Structural Basis of Membrane-induced Cardiotoxin A3 Oligomerization

Cobra cardiotoxins (CTXs) have previously been shown to induce membrane fusion of vesicles formed by phospholipids such as cardiolipin or sphingomyelin. CTX can also form a pore in membrane bilayers containing a anionic lipid such as phosphatidylserine or phosphatidylglycerol. Herein, we show that the interaction of CTX with negatively charged lipids causes CTX dimerization, an important intermediate for the eventual oligomerization of CTX during the CTX-induced fusion and pore formation process. The structural basis of the lipid-induced oligomerization of CTX A3, a major CTX from Naja atra, is then illustrated by the crystal structure of CTX A3 in complex with SDS; SDS likely mimics anionic lipids of the membrane under micelle conditions at 1.9-Å resolution. The crystal packing reveals distinct SDS-free and SDS-rich regions; in the latter two types of interconnecting CTX A3 dimers, D1 and D2, and several SDS molecules can be identified to stabilize D1 and D2 by simultaneously interacting with residues at each dimer interface. When the three CTXSDS complexes in the asymmetric unit are overlaid, the orientation of CTX A3 monomers relative to the SDS molecules in the crystal is strikingly similar to that of the toxin with respect to model membranes as determined by NMR and Fourier transform infrared methods. These results not only illustrate how lipid-induced CTX dimer formation may be transformed into oligomers either as inverted micelles of fusion intermediates or as membrane pore of anionic lipid bilayers but also underscore a potential role for SDS in x-ray diffraction study of protein-membrane interactions in the future. Cobra cardiotoxins (CTXs) have previously been shown to induce membrane fusion of vesicles formed by phospholipids such as cardiolipin or sphingomyelin. CTX can also form a pore in membrane bilayers containing a anionic lipid such as phosphatidylserine or phosphatidylglycerol. Herein, we show that the interaction of CTX with negatively charged lipids causes CTX dimerization, an important intermediate for the eventual oligomerization of CTX during the CTX-induced fusion and pore formation process. The structural basis of the lipid-induced oligomerization of CTX A3, a major CTX from Naja atra, is then illustrated by the crystal structure of CTX A3 in complex with SDS; SDS likely mimics anionic lipids of the membrane under micelle conditions at 1.9-Å resolution. The crystal packing reveals distinct SDS-free and SDS-rich regions; in the latter two types of interconnecting CTX A3 dimers, D1 and D2, and several SDS molecules can be identified to stabilize D1 and D2 by simultaneously interacting with residues at each dimer interface. When the three CTXSDS complexes in the asymmetric unit are overlaid, the orientation of CTX A3 monomers relative to the SDS molecules in the crystal is strikingly similar to that of the toxin with respect to model membranes as determined by NMR and Fourier transform infrared methods. These results not only illustrate how lipid-induced CTX dimer formation may be transformed into oligomers either as inverted micelles of fusion intermediates or as membrane pore of anionic lipid bilayers but also underscore a potential role for SDS in x-ray diffraction study of protein-membrane interactions in the future. Cobra cardiotoxins (CTXs) 1The abbreviations used are: CTX, cardiotoxin; PC, phosphatidylcholine; PS, phosphatidylserine; PG, phosphatidylglycerol; PA, phosphatidic acid; ATR, attenuated total reflection; FTIR spectroscopy, Fourier transform infrared spectroscopy; Rh, rhodamine; HPLC, high performance liquid chromatography; 6-CF, 6-carboxyfluorescein; ATR, attenuated total reflection. are amphiphilic three-finger (L1-L3) basic polypeptides that bind to cell membranes and depolarize cardiomyocytes to cause systolic heart arrest in the envenomed victim (1Housset D. Fontecilla-Camps J.C. Parker M.W. Protein Toxin Structure. Springer-Verlag, Berlin1996: 271-290Crossref Google Scholar). CTX has also been named cytotoxin because it brings about membrane leakage against many cells including red blood cells and phospholipid membrane vesicles (2Dufton M.J. Hider R.C. Pharmacol. Ther. 1987; 36: 1-40Crossref Scopus (134) Google Scholar). This effect is due in part to the interaction of CTX with phospholipid bilayer. For instance, CTX-induced fusions of zwitterionic sphingomyelin vesicles and negatively charged cardiolipin model membranes have been reported, respectively, for CTXs from Taiwan cobra (Naja atra) and African cobra (Naja mossambica) venom (3Aripov T.F. Gasanov S.E. Salakhutdinov B.A. Rozenshtein I.A. Kamaev F.G. Gen. Physiol. Biophys. 