119—Gly122residues in the C-terminal region (Fig. 4 c). The former conformational change may be caused by binding of the calcium(II) ion to the protein, and the latter may reflect the potential flexibility of this region, as described below. In the calcium-free S. violaceoruber PLA2structure, although calcium(II) ion is contained in a buffer to grow the crystal, the ion is absent in the model. This may be due to the fact that the crystal is grown in the presence of sulfate ion at near pH 6. Interestingly, even without calcium(II) ion, the current model maintains the conformational rigidity. The mean temperature factors are 11.0 Å2 for all protein atoms and 9.8 Å2 for the main-chain atoms. On the other hand, the mean temperature factors of the calcium-bound form structure increase to 30.1 Å2for all protein atoms and 28.0 Å2 for the main-chain atoms. Judging from the fact that VM (29Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7909) Google Scholar) values are extremely low (1.85 Å3 Da−1) for calcium-free crystal and moderate (2.35 Å3Da−1) for calcium-bound crystal, it can easily be speculated that the differences of mean temperature factors are mainly caused by the crystal packing effe" @default.
• W2004891458 created "2016-06-24" @default.
• W2004891458 creator A5026668762 @default.
• W2004891458 creator A5039380373 @default.
• W2004891458 creator A5062808952 @default.
• W2004891458 date "2002-05-01" @default.
• W2004891458 modified "2023-10-14" @default.
• W2004891458 title "The Crystal Structure of Prokaryotic Phospholipase A2" @default.
• W2004891458 cites W1573258040 @default.
• W2004891458 cites W1608557156 @default.
• W2004891458 cites W1710565104 @default.
• W2004891458 cites W1835278113 @default.
• W2004891458 cites W1972322003 @default.
• W2004891458 cites W1975914851 @default.
• W2004891458 cites W1976422615 @default.
• W2004891458 cites W1979740497 @default.
• W2004891458 cites W1990808614 @default.
• W2004891458 cites W1993523153 @default.
• W2004891458 cites W1994001535 @default.
• W2004891458 cites W1996607909 @default.
• W2004891458 cites W2014694459 @default.
• W2004891458 cites W2015023165 @default.
• W2004891458 cites W2015942817 @default.
• W2004891458 cites W2024152410 @default.
• W2004891458 cites W2026725988 @default.
• W2004891458 cites W2028231353 @default.
• W2004891458 cites W2031466871 @default.
• W2004891458 cites W2032408951 @default.
• W2004891458 cites W2037909798 @default.
• W2004891458 cites W2042987185 @default.
• W2004891458 cites W2049902088 @default.
• W2004891458 cites W2066864165 @default.
• W2004891458 cites W2074362388 @default.
• W2004891458 cites W2074954129 @default.
• W2004891458 cites W2078248419 @default.
• W2004891458 cites W2080550444 @default.
• W2004891458 cites W2106150672 @default.
• W2004891458 cites W2106675944 @default.
• W2004891458 cites W2116275717 @default.
• W2004891458 cites W2129595368 @default.
• W2004891458 cites W2132305492 @default.
• W2004891458 cites W2155836823 @default.
• W2004891458 cites W2156946828 @default.
• W2004891458 cites W2165334001 @default.
• W2004891458 cites W2165419072 @default.
• W2004891458 cites W4239102874 @default.
• W2004891458 doi "https://doi.org/10.1074/jbc.m200263200" @default.
• W2004891458 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11897785" @default.
• W2004891458 hasPublicationYear "2002" @default.
• W2004891458 type Work @default.
• W2004891458 sameAs 2004891458 @default.
• W2004891458 citedByCount "41" @default.
• W2004891458 countsByYear W20048914582012 @default.
• W2004891458 countsByYear W20048914582013 @default.
• W2004891458 countsByYear W20048914582014 @default.
• W2004891458 countsByYear W20048914582015 @default.
• W2004891458 countsByYear W20048914582017 @default.
• W2004891458 countsByYear W20048914582018 @default.
• W2004891458 countsByYear W20048914582019 @default.
• W2004891458 countsByYear W20048914582020 @default.
• W2004891458 countsByYear W20048914582022 @default.
• W2004891458 countsByYear W20048914582023 @default.
• W2004891458 crossrefType "journal-article" @default.
• W2004891458 hasAuthorship W2004891458A5026668762 @default.
• W2004891458 hasAuthorship W2004891458A5039380373 @default.
• W2004891458 hasAuthorship W2004891458A5062808952 @default.
• W2004891458 hasBestOaLocation W20048914581 @default.
• W2004891458 hasConcept C115624301 @default.
• W2004891458 hasConcept C12554922 @default.
• W2004891458 hasConcept C181199279 @default.
• W2004891458 hasConcept C185592680 @default.
• W2004891458 hasConcept C2776551241 @default.
• W2004891458 hasConcept C55493867 @default.
• W2004891458 hasConcept C70721500 @default.
• W2004891458 hasConcept C8010536 @default.
• W2004891458 hasConcept C86803240 @default.
• W2004891458 hasConceptScore W2004891458C115624301 @default.
• W2004891458 hasConceptScore W2004891458C12554922 @default.
• W2004891458 hasConceptScore W2004891458C181199279 @default.
• W2004891458 hasConceptScore W2004891458C185592680 @default.
• W2004891458 hasConceptScore W2004891458C2776551241 @default.
• W2004891458 hasConceptScore W2004891458C55493867 @default.
• W2004891458 hasConceptScore W2004891458C70721500 @default.
• W2004891458 hasConceptScore W2004891458C8010536 @default.
• W2004891458 hasConceptScore W2004891458C86803240 @default.
• W2004891458 hasIssue "22" @default.
• W2004891458 hasLocation W20048914581 @default.
• W2004891458 hasOpenAccess W2004891458 @default.
• W2004891458 hasPrimaryLocation W20048914581 @default.
• W2004891458 hasRelatedWork W1969215021 @default.
• W2004891458 hasRelatedWork W1969352129 @default.
• W2004891458 hasRelatedWork W1984275137 @default.
• W2004891458 hasRelatedWork W2063698282 @default.
• W2004891458 hasRelatedWork W2082823139 @default.
• W2004891458 hasRelatedWork W2083548216 @default.
• W2004891458 hasRelatedWork W2315457165 @default.
• W2004891458 hasRelatedWork W2711806730 @default.
• W2004891458 hasRelatedWork W2890061065 @default.

• next