e at SciVerse ScienceDirect Toxicon 72 (2013) 52–63 Contents lists availabl Toxicon journal homepage: www.elsevier .com/locate/ toxicon Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like Guilherme H.M. Salvador a,1, Walter L.G. Cavalcante a,b,1, Juliana I. dos Santos a,1, Márcia Gallacci b, Andreimar M. Soares c, Marcos R.M. Fontes a,* aDepto. de Física e Biofísica, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu, SP, Brazil bDepto. de Farmacologia, Instituto de Biociências, UNESP – Univ Estadual Paulista, Botucatu, SP, Brazil cCentro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ Rondônia e Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho, RO, Brazil a r t i c l e i n f o Article history: Received 15 February 2013 Received in revised form 16 May 2013 Accepted 11 June 2013 Available online 25 June 2013 Keywords: Lys49-phospholipase A2 Bothrops moojeni venom Oligomeric assembly X-ray crystallography Myographic studies * Corresponding author. Tel.: þ55 14 38800271; fa E-mail address: fontes@ibb.unesp.br (M.R.M. Fon 1 These authors contributed equally to this work 0041-0101/$ – see front matter � 2013 Elsevier Ltd http://dx.doi.org/10.1016/j.toxicon.2013.06.013 a b s t r a c t Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms ofmedical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved con- cerning their functional–structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin’s hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s. � 2013 Elsevier Ltd. All rights reserved. 1. Introduction Envenoming by snakebites represents a relevant and neglected global health problem, particularly in tropical x: þ55 14 38153744. tes). . . All rights reserved. regions (Gutierrez et al., 2006; Harrison et al., 2009; Russell, 1991). Recent estimates indicate that at least 421,000 envenomations and 20,000 deaths related to ophidian accidents occur each year, mainly in Latin Amer- ica, Asia and Africa (Kasturiratne et al., 2008); however, this same study suggests that these numbers can be as high as 1,841,000 envenomations and 94,000 deaths (Kasturiratne et al., 2008). Even so, the mortality caused by snakebite is mailto:fontes@ibb.unesp.br http://crossmark.dyndns.org/dialog/?doi=10.1016/j.toxicon.2013.06.013&domain=pdf www.sciencedirect.com/science/journal/00410101 http://www.elsevier.com/locate/toxicon http://dx.doi.org/10.1016/j.toxicon.2013.06.013 http://dx.doi.org/10.1016/j.toxicon.2013.06.013 G.H.M. Salvador et al. / Toxicon 72 (2013) 52–63 53 much higher than the given by several neglected tropical diseases, such as dengue haemorrhagic fever, leishmani- asis, cholera, schistosomiasis and Chagas disease, which leads the World Health Organization to include the ophidian accidents in the list of neglected tropical diseases (Williams et al., 2010). Snakes from Viperidae family are found in many parts of the world causing several accidents every year (Gutierrez and Lomonte, 1995; Kasturiratne et al., 2008). Particularly in Brazil, the majority of ophidian accidents occur with the Bothrops genus (Viperidae family) (Rosenfeld and Kelen, 1971; Saúde, 2001) that are characterized by pronounced local effects, including hemorrhage, edema, pain and myonecrosis (Gutierrez and Chaves, 1980; Gutierrez and Ownby, 2003; Homsi-Brandeburgo et al., 1988; Mebs et al., 1983; Queiroz and Petta, 1984; Rosenfeld and Kelen, 1971). These local effects are very relevant in terms of medical and scientific interest since the proteins respon- sible for the toxic process which may lead to permanent tissue loss, disability and, in some cases may require the amputation of the victim’s affected limb are not efficiently neutralized by antivenom administration (Gutierrez and Lomonte, 1995). Phospholipases A2 (PLA2s) are enzymes that catalyze the hydrolysis of glycerophospholipids, in a calcium- dependent manner, and represent the most abundant myotoxic components in Viperidae snake venoms (Gutierrez and Ownby, 2003). These proteins can be clas- sified into two groups according to their evolutionary pathway: i) the catalytically active enzymes, such as Asp49- , Asn49- and Gln49-PLA2s and ii) the catalytically inactive PLA2 variants (Lys49-, Arg49-, and some Asp49-PLA2s) (dos Santos et al., 2011b). In this latter group, the most studied toxins are the basic and homodimeric Lys49-PLA2s that induce noticeable local myonecrosis bymeans of a calcium- independent mechanism (Lomonte and Rangel, 2012). In addition, Lys49-PLA2s exhibit some effects found exclu- sively in vitro, as the blockade of neuromuscular trans- mission in isolated preparations, which has been directly associated to their ability in destabilizing cell membranes (Gallacci and Cavalcante, 2010; Correia-de-Sa et al., 2013). Although these toxins are not able to block neuromuscular transmission in vivo, it may be a useful experimental approach to investigate both the mechanism of action and the structural–activity relationship of the myotoxic Lys49- PLA2s. In this work we report structural and functional studies with a basic Lys49-PLA2 from Bothrops moojeni, known as Myotoxin II or MjTX-II. B. moojeni snakes are found in central and southeastern part of the Brazil and also in some parts of Argentina, Paraguay and Bolivia, living mainly in “cerrado” and “araucaria forests” ecosystems (Borges and Araujo, 1998). Their study have clinical and scientific importance