1989; 8: 459-474PubMed Google Scholar, 4Chien K.-Y. Huang W.N. Jean J.-H. Wu W. J. Biol. Chem. 1991; 266: 3252-3259Abstract Full Text PDF PubMed Google Scholar, 5Batenburg A.M. Bougis P.E. Rochat H. Verkleij A.J. de Kruijff B. Biochemstry. 1985; 24: 7101-7110Crossref PubMed Scopus (89) Google Scholar). After a deep penetration of CTX II of N. mossambica into the acyl chain region of anionic lipid bilayers, an enhanced lipid mixing, as detected by fluorescence fusion essay and the appearance of fusion intermediate of well defined particles, presumably inverted micelles, as observed by freeze-fracture electron microscopy, have been shown to occur during the CTX-induced fusion process of cardiolipin vesicles (5Batenburg A.M. Bougis P.E. Rochat H. Verkleij A.J. de Kruijff B. Biochemstry. 1985; 24: 7101-7110Crossref PubMed Scopus (89) Google Scholar). Apparently the reorganization of CTX-lipid complex plays an important role in the aforementioned CTX-induced membrane-related activity. The lytic property of CTXs is attributed to the coexistence of an exposed hydrophobic patch and a cluster of basic residues forming a cationic zone (6Rees B. Bilwes A. Chem. Res. Toxicol. 1993; 6: 285-405Google Scholar). Based on the phosphatidylcholine (PC) membrane binding activities of CTXs, two distinct types of CTX, P-(Pro-30-containing) and S-(Ser-28-containing), have been identified (7Chien K.Y. Chiang C.M. Hseu Y.C. Vyas A.A. Rule G.S. Wu W. J. Biol. Chem. 1994; 269: 14473-14483Abstract Full Text PDF PubMed Google Scholar, 8Efremov R.G. Volynsky P.E. Nolde D.E. Dubovskii P.V. Arseniev A.S. Biophys J. 2002; 83: 144-153Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), of which the P-type CTX interacts more strongly than the S-type with membranes. The presence of the proline in P-type CTX imposes a ω-like conformation on L2 that tightly binds to a water molecule, which plays an important role in the CTX membrane binding activity (9Bilwes A. Rees B. Moras D. Menez R. Menez A. J. Mol. Biol. 1994; 239: 122-136Crossref PubMed Scopus (101) Google Scholar, 10Sun Y.J. Wu W.G. Chiang C.M. Hsin A.Y. Hsiao C.D. Biochemistry. 1997; 36: 2403-2413Crossref PubMed Scopus (56) Google Scholar, 11Sue S.C. Jarrell H.C. Brisson J.R. Wu W. Biochemistry. 2001; 40: 12782-12794Crossref PubMed Scopus (20) Google Scholar). The presence of low sequence homology in L1 regions among CTXs (Fig. 1A) also hints at toxic specificity of these polypeptides. For instance, local conformational changes in L1 (Val-7–Pro-8 peptide bond) of cytotoxin II from Naja oxiana strongly reduce the binding of its minor (with cis-Pro-8) form as compared with the major (with trans-Pro-8) one to membranes (8Efremov R.G. Volynsky P.E. Nolde D.E. Dubovskii P.V. Arseniev A.S. Biophys J. 2002; 83: 144-153Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 12Dubovskii P.V. Dementieva D.V. Bocharov E.V. Utkin Y.N. Arseniev A.S. J. Mol. Biol. 2001; 305: 137-149Crossref PubMed Scopus (61) Google Scholar). CTX A3, a major component (>50% dried weight of all CTXs) of the venom of Taiwan cobra (2Dufton M.J. Hider R.C. Pharmacol. Ther. 1987; 36: 1-40Crossref Scopus (134) Google Scholar, 4Chien K.-Y. Huang W.N. Jean J.-H. Wu W. J. Biol. Chem. 1991; 266: 3252-3259Abstract Full Text PDF PubMed Google Scholar), is a 60-residue P-type CTX (Fig. 1A). Like other CTXs, CTX A3 is a basic protein (pI = 9.38) that is capable of depolarizing cardiomyocytes and possesses lytic activity on many other cells. CTX A5, a minor component of the venom of the Taiwan cobra, is a 62-residue P-type CTX with strong lipid binding capability. Although it lacks cardiotoxicity, CTX A5 is also called cardiotoxin because its amino acid sequence is homologous to that of CTX A3 (4Chien K.