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• W2019166384 abstract "Agkistrodon contortrix laticinctus myotoxin is a Lys49-phospholipase A2 (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe121 and Phe124 and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118–125. Comparison with other Lys49-phospholipase A2 myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase “active site” and the C-terminal “myotoxic site,” justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules. Agkistrodon contortrix laticinctus myotoxin is a Lys49-phospholipase A2 (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe121 and Phe124 and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118–125. Comparison with other Lys49-phospholipase A2 myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase “active site” and the C-terminal “myotoxic site,” justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules. Phospholipases A2 (PLA2, 1The abbreviations used are: PLA2, phospholipase A2; ACL, A. contortrix laticinctus; SAD, single wavelength anomalous dispersion; PEG, polyethylene glycol; BthTX-I, bothropstoxin-I; PrTX-I, piratoxin-I; PDB, Protein Data Bank. EC 3.1.1.4) constitute a large family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of phospholipids, producing free fatty acids and lysophospholipids (1Davidson F.F. Dennis E.A. J. Mol. Evol. 1990; 31: 228-238Crossref PubMed Scopus (296) Google Scholar, 2Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 4Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 175-189Crossref PubMed Scopus (531) Google Scholar). The enzymatic activity of PLA2s plays an important role in many biological processes, and the fatty acids released by PLA2 can function as energy stores, second messengers (5Gijon M.A. Leslie C.C. J. Leukocyte Biol. 1999; 65: 330-336Crossref PubMed Scopus (251) Google Scholar, 6Berk P.D. Stump D.D. Mol. Cell. Biochem. 1999; 192: 17-31Crossref PubMed Google Scholar), and as precursors of eicosanoids, which are potent mediators of inflammation (7Austin S.C. Funk C.D. Prostaglandins Other Lipid Mediat. 1999; 58: 231-252Crossref PubMed Scopus (36) Google Scholar, 8Bingham III, C.O. Austen K.F. Proc. Assoc. Am. Physicians. 1999; 111: 516-524Crossref PubMed Scopus (61) Google Scholar). The lysophospholipids on the other hand are involved in cell signaling and phospholipid remodeling and are associated with membrane perturbation (9Moolenaar W.H. Kranenburg O. Postma F.R. Zondag G.C. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar, 10Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Some members of the PLA2 superfamily also present a wide variety of pharmacological effects, such as pre- and post-synaptic neurotoxicity (11Chang C.C. Lee C.Y. Eaker D. Fohlman J. Toxicon. 1977; 15: 571-576Crossref PubMed Scopus (69) Google Scholar, 12Bon C. Changeux J.-P. Jeng T.W. Fraenkel-Conrat H. Eur. J. Biochem. 1979; 99: 471-481Crossref PubMed Scopus (183) Google Scholar), myotoxicity (13Mebs D. Toxicon. 1986; 24: 161-168Crossref PubMed Scopus (56) Google Scholar, 14Ownby C.L. Mebs D. Shier T. Handbook of Toxicology. Marcel Dekker, Inc., New York1990: 601-654Google Scholar, 15Mebs D. Ownby C.L. Pharmacol. Ther. 1990; 48: 223-236Crossref PubMed Scopus (178) Google Scholar, 16Johnson E.K. Ownby C.L. Toxicon. 1993; 31: 243-255Crossref PubMed Scopus (56) Google Scholar, 17Fletcher J.E. Araujo H.S.S. Ownby C.L. Kini R.M. Venom Phospholipase A2 Enzymes: Structure, Function, and Mechanism. John Wiley & Sons, Ltd., Chichester, UK1997: 455-497Google Scholar, 18Ownby C.L. J. Toxicol. Toxin Rev. 1998; 17: 213-239Crossref Scopus (74) Google Scholar, 19Gutierrez J.M. Ownby C.L. Toxicon. 2003; 42: 915-931Crossref PubMed Scopus (333) Google Scholar), cardiotoxicity (20Fletcher J.E. Rapuano B.E. Condrea E. Yang C.C. Rosenberg P. Toxicol. Appl. Pharmacol. 