because of the number of accidents caused by these snakes due to their aggressive behavior, their large size compared to other snakes from the same genus and because their adaptive capacity against environmental changes (Melgarejo, 2003). MjTX-II has 122 amino acids, molecular weight of approximately 13.5 kDa (Lomonte et al., 1990; Watanabe et al., 2005), and presents myo- toxic activity that is characterized by increase of serum creatine kinase and morphologic changes in mice muscles when studied in vivo and in vitro (Stabeli et al., 2006; Cavalcante et al., 2007). In addition, it was demonstrated that this protein presents antimicrobial, antitumoral and antiparasitic effects, having therefore potential to ther- apeutical applications (Stabeli et al., 2006). Although the crystal structure of MjTX-II had been re- ported in the literature in 1997 (de Azevedo et al., 1997), the article just presents the comparison of this structure with BaspTX-II (myotoxin II from Bothrops asper) that was the only Lys49-PLA2 structure known at that data. Further- more, the authors did not deposit the coordinates of MjTX- II structure in PDB data bank making any comparison with other structures impossible. In 2005, the structure of the complex formed between MjTX-II and stearic acid was solved (Watanabe et al., 2005), revealing the ligand binding sites and comparing it to PrTX-II/fatty acid structure that was solved in 2001 (Lee et al., 2001). Since then, several structures of native and complexed Lys49-PLA2s have been solved revealing some consensual features of these pro- teins (e.g. homodimeric conformation) but bringing many controversial and intriguing issues (e.g. biological assem- bly, myotoxic site, the role of Lys122 residue) (Murakami et al., 2005; dos Santos et al., 2009; Fernandes et al., 2010; Marchi-Salvador et al., 2009; dos Santos et al., 2011b). Then, in this article we try to definitively address these issues for Lys49-PLA2s in general and to highlight some specific characteristics of MjTX-II which may be very important considering the medical and scientific impor- tance of Lys49-PLA2s proteins for the establishment of myonecrosis. 2. Materials and methods 2.1. Crystallization, X-ray data collection and data processing A lyophilized sample of MjTX-II was dissolved in ultra- pure water at a concentration of 11 mg mL�1. MjTX-II crystals were obtained by the hanging-drop vapor-diffu- sion method (Ducruix and Giegé, 1992) in which 1 ml pro- tein solution and 1 ml reservoir solution were mixed and equilibrated against 500 ml of the precipitant solution. Single crystals were obtained using a solution containing 20% (v/v) 2-propanol, 20% (w/v) polyethylene Glycol 4000 and 1.0 M Sodium Citrate pH 5.6. The crystals measured 0.30 � 0.25 � 0.15 mm after growing approximately one month at 291 K. X-ray diffraction data were collected using wavelength of 1.423 �A at a synchrotron-radiation source (MX2 beam- line – Laboratório Nacional de Luz Síncrotron, LNLS, Cam- pinas, Brazil) using a MAR CCD imaging-plate detector (MAR Research�). The crystals submitted to X-ray diffrac- tion experiments were held in appropriate nylon loops and flash-cooled in a stream of nitrogen at 100 K without cryoprotectant. The best data set was collected with a crystal-to-detector distance of 75 mm and an oscillation range of 1� resulting in 104 images collected. The data were processed at 1.92 �A resolution using the HKL program package (Otwinowski and Minor, 1997) showing the crys- tals belong to P212121 space group and that they are isomorphous to the crystals of MjTX-II complexed to stearic G.H.M. Salvador et al. / Toxicon 72 (2013) 52–6354 acid (Watanabe et al., 2005). X-ray diffraction data pro- cessing and refinement statistics are shown in Table 1. 2.2. Structure determination and refinement The crystal structure was solved by the Molecular Replacement Method using the program MOLREP (Vagin and Teplyakov, 1997) from CCP4 package v.6.1.13 (Potterton et al., 2004) and atomic coordinates of MjTX-II/ stearic acid complex (monomer A with the stearic acid ligand omitted was used - PDB access code 1XXS) (Watanabe et al., 2005). Rounds of crystallographic refine- ment with CNS v.1.3 (Brunger et al., 1998) and manual modeling using the programCoot v.0.7 (EmsleyandCowtan, 2004) were used to improve the model, considering Rcryst and free R-factors. Polyethylene glycol (PEG) 4000, iso- propanol and solvent molecules were added by CNS v.1.3 and Coot v.0.7 programs. Due to the lack of electron density in some regions of the model, the following amino acid side chainswerenotmodeled:monomerA– Lys16, Lys36, Lys70, Glu86, Asn88 and Lys128; monomer B – Lys16, Lys57, Lys69, Lys70 and Lys128. The final model was checked in MolPro- bity program (http://molprobity.biochem.duke.edu/) (Chen et al., 2010). The coordinates were deposited in the Protein Data Bank with identification code 4KF3. 