-Y. Huang W.N. Jean J.-H. Wu W. J. Biol. Chem. 1991; 266: 3252-3259Abstract Full Text PDF PubMed Google Scholar, 7Chien K.Y. Chiang C.M. Hseu Y.C. Vyas A.A. Rule G.S. Wu W. J. Biol. Chem. 1994; 269: 14473-14483Abstract Full Text PDF PubMed Google Scholar). Fluorescence and NMR studies of CTXs in the presence of zwitterionic PC micelles indicate that L1-L3 become perturbed (7Chien K.Y. Chiang C.M. Hseu Y.C. Vyas A.A. Rule G.S. Wu W. J. Biol. Chem. 1994; 269: 14473-14483Abstract Full Text PDF PubMed Google Scholar, 12Dubovskii P.V. Dementieva D.V. Bocharov E.V. Utkin Y.N. Arseniev A.S. J. Mol. Biol. 2001; 305: 137-149Crossref PubMed Scopus (61) Google Scholar, 13Dauplais M. Neumann J.M. Pinkasfeld S. Menez A. Roumestand C. Eur. J. Biochem. 1995; 230: 213-220Crossref PubMed Scopus (49) Google Scholar, 14Sue S.C. Chien K.Y. Huang W.N. Abraham Wu W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). studies that of negatively charged such as and phosphatidic acid induce a in the of CTX A3 (7Chien K.Y. Chiang C.M. Hseu Y.C. Vyas A.A. Rule G.S. Wu W. J. Biol. Chem. 1994; 269: 14473-14483Abstract Full Text PDF PubMed Google Scholar, 13Dauplais M. Neumann J.M. Pinkasfeld S. Menez A. Roumestand C. Eur. J. Biochem. 1995; 230: 213-220Crossref PubMed Scopus (49) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, C.M. Wu W. Biochemistry. PubMed Scopus Google Scholar). these and to an into the of CTX-induced membrane leakage and fusion we CTX A3 with SDS and determined its structure under micelle The results also the high model of CTX-lipid complex to how lipid-induced CTX may to the CTX oligomerization for the formation of fusion intermediate and membrane in the previously CTX-induced membrane and and and from The phospholipids of PC, phosphatidylserine PG, and used in study from these phospholipids acyl are named and CTX A3 and CTX A5 by by on a from venom of N. previously (7Chien K.Y. Chiang C.M. Hseu Y.C. Vyas A.A. Rule G.S. Wu W. J. Biol. Chem. 1994; 269: 14473-14483Abstract Full Text PDF PubMed Google Scholar). dried under and then with containing The and several and a with the pore of for For the pore the and or A.S. Biophys J. 1997; Full Text PDF PubMed Scopus Google Scholar). used in formed in the presence of and used to the molecules of the and the lipid determined by as Biochem. PubMed Scopus Google Scholar). of of residues as J. Biochem. 1987; PubMed Scopus Google with a CTX A3 in containing with at The by by and one be from by and of The identified of CTXs and by of detected by fluorescence Although low fluorescence at high its at low containing at a of of in a After the of CTX, the fluorescence as a of for the CTX-induced leakage process. The leakage the leakage = is the fluorescence is the fluorescence at and is the fluorescence determined by (4Chien K.-Y. Huang W.N. Jean J.-H. Wu W. J. Biol. Chem. 1991; 266: 3252-3259Abstract Full Text PDF PubMed Google Scholar). The and and and either with to the of or with CTX A3, to After to a with an of The determined by a fluorescence and and the of a used to the of When the leakage and the to the leakage determined by the of the to = and the total and leakage of The defined by the of leakage of and by with of CTX A3 and of CTX A3 in complex with to with cell of a = = and = by the of the protein with of containing and which is well its micelle in liquid by on an on a and W. 1997; PubMed Scopus Google Scholar). The structure by the the crystal structure of as the J. A. 1994; Scopus Google Scholar), the containing three molecules in the asymmetric unit an of After the of which from that of the model as by of residues and the of SDS and water the to about the L2 of each in the asymmetric unit a not during for which J. M. D. PubMed Scopus Google and D.E. J. Biol. PubMed Scopus Google The model with and of and from to 1.9-Å several of M.W. J.M. J. 1993; Google that of the residues are in the region with the residues in the have been in the Protein under and of total of = is the of the of protein water of = is the of the of in a Fluorescence with in the presence