1981; 59: 375-382Crossref PubMed Scopus (83) Google Scholar), and platelet aggregation (21Gerrard J.M. Robinson P. Narvey M. McNicol A. Biochem. Cell Biol. 1993; 71: 432-439Crossref PubMed Scopus (27) Google Scholar, 22Yuan Y. Jackson S.P. Mitchell C.A. Salem H.H. Thromb. Res. 1993; 70: 471-481Abstract Full Text PDF PubMed Scopus (45) Google Scholar) among others. According to their common source, size, pathological effects, amino acid sequence similarity, structural homology, disulfide bridge pattern, and other criteria, the PLA2 superfamily has been classified into 11 groups and 23 subgroups (23Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1220) Google Scholar). Groups I and II comprise secreted phospholipases A2 (of about 14 kDa) typically found in mammalian secretory fluids and snake venoms (24van den Bosch H. Biochim. Biophys. Acta. 1980; 604: 191-246Crossref PubMed Scopus (828) Google Scholar, 25Gutiérrez J.M. Cerdas L. Rev. Biol. Trop. 1984; 32: 213-222PubMed Google Scholar). They show a high degree of sequence and structural similarity and are believed to have a common calcium-dependent catalytic mechanism. The hydrolytic activity displayed by these groups of enzymes requires the presence of His48 and Asp99 residues (following the numbering system of Renetseder et al. (26Renetseder R. Brunie S. Dijkistra B.W. Drenth J. Sigler P. J. Biol. Chem. 1985; 260: 11627-11634Abstract Full Text PDF PubMed Google Scholar)), the former coordinating a conserved water molecule that acts as a nucleophile during hydrolysis. An aspartic acid at position 49, also described to be essential for the catalytic activity, binds the Ca2+ cofactor and aids in the stabilization of the tetrahedral intermediate during catalysis (27Verheij H.M. Volwerk J.J. Jasen E.H.J.M. Puyk W.C. Dijkistra B.W. Drenth J. Haas G.H. Biochemistry. 1980; 19: 743-750Crossref PubMed Scopus (292) Google Scholar, 28Scott D.L. White S.P. Otwinowski Z. Yuan W. Gelb M.H. Sigler P.B. Science. 1990; 250: 1541-1546Crossref PubMed Scopus (682) Google Scholar). A subfamily of group II PLA2s, where Asp49 is replaced by a lysine residue (known as Lys49-PLA2), has been described (29Selistre-de-Araujo H.S. White S.P. Ownby C.L. Arch. Biochem. Biophys. 1996; 326: 21-30Crossref PubMed Scopus (78) Google Scholar, 30Maraganore J.M. Merutka G. Cho W. Werlches W. Kezdy F.J. Heinrickson R.L. J. Biol. Chem. 1984; 259: 13839-13843Abstract Full Text PDF PubMed Google Scholar). Initially Lys49-PLA2s were believed to be catalytically inactive because of flipping of the Cys29–Gly30 peptide bond and their stereochemical incapacity to bind the cofactor calcium ion and thus stabilize the tetrahedral intermediate observed in the calcium-dependent catalytic reaction promoted by Asp49-PLA2s (31van den Bergh L.L.M. Slotboom A.J. Verheij H.M. de Haas G.H. J. Cell. Biochem. 1989; 39: 379-390Crossref PubMed Scopus (121) Google Scholar). This hypothesis was supported by structural analyses that have shown that the ϵ-amino group of Lys49 is located in the position occupied by Ca2+ in Asp49-PLA2 (32Holland D.R. Clancy L.L. Muchmore S.W. Ryde T.J. Einspahr H.M. Finzel B.C. Heinrickson R.L. Watenpaugh K.D. J. Biol. Chem. 1990; 265: 17649-17656Abstract Full Text PDF PubMed Google Scholar, 33Scott D.L. Achari A. Vidal J.C. Siegler P.B. J. Biol. Chem. 1992; 267: 22645-22657Abstract Full Text PDF PubMed Google Scholar, 34Arni R.K. Ward R.J. Gutiérrez J.M. Tulinsky A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1995; 51: 311-317Crossref PubMed Scopus (123) Google Scholar). However, in vitro assays have provided evidence of a limited catalytic activity for Lys49-PLA2 (35Shimohigashi Y. Tani A. Matsumoto H. Nakashima K. Yamaguchi Y. Oda N. Takano Y. Kamiya H. Kishino J. Arita H. Ohno M. J. Biochem. (Tokyo). 