2.3. Comparative analysis Molecular comparisons of the structures were per- formed using the Coot v.0.7 program (Emsley and Cowtan, 2004) with only Ca coordinates. The structures of MjTX-II/ stearic acid (PDB ID 1XXS) (Watanabe et al., 2005), BaspTX-II (PDB ID 1CLP) in its native form (Arni et al., 1995) Table 1 X-ray data collection and refinement statistics for the MjTX-II crystallo- graphic structure. Unit cell (�A) a ¼ 50.0; b ¼ 62.2; c ¼ 86.0 Space group P212121 Resolution (�A) 50.00–1.92 (1.99–1.92)a Unique reflections 19571 (2036)a Completeness (%) 92.4 (97.6)a I/s (I) 10.4 (2.0)a Redundancy 3.9 (3.7)a Molecules in ASU 2 Matthews coefficient VM (�A3Da�1) 2.5 Rmerge b (%) 11.0 (41.9)a Rcryst 22.8 Rfree 25.7 Number of non-hydrogen atoms Protein 1916 Waters 186 PEG molecules 4 Isopropanol molecules 6 Mean B-factor (�A2)c Overall 56.12 Ramachandran plot (%)d Residues were in favored 96.7 Residues were in allowed 3.3 a Numbers in parenthesis are for the highest resolution shell. b Rmerge ¼ Shkl[Si(Ihkl,i �j)]/Shkl, , where Ihkl,i is the intensity of an individual measurement of the reflection with Miller indices h, k and l, and s the mean intensity of that reflection. Calculated for I > �3s (I). c Calculated with CNS program. d Calculated with MolProbity program. and complexed to suramin (PDB ID 1Y4L) (Murakami et al., 2005), BthTX-I (PDB ID 3HZD), BthTX-I/PEG4000 (PDB ID 3IQ3), BthTX-I/BPB (PDB ID 3HZW), PrTX-I/BPB (PDB ID 2OK9) (Fernandes et al., 2010), BthTX-I/a – tocopherol (PDB ID 3CXI), PrTX-I (PDB ID 2Q2J), PrTX-I/a – tocopherol (PDB ID 3CYL) (dos Santos et al., 2009), PrTX-I/Rosmarinic acid (PDB ID 3QNL) (dos Santos et al., 2011a), PrTX-II/fatty acid (PDB ID 1QLL) (Lee et al., 2001), BnSP-6 and BnSP-7 (PDB ID 1PC9 and 1PA0 respectively) (Magro et al., 2003) and BnIV/ Myristic acid (PDB ID 3MLM) (Delatorre et al., 2011) were used in the comparative analysis. All the structuralfiguresweregenerated using the Pymol program (DeLano, 2002). Analysis of the quaternary as- semblies and interfacial contacts of the crystallographic models were performed using the online interactive tool PISA (Krissinel andHenrick, 2007) available at the European Bioinformatics Institute server (http://www.ebi.ac.uk). 2.4. Dynamic light scattering Dynamic light scattering (DLS) experiments were executed at 283 K using a DynaPro TITAN� (Wyatt Tech- nology�) device.Onehundredmeasurementswere acquired with the protein dissolved inultra-purewater at 3.5mgmL�1 concentration. Analyses of these data were performed with Dynamics v.6.10 program (Wyatt Technology�). 2.5. Myographic study Adult male Swiss mice (20–25 g) were killed by exsan- guination after cervical dislocation. The mouse phrenic nerve-diaphragm muscle was removed and mounted vertically under a tension of 5 g in a conventional isolated organ bath chamber containing 15 ml of Ringer solution, with the following composition (mol/l): NaCl, 135; KCl, 5; MgCl2, 1; CaCl2, 2; NaHCO3, 15; Na2HPO4, 1; glucose, 11. This solution was gassed with O2 (95%) þ CO2 (5%) and kept at 35 � 2 �C. The preparation was attached to an isometric force transducer (Grass, FT03) coupled to a signal amplifier (Gould Systems,13-6615-50). The recordings weremade on a computer through data acquisition system (Gould Sytems, Summit ACQuire and Summit DataViewer). The preparation was stabilized for at least 45 min before the toxin addition. Indirect contractions were evoked by supramaximal strength pulses (0.2 Hz; 0.5 ms; 3 V), delivered by an electronic stimulator (Grass S88K) and applied on the phrenic nerve by suction electrode. Direct contractions were evoked by supramaximal pulses (0.2 Hz; 5 ms; 13 V) through a bipolar electrode positioned on opposite sides of the muscle. Experiments of direct con- tractions were performed in the presence of D-tubocurarine (5 mg/ml) previously to toxin addition. The amplitudes of indirect and direct twitches were evaluated during 90 and 120 min respectively and the time required to reach 50% paralysis (t1/2) was determined in each situation. 2.6. Electrophysiological study The mouse phrenic diaphragm muscle was removed and fixed in an isolated organ bath chamber containing 5 ml of Ringer solution. The resting membrane potentials http://molprobity.biochem.duke.edu/ http://www.ebi.ac.uk G.H.M. Salvador et al. / Toxicon 72 (2013) 52–63 55 (MP) and miniature endplate potentials (MEPP) were measured by standard microelectrode techniques (Fatt and Katz, 1951). The glass microelectrodes were filled with 3 M KCl and introduced intracellularly in the muscle fibers with a micromanipulator (Leitz). Microelectrodes were attached to a preamplifier (World Precision Instruments, Electro 70s) coupled to an amplification system (Biopac Systems, MP450) and monitored on an oscilloscope (Tektronix, 2232) and on a computer with a data acquisition and analysis system (AcqKnowledge�, version 3.8.2). The resting MP was recorded at times 5, 15, 30, 60 and 90 min and MEPPs at 5, 30, 60 and 90 min after MjTX-II adminis- tration. Recording sites were rejected if the membrane potential was less than – 65 mV on the initial impalement. 2.7. Ethics Institutional Animal Care andUseCommittee (Institute of Biosciences – Sao Paulo State University – UNESP) approved this study under the number 033/05. Animal procedures were in accordance with the guidelines for animal care prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. 2.8. Statistical analysis Results are expressed asmean� S.E. Datawere analyzed by ANOVA complemented by the Tukey–Kramer test. Values of P < 0.05 were considered significant. Fig. 1. Crystal structure of MjTX-II (A) and its superposition with MjTX-II/stearic aci ligand molecules are shown in sticks: in yellow, PEG4K from MjTX-II structure, in g PEG4K from BthTX-I/PEG4K structure. Drawn using PyMol program (DeLano, 2002). is referred to the web version of this article.) 