of containing The at for and the CTX by CTX identified For of the on CTX, the in acid at for to amino of CTX and the weight by of and CTX determined of = for CTX A3, = for CTX A3, and = for CTX For each only CTXs Fourier at a FTIR with a liquid The a with an of The with and water and by for the of and CTX in in the and presence of lipids dried on the of the and in a The at a of with Fourier with the of for the of and for the as previously Huang W.N. S.C. Wu W. J. Biol. PubMed Scopus Google Scholar). Fluorescence fluorescence for the of CTX oligomerization binding to anionic lipids on an fluorescence with and at and and CTX in an as shown in in the presence of containing The of at the of anionic lipids vesicles to CTX, the fluorescence as a of the fluorescence during the oligomerization process. The effect of the CTX to the CTX Biophys. J. 1995; Full Text PDF PubMed Scopus Google Scholar, J.C. J. Biol. PubMed Scopus Google Scholar). at and of CTX P-type CTXs are to bind to micelles of zwitterionic lipid or membrane vesicles of interaction with PC membranes at liquid causes lytic effect of lipids such as into the model membrane to a CTX leakage of fluorescence and interaction of CTX A5 than CTX A3 with the PC is CTX leakage in vesicles with similar CTX-induced leakage can also be observed for vesicles formed by PG, PA, or interactions anionic lipids and cationic CTX A3 a role in the CTX-induced leakage of negatively charged membranes. study of the effect of CTX leakage reveals a interaction of CTX A3 be the leakage of the process on the of the CTX A3 (Fig. and the CTX membrane leakage may from formation of a toxin pore its lytic on toxin the CTX A3 and CTX A5 molecules in the presence of anionic lipid fluorescence in the presence of CTX A3 or CTX A5 is a the membrane of the with the CTX molecules cause in the fluorescence in as either dimer or of with CTX, the of fluorescence among of the effect of fluorescence a for the CTX dimer and an for the CTX (Fig. of the that CTX A3 in the presence of negatively charged lipids in CTX A5 only dimer (Fig. This in an oligomerization and membrane leakage in the of CTX membrane pore formation of CTX A3 the anionic lipid membrane we the CTX-induced leakage of the fluorescence molecules with Although vesicles the leakage of the of = and = in CTX vesicles more of has been to be a J. Gen. Physiol. 1997; Scopus Google Scholar). Based on the of it is that the of the pore is A.S. Biophys J. 1997; Full Text PDF PubMed Scopus Google Scholar). The of CTX-induced pore determined to be which than that of in This that the or of the CTX-induced pore is or than that of the all the CTX vesicles (Fig. the an for the of the pore is which to the of an J. Biol. Chem. Full Text PDF PubMed Google Scholar). Based on the aforementioned we that the anionic lipid may induce the oligomerization of CTX A3 the membrane and formation of a pore with a and the interaction of CTX with other anionic lipid such as cardiolipin also the formation of well defined membrane particles, presumably inverted micelles, as its fusion it is to a high structure to the for the CTX on phospholipid membranes. CTX A3 with anionic lipid of SDS and determined its structure at 1.9-Å resolution. of CTX are three CTX A3 molecules in an asymmetric each a dimer with its from an asymmetric The crystal structure of CTX A3 residues and The three are formed by residues and (Fig. 1A). The structural of the three molecules in the asymmetric unit (Fig. reveals that the structural monomers at the of and in similar with each to an SDS the L2 of the CTX A3 in has a conformation that that of the in determined by NMR in S.C. Jarrell H.C. Brisson J.R. Wu W. Biochemistry. 2001; 40: 12782-12794Crossref PubMed Scopus (20) Google Scholar, R. Huang C. J. Mol. Biol. 1994; PubMed Scopus Google Scholar). This that L2 a local and conformational in the presence of CTX A3 only two and of which the chain of is exposed to the and the of with the of The latter is with fluorescence from a study into the effect of the of to on the process of CTXs Y.C. Chiang C.M. Wu W. W. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). of a CTX A3 with SDS the SDS molecules to with CTX A3 monomers in the asymmetric Although of the charged amino the of CTX A3 can with the SDS on or the three are to be in the CTX interactions and the toxin of amino acid and from the L1 and L2 are also important in CTX as each a conformation that the SDS SDS and in with and NMR studies in the of the CTX interaction observed in the crystal that the three the CTX the structure a model on how negatively charged lipids may with the charged amino the three hydrophobic that the Although all the three with in the of with L2 the interaction with SDS molecules and the the of of the total SDS with of L2 of the CTX that is the important for the it is not in all the of a CTX A3, it is to has a role in the of CTX the major component of the venom of the cobra that CTX A3 has a major role in the of the that may also be important for its activity. which is only in P-type CTXs, the interaction with the peptide of similar to that of cytotoxin II of N. oxiana P.V. Dementieva D.V. Bocharov E.V. Utkin Y.N. Arseniev A.S. J. Mol. Biol. 2001; 305: 137-149Crossref PubMed Scopus (61) Google Scholar), a from interactions CTX A3 and the SDS the hydrophobic residues of CTX A3 interactions with the acyl of SDS as The water molecule, the (Fig. at the of the of it with the of and and the of shown previously by NMR studies and it plays a role in the of L2 S.C. Jarrell H.C. Brisson J.R. Wu W. Biochemistry. 2001; 40: 12782-12794Crossref PubMed Scopus (20) Google Scholar). other water molecules low can also be identified the of three of which are in CTX interactions (Fig. to have an important role in the CTX interactions an into the orientation on which CTX A3 its interaction with the cell we the three CTX complexes in the asymmetric This that the toxin interacts with SDS in an (Fig. FTIR of CTX A3 in the presence of the negatively charged also indicate a similar toxin orientation relative to the model membrane (Fig. similar binding the three hydrophobic of CTX A3 have previously been NMR S.C. Wu W. Biochemistry. 1997; 36: PubMed Scopus Google and NMR S.C. Chien K.Y. Huang W.N. Abraham Wu W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google studies of CTXs with zwitterionic PC that the binding of CTX A3 with negatively charged membranes similar with phospholipid membranes. the CTX A3 monomers in the asymmetric unit with and SDS which is with the of the fluorescence studies J. Biochemistry. PubMed Scopus Google Scholar), that CTX interacts with negatively all the that CTX A3, its three interacts with of and negatively charged membranes in an to be is how such an interaction to the formation of a toxin pore or inverted micelles fusion intermediates the membrane the the CTX A3 molecules with one and form two types of dimers, D1 and D2 D1 (Fig. is formed by interactions of residues on the three it and hydrophobic interactions a of D2 (Fig. or (Fig. which is in a SDS than that of and and hydrophobic The of D2 or is by the residues forming and of C. J. J. Mol. Biol. PubMed Scopus Google Scholar, S. J.M. S. A. PubMed Scopus Google Scholar), it is that D1 and D2 can form in as by NMR studies the of only S.C. Jarrell H.C. Brisson J.R. Wu W. Biochemistry. 2001; 40: 12782-12794Crossref PubMed Scopus (20) Google Scholar, R. Huang C. J. Mol. Biol. 1994; PubMed Scopus Google Scholar). are likely to form either SDS molecules or model membranes are in the shown in SDS molecules and hydrophobic interactions at the D1 the of SDS molecules at the D2 which results in the of D2 The interactions of the SDS molecules with the of D1 and D2 are of of with membrane such as Biochem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), as the acyl of several of in the of the toxin The is CTX A3 D1 and D2 in model membranes. the D1 the of and of each forming D1 are in to each other These residues have been in interactions S.C. Chien K.Y. Huang W.N. Abraham Wu W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and J. Biochem. 