1995; 118: 1037-1044Crossref PubMed Scopus (42) Google Scholar, 36Yamaguchi Y. Shimohigashi Y. Chiwata T. Tani A. Chijiwa T. Lomonte B. Ohno M. Biochem. Mol. Biol. Int. 1997; 43: 19-26PubMed Google Scholar). These studies have often been criticized on the basis that they were performed by using toxins extracted directly from the snake venom, where the presence of trace amounts of contaminating Asp49-PLA2 would be sufficient to yield the low levels of activity observed. Recently, the use of refolded recombinant enzyme, where this risk has been eliminated (37Ward R.J. Chioato L. Oliveira A.H.C. Ruller R. Sá J.M. Biochem. J. 2002; 362: 89-96Crossref PubMed Scopus (78) Google Scholar), appears to confirm the original hypothesis that Lys49-PLA2s are indeed catalytically inactive. Nevertheless, a global consensus on this point has yet to be reached as emphasized by recent review articles (38Bruno Lomonte B. Angulo Y. Calderón L. Toxicon. 2003; 42: 885-901Crossref PubMed Scopus (260) Google Scholar, 39Murakami M.T. Arni R.K. Toxicon. 2003; 42: 903-913Crossref PubMed Scopus (36) Google Scholar). Despite having little or no enzymatic activity, Lys49-PLA2s are very active in induction of myonecrosis (16Johnson E.K. Ownby C.L. Toxicon. 1993; 31: 243-255Crossref PubMed Scopus (56) Google Scholar, 40Homsi-Brandeburgo M.I. Queiroz L.S. Neto H.S. Simioni L.R. Giglio J.R. Toxicon. 1988; 26: 615-627Crossref PubMed Scopus (227) Google Scholar, 41Francis B. Gutiérrez J.M. Lomonte B. Kaiser I.I. Arch. Biochem. Biophys. 1991; 284: 352-359Crossref PubMed Scopus (184) Google Scholar). If these molecules are genuinely catalytically inactive, this raises the intriguing question of why their “active” sites are so well conserved. “SequenceSpace” analysis (42Ward R.J. Alves A.R. Netto J.R. Arni R.K. Casari C. Protein Eng. 1998; 11: 285-294Crossref PubMed Google Scholar) and x-ray diffraction studies (32Holland D.R. Clancy L.L. Muchmore S.W. Ryde T.J. Einspahr H.M. Finzel B.C. Heinrickson R.L. Watenpaugh K.D. J. Biol. Chem. 1990; 265: 17649-17656Abstract Full Text PDF PubMed Google Scholar, 33Scott D.L. Achari A. Vidal J.C. Siegler P.B. J. Biol. Chem. 1992; 267: 22645-22657Abstract Full Text PDF PubMed Google Scholar, 34Arni R.K. Ward R.J. Gutiérrez J.M. Tulinsky A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1995; 51: 311-317Crossref PubMed Scopus (123) Google Scholar, 43de Azevedo Juniorperiod W.F. Ward R.J. Lombardi F.R. Giglio J.R. Soares A. Fontes M.R.M. Arni R.K. Protein Pept. Lett. 1997; 4: 329-334Google Scholar, 44de Azevedo Juniorperiod W.F. Ward R.J. Canduri F. Soares A. Giglio J.R. Arni R.K. Toxicon. 1998; 36: 1395-1406Crossref PubMed Scopus (36) Google Scholar, 45de Azevedo Juniorperiod W.F. Ward R.J. Gutiérrez J.M. Arni R.K. Toxicon. 1999; 37: 371-384Crossref PubMed Scopus (39) Google Scholar, 46da Silva Giotto M.T. Garratt R.C. Oliva G. Mascarenhas Y.P. Giglio J.R. Cintra A.C. de Azevedo Jr., W.F. Arni R.K. Ward R.J. Proteins. 1998; 30: 442-454Crossref PubMed Scopus (100) Google Scholar) have both clearly shown that, with the exception of Asp49 itself, the Lys49-PLA2s conserve all the important residues of the catalytic machinery, as well the nucleophilic water molecule and a hydrogen-bonding network that involves Tyr52 and Tyr73 and the N terminus. In addition, crystallographic structures in complex with naturally bound fatty acid molecules (interpreted as the product of catalysis) have been described (46da Silva Giotto M.T. Garratt R.C. Oliva G. Mascarenhas Y.P. Giglio J.R. Cintra A.C. de Azevedo Jr., W.F. Arni R.K. Ward R.J. Proteins. 1998; 30: 442-454Crossref PubMed Scopus (100) Google Scholar, 47Lee W.-H. da Silva Giotto M.T. Marangoni S. Toyama M.H. Polikarpov I. Garratt R.C. Biochemistry. 2001; 40: 28-36Crossref PubMed Scopus (79) Google Scholar). Based on these results, Lee and co-workers (47Lee W.-H. da Silva Giotto M.T. Marangoni S. Toyama M.H. Polikarpov I. Garratt R.C. Biochemistry. 