3. Results and discussion 3.1. Overall crystallographic structure The crystal structure of MjTX-II was solved at 1.92 �A resolution reveling an asymmetric unit containing two monomers. As shown in Table 1, the refinement of the model converged to a final Rcryst of 22.8% and an Rfree of 25.7%. The final model is constituted by 1916 non-hydrogen protein atoms, 186 water, four polyethylene glycol 4000 (PEG4K) and six isopropanol molecules. The overall ste- reochemical quality of the final MjTX-II structure was judged as satisfactory since 96.7% and 100% of the total number of amino acid residues are located in the favored and allowed regions of the Ramachandran plot respec- tively, according to their 4/j angle combinations. MjTX-II structure is stabilized by seven disulfide bridges and pre- serves the classical secondary structure elements found in this group of proteins, i.e., an N-terminal a-helix, a “short” helix, a non-functional Ca2þ-binding loop, two anti-parallel a-helices (2 and 3), two short strands of anti-parallel b- sheet (known as b-wing), and a C-terminal loop (Fig. 1A). MjTX-II structure presents four PEG4K molecules interact- ing with it (Fig. 2): (i) two PEG4K (PEG 1 and 2) molecules are found inside of the hydrophobic channels (one mole- cule in each protein protomer), displaying hydrogen bond with Gly30 and also other interactions with “active site” residues; (ii) one PEG4K (PEG 3) molecule interacts at the same time with the residues Lys49 and Tyr52 from both d (B) and with BthTX-I/PEG4K (C). The protein is represented in cartoon and reen, stearic acid molecules from MjTX-II/stearic acid structure and in cyan, (For interpretation of the references to colour in this figure legend, the reader Fig. 2. Electron density difference maps for PEG4K molecules from the MjTX-II structure: (A) PEG4K (1) in the hydrophobic channel region of monomer A; (B) PEG4K (2) in the hydrophobic channel region of monomer B; (C) PEG4K (3) in the hydrophobic channel region connecting the monomers and (D) PEG4K (4) around Lys7 residue from monomer A. The maps were calculated with coefficients jFobsj�jFcalcj and contoured at 1.0 standard deviations. The ligands molecules were not considered for electron density maps calculation but are drawn for clarity. Drawn using PyMOL program (DeLano, 2002). G.H.M. Salvador et al. / Toxicon 72 (2013) 52–6356 monomers and (iii) one PEG4K (PEG 4) molecule interacts with Lys7, Trp77 and several other residues of monomer A (Fig. 3). Dynamic light scattering experiments indicates a mean hydrodynamic radius (RH) of 2.3 nm with a polydispersity of 12.0%. This RH value corresponds to amolecular weight of approximately 23 kDa and is, thus, equivalent to a dimer. These results are in agreement with other literature data for Lys49-PLA2s since electrophoresis, spectroscopic (Arni et al., 1999; da Silva Giotto et al., 1998), crystallographic (Arni and Ward, 1996; dos Santos et al., 2009; Magro et al., 2003; Murakami et al., 2005), small angle X-ray scattering (Murakami et al., 2007) and dynamic light scattering (Fernandes et al., 2010) experiments demonstrates that bothropic Lys49-PLA2s are dimeric in solution. In addition, the inspection of unit-cell packing showed that there are two possible dimeric configurations for the MjTX-II structure. The first possibility is similar to the so- called “conventional dimer” and the second one is similar to the “alternative dimer” (Murakami et al., 2005). In the conventional dimer, the monomers are stabilized by in- teractions between the tips of b-wings and the residues of the N-terminal helices (Arni and Ward, 1996) while in the alternative dimer they are stabilized by contacts between the putative calcium-binding loops and C-termini forming a connection route between the “active sites” of both monomers (dos Santos et al., 2009). Examination of the unit-cell packing using PISA software (Krissinel and Henrick, 2007) points the alternative dimeric configura- tion is the most probable to occur in solution. According to this analysis, MjTX-II/PEG4K crystallographic structure presents an interfacial area of 552.6�A2, Gint¼�9.8 kcal/mol and Gdiss ¼ 0.145 kcal/mol. Furthermore, this choice is also supported by previous small angle X-ray scattering exper- iments (Murakami et al., 2007) and functional aspects of Lys-PLA2s myotoxins (dos Santos et al., 2009; Murakami et al., 2005). 3.2. Comparison between MjTX-II and MjTX-II complexed to stearic acid structures The crystal structure of MjTX-II co-crystallized with stearic acid (a fatty acid) has been previously solved (Watanabe et al., 2005) and evidenced six stearic acid molecules interactingwith the protein: two of them in each hydrophobic channel (two molecules in each protomer) and other two in the dimeric interface interactingwith Lys7 residue. Contrasting with the co-crystallized structure (MjTX-II/stearic acid), the native MjTX-II (this study) only presents four PEG4K molecules: three of them are inside the hydrophobic channels and the fourth one interacts with Lys7 residue (Fig. 1A). However, the comparison of both structures reveals that all ligands occupy similar positions: (i) PEG 1 and PEG 2 occupy the same sites that two stearic acids from the MjTX-II/stearic acid complex (inside of the hydrophobic channels) (Fig. 1B); (ii) PEG 3 is at the hy- drophobic channels entrance (N-terminal face of the dimeric structure), connecting both protomers of the dimeric structure and is located approximately at the same position that two stearic acids molecules in the MjTX-II complexed structure (Fig. 1B); (iii) PEG 4 occupies approximately the same position of two stearic acids in the dimeric interface of MjTX-II/stearic acid structure which presents 50% occupancy values and are sited in a tail-to-tail conformation (Fig. 1B) (Watanabe et al., 2005). Due to the alternative dimeric configuration adopted for the native Fig. 3. Interaction of PEG4K molecules in MjTX-II structure. (A) and (B) represents the interactions of PEG4K (1 and 2) molecules that occupies the hydrophobic channel of the structure. (C) Hydrophobic interactions of PEG4K (3) molecule that connects both monomers. (D) Hydrophobic interactions of PEG4K (4) molecule in the protein surface (around Lys7 from monomer A). Drawn using Ligplot program (Wallace et al., 1995). G.H.M. Salvador et al. / Toxicon 72 (2013) 52–63 57 G.H.M. Salvador et al. / Toxicon 72 (2013) 52–6358 MjTX-II only one PEG ligand with 100% occupancy was modeled at this site. Therefore, despite the differences between both ligands (PEG4K and stearic acid), both structures are essentially identical as evidenced by the root-mean-square deviation (r.m.s.d.) of 0.52 �A for their Ca atoms superposition. Important regions for this toxin biological functions (e.g. the active site, Ca2þ-binding loop, C-termini (dos Santos et al., 2009; Fernandes et al., 2010; Magro et al., 2003)) are conserved indicating that PEG4K may structurally simulate a fatty acid molecule bound to toxin’s hydrophobic channels since its backbone is structurally similar to the protein substrate (Watanabe et al., 2005). For this reason, we can state that the MjTX-II structure may represent the protein in its active state (attached to the membrane) (dos Santos et al., 2009). 3.3. Comparison of MjTX-II and other Lys49-PLA2 structures Several myotoxic Lys49-PLA2s in the apo and complexed forms have been solved (Arni et al., 1999; dos Santos et al., 2011a; dos Santos et al., 2009; Fernandes et al., 2010; Lee et al., 2001; Magro et al., 2003; Marchi-Salvador et al., 2009; Murakami et al., 2005, 2007; Watanabe et al., 2005). Table 2 shows a structural comparison between the monomers of MjTX-II and the same analysis for several other apo and complexed Lys49-PLA2s. As previously observed (dos Santos et al., 2009), all complexed structures present lower r.m.s.d. values compared to their respective apo structures. In other words, there is a clear structural pattern for Lys49-PLA2s whose apo and complexed states can also be distinguished by the “two angle” model previ- ously suggested (dos Santos et al., 2009). Applying this model to MjTX-II structure, the aperture and torsional an- gles between its monomers are 55� and 25�, respectively. These values are in agreement to those calculated for MjTX- II/stearic acid structure (52� and 20�) and are also similar to values found for other complexed Lys49-PLA2s (Table 3) (dos Santos et al., 2009). In 2001, Lee and colleagues solved the PrTX-II/fatty acid structure and suggested an important role played by Lys122. According to the authors, Lys122 interacts with the Table 2 Superposition between Lys49-PLA2s monomers (root mean square devi- ation of Ca atoms). Protein (PDB id) r.m.s.d. (�A) MjTX-II/PEG4000 0.23 MjTX-II/Stearic acid (1XXS) 0.26 BaspTX-II/Suramin (1Y4L) 0.68 BthTX-I/a-tocopherol (3CXI) 0.76 PrTX-I/a-tocopherol (3CYL) 0.82 BthTX-I/BPB (3HZW) 0.67 PrTX-I/BPB (2OK9) 0.47 BthTX-I/PEG4000 (3IQ3) 0.79 PrTX-I/Rosmarinic acid (3QNL) 0.32 BnIV/Myristic acid (3MLM) 0.55 PrTX-II/Fatty acid (1QLL) 0.25 BaspTX-II (1CLP) 0.60 BnSP-6 (1PC9) 0.96 BnSP-7 (1PA0) 0.96 PrTX-I (2Q2J) 1.04 BthTX-I (3HZD) 1.05 main chain carbonyl of Cys29 causing hyperpolarization of the Cys29/Gly30 peptide bond and, consequently, increases the affinity of the toxin for fatty acids (Lee et al., 2001). This hypothesis suggested that Lys49-PLA2s are enzymes that are able to hydrolyze phospholipids but fail to release the products of its action. The fatty acid would stay retained in the hydrophobic channel of the toxin consequently inhib- iting it, therefore explaining why Lys49-PLA2s toxins do not display significant catalytic activity. In contrast with this hypothesis, Fernandes and colleagues (Fernandes et al., 2010) performed a very comprehensive study using 16 different dimeric Lys49-PLA2s and showed that Lys 122 is a very flexible residue that may adopt random configurations even though it usually interacts with different negative charged sites. Despite the highlighted absence of pattern for Lys122 interaction, PrTX-II complexed to fatty acid and MjTX-II complexed to stearic acid structures are two observed exceptions (Lee et al., 2001; Watanabe et al., 2005). The Lys122 side chain interacts with main chain carbonyl of Cys29 in both monomers of these dimeric complexes indicating that the phenomena just occur when fatty acids are present in the hydrophobic channels (Fernandes et al., 2010). Interesting, the results presented here for the MjTX-II can clarify this issue. As discussed in the item 3.2, the structure of MjTX-II presents PEG4K molecules in its hydrophobic channels at the same regions where fatty acids are found in the MjTX-II/stearic acid structure. Therefore, this finding suggests