1987; PubMed Scopus Google Scholar). The of and respectively, and at the dimer of important role in the D1 are important for the property of CTX A3, we fluorescence to the effect of on CTX leakage of in the of with a in leakage compared with CTX A3, that of it by The of and it by (Fig. CTX A5, which lacks residues at the not cause leakage (Fig. to the D2 and are formed by the two D2 in the presence of or we FTIR on CTX A3 in the presence of and with FTIR studies J. Biol. Chem. Full Text PDF PubMed Google Scholar), the of CTX A3 enhanced toxin interaction with negatively charged lipids (Fig. The in the of CTX A3 is because of the of of each interacting forming D2 (Fig. as a lipid-induced conformational of the CTX A3 S.C. Chien K.Y. Huang W.N. Abraham Wu W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). is in the of the CTX A3 from the NMR and the one in the presence of SDS (Fig. CTXs are as of activities M.J. A.J. M.J. PubMed Scopus Google Scholar). that CTX A3 a conformation that in an of is also for which is the CTX to cause an in with the of it its conformation (Fig. and in the presence of the model membrane in the S.C. Chien K.Y. Huang W.N. Abraham Wu W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and the SDS molecules in the The crystal an for the in the (Fig. of the CTX that the formation of D2 by the of two to CTX A3, is in the for CTX A5 not we have the crystal structure of CTX A5 in complex with K.-Y. and the the CTX A5 molecules form only one of dimer that the one in the of SDS Y.J. Wu W.G. Chiang C.M. Hsin A.Y. Hsiao C.D. Biochemistry. 1997; 36: 2403-2413Crossref PubMed Scopus (56) Google Scholar), at the of which and of each interacting are in and hydrophobic the of each CTX A5 forming the dimer is not to each as in D2 of CTX A3, it that the detected of the of CTX A3 is due to the formation of packing of the toxin and SDS molecules in the crystal also an as to how SDS molecules strong interactions in When the crystal is the the SDS molecules are in SDS that have a of and are by The CTX to a role in each SDS (Fig. the crystal is to the a of SDS molecules is at each of the unit at the of which a D2 dimer is formed (Fig. the SDS (Fig. the CTX A3 molecules form D2, and two types of interactions not the SDS only are formed and This structural strongly that the SDS molecules the packing of the toxin molecules the because the packing of the is and has a of as compared with the SDS-rich regions (Fig. as in the of the structure how the formation of dimer may the membrane of D1 can be not only in its L2 of each interacting but also in its two and in the formation of the CTX A3 and in of CTX A3 This in the crystal is with the (Fig. that CTX A3 an in the presence of negatively charged the interactions of the CTX A3 molecules a D2, and and SDS (Fig. may the for CTX A3 oligomerization and eventual pore formation of CTX the crystal packing also the structure formed by D2, and (Fig. to the interaction of anionic lipid with D1 and D2, the pore can be into lipid is into D2 by binding to more SDS for the CTX on the of interacting CTX A3 monomers in the we the chain of CTX A3 interacts with the cell membrane with anionic For the CTX-induced and fusion of anionic lipid vesicles such as the of the L1-L3 with the negatively charged by the dimer formation of After the formation of D2 dimer the binding of lipids to D2 dimer may to the formation of inverted micelles as fusion For the CTX-induced pore formation of anionic lipid membrane such as the of the L1-L3 with the negatively charged of the cell membrane the formation of D1 (Fig. to of the This process is by interactions of the hydrophobic and from and from and and from with acyl of the phospholipids (Fig. The cluster and from each in the of D1 to the D2 with D1 to CTX A3 oligomerization and a pore formation (Fig. pore observed by C. B. M. F.G. Mol. Biol. 1997; PubMed Scopus Google Scholar), the of CTX A3 not that the toxin molecules can a is of CTX pore formation in negatively charged and in W. J. Toxicol. Toxin 1997; Scopus Google Scholar), which is and a structural and a for CTX A3 oligomerization anionic lipid-induced dimer which likely to the formation of a pore or inverted micelles, on the of negatively charged phospholipid membranes used for the The also the of the from which the CTX interaction has been and of for

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