2001; 40: 28-36Crossref PubMed Scopus (79) Google Scholar) have suggested that the apparent low level or lack of catalytic activity for Lys49-PLA2 observed in vitro could be the result of the failure of product release from the active site after one cycle of catalysis, leading to enzyme inhibition. Despite the abundance of crystallographic data on the Lys49-PLA2s, the mechanism of action that leads to their pharmacological effects has still to be fully elucidated. This is due at least in part to the fact that it has proved difficult to capture the same myotoxin in different conformational states. Inferences such as those made by Lee et al. (47Lee W.-H. da Silva Giotto M.T. Marangoni S. Toyama M.H. Polikarpov I. Garratt R.C. Biochemistry. 2001; 40: 28-36Crossref PubMed Scopus (79) Google Scholar) have had to be made on the basis of comparisons between different Lys49-PLA2s crystallized under different conditions, some in the presence of a bound active-site ligand and others not. There is, however, a growing consensus that both the myotoxic and calcium-independent membrane damaging activities (48Diaz C. Gutiérrez J.M. Lomonte B. Gene J.A. Biochim. Biophys. Acta. 1991; 1070: 455-460Crossref PubMed Scopus (117) Google Scholar, 49Rufini S. Cesaroni P. Desideri A. Farias R. Gubensek F. Gutiérrez J.M. Luly P. Massoud R. Morero R. Pedersen J.Z. Biochemistry. 1992; 31: 12424-12430Crossref PubMed Scopus (111) Google Scholar) of these molecules involve a mixture of basic and hydrophobic residues (particularly aromatics) concentrated toward the C terminus (37Ward R.J. Chioato L. Oliveira A.H.C. Ruller R. Sá J.M. Biochem. J. 2002; 362: 89-96Crossref PubMed Scopus (78) Google Scholar, 38Bruno Lomonte B. Angulo Y. Calderón L. Toxicon. 2003; 42: 885-901Crossref PubMed Scopus (260) Google Scholar, 50Lomonte B. Moreno E. Tarkowski A. Hanson L.A ̊. Maccarana M. J. Biol. Chem. 1994; 269: 29867-29873Abstract Full Text PDF PubMed Google Scholar, 51Lomonte B. Tarkowski A. Bagge U. Hanson L.A ̊. Biochem. Pharmacol. 1994; 47: 1509-11518Crossref PubMed Scopus (85) Google Scholar, 52Calderón L. Lomonte B. Arch. Biochem. Biophys. 1998; 358: 343-350Crossref PubMed Scopus (52) Google Scholar). One of the principal mechanistic mysteries surrounding Lys-PLA2 is the way in which ligand binding or catalysis at the active site is coupled to, or independent from, events involving this C-terminal region. In the present work we address this question directly via a description of the crystal structure of ACL myotoxin, a Lys49-PLA2 isolated from Agkistrodon contortrix laticinctus (broad-banded copperhead) venom. For the first time, three different crystal forms are observed for a Lys49-PLA2 originating from the same sample. Most interestingly, one of the crystal forms reveals the presence of a non-protein electron density within the hydrophobic substrate-binding channel, whereas the other two do not. This has allowed for a direct comparison between three different conformations of the same molecule. These have been used, together with our current knowledge of other PLA2 structures, to propose a coupling mechanism that relates changes in the conformation of the Ca2+ binding loop, mediated by fatty acid binding, to the exposure of a significant additional hydrophobic surface close to the C terminus. This may aid in explaining the conundrum of the conservation of the PLA2 active site in apparently catalytically inactive molecules. ACL myotoxin from the venom of A. contortrix laticinctus was isolated and purified as reported previously (16Johnson E.K. Ownby C.L. Toxicon. 