that the Cys29- Lys122 interaction is not exclusive for Lys49-PLA2s-fatty acid bound structures. Additionally, comparison between BthTX-I/PEG4K (Fernandes et al., 2010) and MjTX-II/PEG4K (this work) structures reveals important features of MjTX-II in com- parison to BthTX-I and other bothropic Lys49-PLA2s myo- toxins. Despite the high sequential and structural similarity between MjTX-II and BthTX-I (they share 95% of identity), and the presence of two PEG4K molecules in the hydro- phobic channels of both structures, their superposition clearly demonstrates the ligand way of binding at these proteins are somewhat different (Fig.1C). The reason seems to be the insertion of the residue Asn120 and the Leu32Gly mutation since these characteristics apparently shifts the orientation of PEG molecules inside the hydrophobic channel by modification of the channel ends. Interestingly, Asn120 insertion and Leu32Gly mutation are only found in MjTX-II when comparing this protein to all the other bothropic Lys49-PLA2s whose structures are solved to date (Fig. 4). Therefore, the Asn120 insertion is probably the responsible for the binding of the interchain PEG 3 mole- cule since this insertion leads to a distortion of the protein protomers (Table 3). This evidence is enhanced by the fact that other Lys49-PLA2s toxins that presents 121 amino acids do not bind this interchain PEG4K or PEG3350 (Fernandes et al., 2010) whereas MjTX-II (122 residues) allow a PEG4K interaction at this region. It is important to highlight that bothropic Lys49-PLA2s that do not have structures solved yet but present mutations and/or in- sertions at the positions 32 and 120 are known (Fig. 4); however their structural characteristics upon ligand bind- ing will just be evidenced when these structures are elucidated. Table 3 Torsional angles (qT), aperture angles (qA) and hydrogen bonds at the interface of Lys49-PLA2s from Bothrops genus. Protein (PDB id) qT (torsional angle)a qA (aperture angle)a Hydrogen bonds at the interfacesb Monomer A Monomer B Distance �A MjTX-II 55� 25� Ala19 [N] Tyr121 [OH] 2.88 Asn17 [ND2] Tyr121 [OH] 2.29 MjTX-II/Stearic acid (1XXS) 52� 20� Tyr121 [OH] Asn17 [ND2] 2.36 Tyr121 [OH] Ala19 [N] 2.58 Ala19 [N] Tyr 121 [OH] 3.64 BaspTX-II/Suramin (1Y4L) 51� 15� – – – BthTX-I/a-tocopherol (3CXI) 40� 11� Tyr119 [O] Asn17 [ND2] 2.84 Tyr 119 [OH] Tyr119 [OH] 2.57 PrTX-I/a-tocopherol (3CYL) 41� 12� Tyr119 [O] Asn17 [ND2] 2.84 Tyr119 [OH] Tyr119 [OH] 2.48 BthTX-I/BPB (3HZW) 53� 22� Tyr119 [OH] Asn17 [ND2] 3.25 Tyr119 [OH] Tyr119 [OH] 3.09 PrTX-I/BPB (2OK9) 41� 29� Tyr119 [O] Asn17 [ND2] 2.82 Tyr119 [OH] Tyr119 [OH] 2.16 BthTX-I/PEG4000 (3IQ3) 41� 29� Tyr119 [O] Asn17 [ND2] 2.82 Tyr119 [OH] Tyr119 [OH] 2.56 PrTX-I/Rosmarinic acid (3QNL) 43� 23� Tyr119 [O] Asn17 [ND2] 3.16 Tyr119 [OH] Tyr119 [OH] 2.18 Asn17 [ND2] Tyr119 [O] 3.22 BnIV/Myristic acid (3MLM) 41� 27� Tyr119 [OH] Tyr119 [OH] 2.42 Tyr119 [O] Asn17 [ND2] 2.49 PrTX-II/fatty acid (1QLL)c 81� 23� Tyr119 [OH] Tyr119 [OH] 3.09 BaspTX-II (1CLP)d 44� 22� Tyr119 [OH] Tyr119 [OH] 2.35 Tyr119 [OH] Lys20 [NZ] 3.86 BnSP-6 (1PC9) 60� 6� Tyr119 [O] Asn17 [ND2] 2.51 BnSP-7 (1PA0) 60� 6� Lys69 [NZ] Val31 [O] 2.74 Tyr119 [O] Asn17 [ND2] 2.68 PrTX-I (2Q2J) 60� 7� Val31 [O] Lys69 [NZ] 2.57 Asn17 [ND2] Tyr 119 [O] 2.91 Asn17 [ND2] His120 [O] 3.36 BthTX-I (3HZD) 60� 14� Val31 [O] Lys69 [NZ] 2.72 a Torsional and aperture angles were calculated according to the proposal of dos Santos et al., 2009. b Hydrogen bonds were inferred using the online interactive tool PISA (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). c PrTX-II/fatty acid (PDB id 1QLL) crystallographic structure do not follow the torsional/aperture angle pattern for complexed structures as pointed by dos Santos et al., 2009. d BaspTX-II (PDB id1CLP) crystallographic structure was deposited as an apo structure but according to dos Santos et al., 2009 it can be a complex. G.H.M. Salvador et al. / Toxicon 72 (2013) 52–63 59 The C-termini of Lys49-PLA2s toxins has been pointed as containing their myotoxic site (Arni andWard, 1996; Magro et al., 2003; Ward et al., 2002). Afterward, dos Santos and colleagues reviewed several native and complexed Lys49- PLA2s and proposed that Arg118, Lys20 and Lys115 residues constitute these toxins myotoxic site (dos Santos et al., 2009). In the same study, the authors propose a two-step mechanism for Lys49-PLA2s which allow the destabiliza- tion of biological membranes. The first step of the mecha- nism would be the interaction of the Lys20, Lys115 and Arg118 of these proteins with the phospholipid head group region (dos Santos et al., 2009); subsequently, a quaternary rearrangement takes place in Lys49-PLA2s monomers allowing long-chain hydrophobic portions of membrane phospholipids to be inserted into the hydrophobic channel of the toxin (dos Santos et al., 2009; dos Santos et al., 2011a). Therefore, the hydrophobic channelwas demonstrated to be involved in one of the steps required for Lys49-PLA2s action mechanism (dos Santos et al., 2009; dos Santos et al., 2011a). It is also interesting to highlight that if the alternative dimer is considered as biological dimer, the myotoxic sites from both monomers are aligned at the same plane (side by side) for the complexed structures (active state) and an interchain Tyr119-Tyr119 hydrogen bond is formed (Table 3) which increasing the toxin potency (dos Santos et al., 2009). The sequence alignment of bothropic Lys49-PLA2s (Fig. 4) shows that the residues of the myotoxic site (Lys20, Lys115 and Arg118) and Tyr119 are conserved in MjTX-II, however, the interchainTyr119–Tyr119hydrogenbond isnot present in its dimeric interface (these residues are at a distance of 4.7 �A). Analyzing MjTX-II sequence (Fig. 4) it is possible to observe that the C-terminal region of this toxin presents some par- ticularities as an insertion of a residue at position 120 and a mutation at position 121 (His/Tyr) if compared to other bothropic Lys49-PLA2s whose structures are known. There- fore, Asn120 insertionmay be the responsible for a diversion of this region as evidenced by the lack of Tyr119-Tyr119 hydrogen bond which is probably compensated by the cre- ation of two new hydrogen bonds with the participation of Tyr121 residue (Table 3). Then, taking into account these facts (Asn120 insertion and mutations of residues 32 and 121) and their conse- quences to PEG4Ks mode of binding, it is reasonable to suggest that MjTX-II may require specific or modified in- hibitors when compared to molecules that are able to inhibit bothropic Lys49-PLA2s by interaction with their hydrophobic channels. This is due to the different profile of ligand binding presented at this region (Fig. 1C.) and may have implications when considering structure-based ligand design for Lys49-PLA2s. http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver Fig. 4. Amino acid sequence alignment of Lys49-PLA2s from Bothrops genus. The natural substitution Gly32 and the Asn120 insertion are highlighted. NCBI GI entry codes for the proteins represented are BnIV (Bothrops pauloensis): 333361256, BnSP-6 (Bothrops pauloensis): 49258448, BnSP-7 (Bothrops pauloensis): 239938675, BthTX-I (Bothrops jararacussu): 51890398, MjTX-II (Bothrops moojeni): 62738542; BaspTX-II (Bothrops asper): 166215047, PrTX-I (Bothrops pirajai): 17433154, PrTX-II (Bothrops pirajai): 17368328, Basp-M1-3-3 (Bothrops asper): 17433168, BaTX (Bothrops alternatus): 292630846, Batr-1(Bothrops atrox): 82201805, BlK-PLA2 (Bothrops leucurus): 353678055, Myo-IVa (Bothrops asper): 166216293. G.H.M. Salvador et al. / Toxicon 72 (2013) 52–6360 3.4. The crystal structure of MjTX-II and the biological dimer of Lys49-PLA2 structures As discussed in the last two sections, MjTX-II structure was solved in the oligomeric assembly known as “alterna- tive dimer” given that it has higher probability of occur- rence in solution due to bioinformatic analyses and also due to several experimental and functional reasons (dos Santos et al., 2011a; dos Santos et al., 2009; Fernandes et al., 2010; Marchi-Salvador et al., 2009; Murakami et al., 2005, 2007). However, as discussed in a recent review in this field (Lomonte and Rangel, 2012), this subject is still controversial for some authors. Although no experiment was able to definitively prove the correct assembly adopted by Lys49-PLA2s toxins, MjTX- II structure added an important experimental evidence for the choice of the alternative dimer as the probable qua- ternary assembly found in solution for these proteins. As shown in the Fig. 2, the PEG 3 binds simultaneously to both monomers of the protein. This mode of binding is only possible for the alternative dimeric conformation because if the conventional dimer had been used to solve this struc- ture a large portion of the ligand would be exposed to the solvent which is energetically unfavorable. Similar results were also obtained for PrTX-I/a-tocopherol and BaspTX-II/ suramin structures (dos Santos et al., 2009; Murakami et al., 2005), leading these authors to choose the alterna- tive assembly when solving both complexed structures. 3.5. Functional studies Myographic studies show that MjTX-II (1.0 mM) pro- duced an irreversible and time-dependent blockade of both directly (t1/2 ¼ 40.0 � 2.3 min, n ¼ 7) and indirectly (t1/ 2 ¼ 32.1 � 4.8 min, n ¼ 5) evoked twitches (Fig. 5A and B). The present findings were consistent with those already described for other Lys49-PLA2s (Cavalcante et al., 2007; Gallacci et al., 2006; Randazzo-Moura et al., 2008; Rodrigues-Simioni et al., 1995; Soares et al., 2000, 2001; Stabeli et al., 2006). Statistical comparison of the indirectly evoked contractions t1/2 of the MjTX-II with those of other Lys49 PLA2s, such as PrTX-I from Bothrops pirajai (t1/ 2 ¼ 49.0 � 6.9 min, n ¼ 8) and BthTX-I from Bothrops jar- aracussu (t1/2 ¼ 40.3 � 3.5 min, n ¼ 6), obtained at same experimental conditions, indicates that these toxins have similar potency (Cavalcante et al., 2007). In contrast, MjTX- II presents a more potent neuromuscular blockade when compared to MjTX-I from B. moojeni, since at 1.0 mM this toxin depressed in only about 20% the twitches amplitude after 90min of contact with the preparation (Salvador et al., 2013). Then, these results indicate MjTX-II has similar or superior neuromuscular blockade action compared to other Lys49-PLA2s. It has been suggested that in vitro neuromuscular blockade effect observed for Lys49-PLA2s may be a conse- quence of their membrane depolarizing activity (Gallacci and Cavalcante, 2010). In order to clarify this issue, we Fig. 5. Effect of MjTX-II on indirectly (A) and directly (B) evoked twitches in mice phrenic-diaphragm preparations. The ordinate represents the % amplitude of twitches relative to the initial