1993; 31: 243-255Crossref PubMed Scopus (56) Google Scholar). The protein, obtained from the same purification batch, was crystallized under three different conditions using the conventional hanging drop vapor diffusion techniques. The structures derived from different crystallization conditions present conformational differences, and here they have been named forms I, II, and III respectively. All crystallization experiments were performed at 291 K with drops containing equal volumes (5 μl) of reservoir and protein solution. The latter was dissolved in water at a concentration that varied between 10 and 16 mg/ml prior to crystallization. X-ray diffraction measurements were performed at 100 K using crystals previously soaked in cryoprotectant solution (see Table I). Data for the first two crystal forms were collected at the Brazilian Synchrotron Light Laboratory, LNLS (53Polikarpov I. Perles L.A. de Oliveira R.T. Oliva G. Castellano E.E. Garratt R.C. Craievich A. J. Synchrotran Rad. 1998; 5: 72-76Crossref PubMed Scopus (102) Google Scholar), using λ = 1.544 Å, on a MAR345 image plate. For the third crystal form, a data set was obtained by using CuKα radiation (λ = 1.5418 Å) from a Rigaku UltraX 18 rotating-anode home source, equipped with Osmic focusing mirrors and measured on a Mar345dtb image plate detector. Data from forms I and II were processed using MOSFLM (54Leslie A.G.W. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography. Daresbury Laboratory, Daresbury, UK1992Google Scholar) and scaled with SCALA (55Evans P.R. Proceedings of CCP4 Study Weekend, on Data Collection & Processing, January 9 –10, Daresbury, UK. Daresbury Laboratory, Daresbury, UK1993: 114-122Google Scholar). For the third form, data were processed using the HKL 1.96 package (56Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar) but retaining the Bijvoet's pairs independently. In all cases the crystals belong to the space group P41212 with slightly different cell parameters.Table IData collection parameters and structures refinement statisticsForm IForm IIForm IIIData collectionCryoprotectant solutionMother liquor + 20% (v/v) glycerolMother liquor + 20% (v/v) glycerolMother liquor + 15% (v/v) ethylene glycolSpace groupP41212P41212P41212Cell parameters (Å)a = b = 81.89a = b = 70.50a = b = 70.57c = 49.9c = 57.14c = 57.45Mosaicity (°)0.600.500.45Resolution range (Å)57.3-2.350.0-1.826.5-1.61(2.36-2.3)(1.85-1.8)(1.65-1.61)Total reflections101,833118,620526,515Unique reflections796013,87235,959Redundancy6.2 (6.2)3.9 (3.7)14.6 (14.1)aAnomalous redundancyRsymm (%)11.8 (38.2)5.9 (36.1)6.1 (31.4)Completeness (%)98.1 (99.7)97.5 (99.6)99.8 (99.8)bCompletenessI/σ(I)3.5 (1.7)8.3 (2.0)49.5 (8.1)Molecules/AU111Solvent content (%)58.649.149.1Matthews volume (A3/Da)3.02.52.5Refinement (r.m.s.d.cr.m.s.d., root mean square deviation from standard geometries)Bond length (Å)0.0050.0070.009Bond angles (°)0.9291.0981.295Ramachandran plotMost favored (%)89.993.695.4Allowed (%)10.16.44.6a Anomalous redundancyb Completenessc r.m.s.d., root mean square deviation Open table in a new tab Forms I and II—The first set of phases for both form I and II crystals were obtained by molecular replacement techniques implemented in the program MOLREP (57Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4152) Google Scholar). For form I, a monomer of the Lys49-PLA2 from A. piscivorus piscivorus was used as the search model (PDB code 1PPA (32Holland D.R. Clancy L.L. Muchmore S.W. Ryde T.J. Einspahr H.M. Finzel B.C. Heinrickson R.L. Watenpaugh K.D. J. Biol. Chem. 1990; 265: 17649-17656Abstract Full Text PDF PubMed Google Scholar)). For form II, a partially refined structure from form I was used as the search model. In both cases, rigid body and simulated annealing refinement were performed using CNS (58Bru ̈nger 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 Y. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar), followed by subsequent cycles of positional and B factor refinement carried out with REFMAC 5.0 (59Murshudov G.N. Vagin A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13869) Google Scholar). The electron density maps were examined in O (60Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) with atomic positions being adjusted manually when necessary. Solvent water molecules, treated as oxygen atoms, were added using the automated refinement procedure routine (61Lamzin V. Wilson K.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 129-147Crossref PubMed Google Scholar). During the refinement of form I, the analysis of Fourier difference maps indicated the presence of a very strong electron density (over 3σ) inside the hydrophobic channel that leads to the active site. This was initially modeled as a single molecule of lauric acid (C12H24O2) that was incorporated into the model prior to the final stages of refinement. As an alternative, the possibility of modeling the additional electron density as a low molecular weight polyethylene glycol (PEG) molecule was also explored. Form III—The high quality data collected for the third crystal form were used for trials in a single anomalous dispersion using sulfur as the anomalous scatterer phasing procedure. The positions of the 18 sulfur sites were determined using SHELXD (62Sheldrick G.M. Hauptman H.A. Weeks C.M. Miller M. Usón I. Arnold E. Rossmann M. International Tables for Crystallography. F. Kluwer Academic Publishers Group, Dordrecht, Netherlands2001: 333-351Google Scholar). Besides the 16 sites expected for the 14 half-cystines and 2 methionine residues of the ACL myotoxin, the two extra sites correspond to a second conformation of one of the methionine residues and a sulfate ion that was derived from the crystallization solution. SHARP (63de la Fortelle E. Bricogne G. Methods Enzymol. 1997; 276 (494): 472Crossref PubMed Scopus (1797) Google Scholar) was used for refinement of the SHELXD sites and phase calculation, followed by density modification with SOLOMON (64Abrahams J.P. Leslie A.G.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar). An initial model was obtained by ARP/wARP (65Perrakis A. Morris R. Lamzin V. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2564) Google Scholar), followed by iterative model building (for 108 of the 121 residues) and side chain docking. Successive rounds of rebuilding and interspersed with torsion angle simulated annealing using CNS, and positional and individual B factor refinements with REFMAC 5.0 were carried out to generate the final model. The overall stereochemical quality of the final models for ACL myotoxin was assessed by the program PROCHECK (66Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), and the agreements between model and experimental data were checked with SF-CHECK (67Vaguine A.A. Richelle J. Wodak S.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 191-205Crossref PubMed Scopus (859) Google Scholar). Atomic coordinates for the three forms have been deposited in the Protein Data Bank (68Berman 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 (27540) Google Scholar) under codes 1S8G (form I), 1S8H (form II), and 1S8I (form III). Crystallization, Data Processing, and Structure Refinement—Crystals of form I were obtained using 30% w/v PEG 8000, 0.1 m sodium cacodylate, pH 6.5, 0.2 m ammonium sulfate, and a protein solution at 10 mg/ml. Form II crystals grow in the presence of 2.0 m ammonium sulfate, 0.1 m Tris-HCl, pH 8.5, and a protein solution at a concentration of 16 mg/ml. These crystals are isomorphous with those described previously by Treharne et al. (69Treharne A.C. White S.P. Ownby C.L. Selistre-de-Araújo H.S. Foundling S.I. Toxicon. 