amplitude. The abscissa indicates the time (min) after the addition of MjTX-II to the organ bath. Vertical bars represent the SEM, *indicates the point from which there are significant differences relative to control (P < 0.05). G.H.M. Salvador et al. / Toxicon 72 (2013) 52–63 61 performed an electrophysiological study to evaluate membrane depolarizing activity induced by MjTX-II. This technique measures the resting membrane potential of the sarcolemma and has been used as a more direct and sen- sitive method to assess the initial events in myotoxicity (Aragao et al., 2009). The results show a progressive in- crease in the restingmembrane potential of skeletal muscle fibers from 15 min onwards (Fig. 6) and the increase of the frequency of miniature endplate potentials after 5 min (data not shown), probably as a consequence of the cell depolarization. Taken together, these results show a direct effect of MjTX-II increasing the permeability of the skeletal muscle fibers plasma membrane. Although the Lys49-PLA2s mechanism of action is not yet fully elucidated, several studies point the sarcolemma Fig. 6. The resting membrane potential of mice diaphragm muscle prepa- rations under MjTX-II action. The ordinate represents the resting membrane potential (mV) and the abscissa indicates the time (min) after the addition of MjTX-II to the organ bath. The data were grouped as mean � SEM (P < 0.05). *indicates the point from which there are significant differences relative to control (P < 0.05). as the initial target of these myotoxins (Gutierrez and Lomonte, 1995; Lomonte et al., 2003; Lomonte and Rangel, 2012). A recent study suggests Arg118, together with Lys20 and Lys115, are the residues responsible for protein-membrane anchorage initial steps (dos Santos et al., 2009; dos Santos et al., 2011a). After the establish- ment of these electrostatic interactions, the protein un- dergoes a quaternary rearrangement that allows hydrophobic portions of membrane phospholipids to be inserted in the protein hydrophobic channels, therefore culminating with membrane destabilization (dos Santos et al., 2011a). The first consequence of this destabilization is the loss of ionic permeability regulation, leading to a reduction of the resting membrane potential, inactivation of sodium channels and blockade of both directly and indirectly evoked contractions (Gallacci and Cavalcante, 2010). In addition, the disruption of the muscle fiber membranes induced by Lys49-PLA2s also promotes an in- crease of cytosolic calcium concentration, initiating a complex series of degenerative mechanisms that culmi- nates with the muscle cell damage (Gutierrez and Ownby, 2003; Lomonte and Rangel, 2012; Montecucco et al., 2008). 4. Conclusions In this article, we fully characterize functionally and structurally the Lys49-PLA2MjTX-II from B.moojeni. Despite the fact that this class of proteins has been extensively studied, several issues regarding the function–structure relationships are still need tobe clarified, as highlightedbya recent review in this field (Lomonte and Rangel, 2012). This requirement is probably due to the high evolutionary pressure process by which snake venom molecules are submitted, since proteins with few natural amino acid mutations may present different oligomeric configurations, variable toxic potency or even different functions when compared to their ancestral toxins (Doley and Kini, 2009; dos Santos et al., 2011b; Kini, 1997, 2003). An interesting example is the MjTX-I, other myotoxic Lys49-PLA2 from B. G.H.M. Salvador et al. / Toxicon 72 (2013) 52–6362 moojeni that presents unusual oligomeric characteristics and displays lower myotoxic activity when compared to all other bothropic Lys49-PLA2s that have already been struc- turally and functionally characterized (Andriao-Escarso et al., 2000; Salvador et al., 2013). As demonstrated in this work, MjTX-II also presents some particularities if compared to other Lys49-PLA2s which seem to influence the mode of ligand binding along the toxin hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s. Acknowledgments This work was supported by Fundação de Amparo à Pes- quisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financia- dora de Estudos e Projetos (FINEP), Coordenação de Aper- feiçoamento deNível Superior (CAPES) – Projeto NanoBiotec, Rede de Biodiversidade e Biotecnologia da Amazônia Legal (BIONORTE/CNPq/MCT), Instituto Nacional para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INCT-INPeTAm/CNPq/MCT) e Instituto Nacional para Pes- quisa em Toxinas (INCT-Tox), Secretary of Development of Rondonia State (SEPLAN/PRONEX/CNPq). Conflict of interest The authors have no conflicts of interest with this work. 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methods 2.1 Crystallization, X-ray data collection and data processing 2.2 Structure determination and refinement 2.3 Comparative analysis 2.4 Dynamic light scattering 2.5 Myographic study 2.6 Electrophysiological study 2.7 Ethics 2.8 Statistical analysis 3 Results and discussion 3.1 Overall crystallographic structure 3.2 Comparison between MjTX-II and MjTX-II complexed to stearic acid structures 3.3 Comparison of MjTX-II and other Lys49-PLA2 structures 3.4 The crystal structure of MjTX-II and the biological dimer of Lys49-PLA2 structures 3.5 Functional studies 4 Conclusions Acknowledgments Conflict of interest References