1997; 35: 613-615Crossref PubMed Scopus (6) Google Scholar). Form III crystals were obtained by using essentially identical conditions to those described for form II but with a reduced protein concentration (2.0 m ammonium sulfate, 0.1 m Tris-HCl, pH 8.5, and protein solution at 10 mg/ml). All three crystal forms belong to the space group P41212, and the difference between form II and form III is essentially structural, becoming evident only after refinement. Crystals of the myotoxin diffracted to 2.3, 1.8, and 1.6 Å, respectively, for forms I, II, and III. In the case of form I, the refinement converged to final residuals of Rfactor = 20.5% and Rfree = 24.7%, based on a model that consists of 121 residues for the polypeptide chain, 78 sites treated as water oxygens, a lauric acid molecule, and a sulfate ion. As with the structures reported previously for the myotoxins from Bothrops nummifer and Bothrops pirajai, which also presented additional density within the hydrophobic channel, the ACL myotoxin was crystallized in the presence of polyethylene glycol (PEG). It therefore seemed prudent to consider the possibility that the ligand density might correspond to a low molecular weight PEG. Attempts to refine the structure with a diethylene glycol unit occupying this site led to essentially identical results to those obtained with lauric acid, both in terms of the B factors for the ligand and its local environment and the final R values. The refinement of crystal form II converged to Rfactor and Rfree values of 19.1 and 22.9%, respectively. In this case the model consists of the same 121 amino acid residues for the polypeptide chain together with 139 solvent sites treated as water oxygens and a sulfate ion. In the case of form III, the final residuals were Rfactor = 18.6% and Rfree = 20.5%. Once again, the final model contained all of the expected atoms for the 121 residues of the polypeptide chain, besides 156 water molecules treated as oxygen atoms and a sulfate ion. Data collection, refinement, and quality statistics for the final models are summarized in Table I. Overall Structures—The three different crystal structures for ACL myotoxin include all of the atoms foreseen by the amino acid sequence (29Selistre-de-Araujo H.S. White S.P. Ownby C.L. Arch. Biochem. Biophys. 1996; 326: 21-30Crossref PubMed Scopus (78) Google Scholar) and present the canonical fold for a class II PLA2, consisting of an N-terminal α-helix (called α1), the calcium binding loop, two long anti-parallel α-helices (α2 and α3, respectively), the β-wing, and the C-terminal loop (see Fig. 1a). Furthermore, the seven disulfide bridges expected for class II PLA2 myotoxins are all present. As has been previously observed for Lys49-PLA2s, the positions of the amino acid residues His48, Tyr52, Tyr73, and Asp99, and their interactions are also conserved (43de Azevedo Juniorperiod W.F. Ward R.J. Lombardi F.R. Giglio J.R. Soares A. Fontes M.R.M. Arni R.K. Protein Pept. Lett. 1997; 4: 329-334Google Scholar). These residues are essential to the formation of the catalytic site of active phospholipases. It has been described for other Lys49-PLA2 myotoxins that residues located in the N-terminal α-helix and in the short anti-parallel β-wing play a role in protein dimerization (47Lee W.-H. da Silva Giotto M.T. Marangoni S. Toyama M.H. Polikarpov I. Garratt R.C. Biochemistry. 2001; 40: 28-36Crossref PubMed Scopus (79) Google Scholar, 48Diaz C. Gutiérrez J.M. Lomonte B. Gene J.A. Biochim. Biophys. Acta. 1991; 1070: 455-460Crossref PubMed Scopus (117) Google Scholar, 70Ward R.J. de Azevedo Juniorperiod W.F. Arni R.K. Toxicon. 1998; 36: 1623-1635Crossref PubMed Scopus (45) Google Scholar). Specific residues believed to be important for stabilizing this dimer and that are directly involved in monomer-monomer contacts include Glu12, Trp77, Asp79, and Lys80. Structural comparison reveals that these residues are conserved in ACL myotoxin and display sim" @default.
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• W2019166384 date "2005-02-01" @default.
• W2019166384 modified "2023-10-16" @default.
• W2019166384 title "A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change" @default.
• W2019166384 cites W1487525039 @default.
• W2019166384 cites W1501888177 @default.
• W2019166384 cites W1520730837 @default.
• W2019166384 cites W1534485474 @default.
• W2019166384 cites W1539796472 @default.
• W2019166384 cites W1573258040 @default.
• W2019166384 cites W1591680814 @default.
• W2019166384 cites W1591927080 @default.
• W2019166384 cites W1964814810 @default.
• W2019166384 cites W1970838759 @default.
• W2019166384 cites W1971746646 @default.
• W2019166384 cites W1975269273 @default.
• W2019166384 cites W1978063620 @default.
• W2019166384 cites W1980098090 @default.
• W2019166384 cites W1982084635 @default.
• W2019166384 cites W1985687874 @default.
• W2019166384 cites W1986191025 @default.
• W2019166384 cites W1991569266 @default.
• W2019166384 cites W1995017064 @default.
• W2019166384 cites W1996548832 @default.
• W2019166384 cites W1997945095 @default.
• W2019166384 cites W1999253885 @default.
• W2019166384 cites W2000291757 @default.
• W2019166384 cites W2004497536 @default.
• W2019166384 cites W2007841643 @default.
• W2019166384 cites W2010800897 @default.
• W2019166384 cites W2013066210 @default.
• W2019166384 cites W2013083986 @default.
• W2019166384 cites W2015942817 @default.
• W2019166384 cites W2016008242 @default.
• W2019166384 cites W2016255772 @default.
• W2019166384 cites W2020929208 @default.
• W2019166384 cites W2026307507 @default.
• W2019166384 cites W2027188394 @default.
• W2019166384 cites W2038349428 @default.
• W2019166384 cites W2038644399 @default.
• W2019166384 cites W2038840577 @default.
• W2019166384 cites W2040170805 @default.
• W2019166384 cites W2040600113 @default.
• W2019166384 cites W2040827725 @default.
• W2019166384 cites W2041541030 @default.
• W2019166384 cites W2053786834 @default.
• W2019166384 cites W2053878306 @default.
• W2019166384 cites W2057403631 @default.
• W2019166384 cites W2059289906 @default.
• W2019166384 cites W2061084405 @default.
• W2019166384 cites W2062685941 @default.
• W2019166384 cites W2066319194 @default.
• W2019166384 cites W2067629117 @default.
• W2019166384 cites W2068762412 @default.
• W2019166384 cites W2070093747 @default.
• W2019166384 cites W2073745805 @default.
• W2019166384 cites W2074466720 @default.
• W2019166384 cites W2075347510 @default.
• W2019166384 cites W2077065351 @default.
• W2019166384 cites W2078386027 @default.
• W2019166384 cites W2078681809 @default.
• W2019166384 cites W2079382831 @default.
• W2019166384 cites W2079528723 @default.
• W2019166384 cites W2080550444 @default.
• W2019166384 cites W2081404276 @default.
• W2019166384 cites W2083357538 @default.
• W2019166384 cites W2084575458 @default.
• W2019166384 cites W2088195456 @default.
• W2019166384 cites W2095543541 @default.
• W2019166384 cites W2097493124 @default.
• W2019166384 cites W2101446086 @default.
• W2019166384 cites W2106150672 @default.
• W2019166384 cites W2106315897 @default.
• W2019166384 cites W2119073319 @default.
• W2019166384 cites W2130479394 @default.
• W2019166384 cites W2132305492 @default.
• W2019166384 cites W2143199916 @default.
• W2019166384 cites W2143397299 @default.
• W2019166384 cites W2148283989 @default.
• W2019166384 cites W2153539273 @default.
• W2019166384 cites W2216330835 @default.
• W2019166384 cites W4210963323 @default.
• W2019166384 cites W4230141118 @default.
• W2019166384 cites W4237414156 @default.
• W2019166384 cites W4239102874 @default.
• W2019166384 cites W4313122328 @default.
• W2019166384 doi "https://doi.org/10.1074/jbc.m410588200" @default.
• W2019166384 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15596433" @default.
• W2019166384 hasPublicationYear "2005" @default.

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