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• W2055158765 abstract "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.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 (124) 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 (86) 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 (86) 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 (51) 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 (99) 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 (51) 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 (59) 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 (124) 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 (59) Google Scholar, 13Dauplais M. Neumann J.M. Pinkasfeld S. Menez A. Roumestand C. Eur. J. Biochem. 1995; 230: 213-220Crossref PubMed Scopus (47) Google Scholar, 14Sue S.C. Chien K.Y. Huang W.N. Abraham J.K Chen K.M. Wu W. J. Biol. Chem. 2002; 277: 2666-2673Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Previous biophysical studies suggest that association of lipids, especially negatively charged ones such as phosphatidylglycerol (PG) and phosphatidic acid (PA), induce a significant increase in the β-sheet content 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 (47) Google Scholar, 15Surewicz W.K. Stepanik T.M. Szabo A.G. Mantsch H.H. J. Biol. Chem. 1988; 263: 786-790Abstract Full Text PDF PubMed Google Scholar, 16Chiang C.M. Chang S.L. Lin H.J. Wu W. Biochemistry. 1996; 35: 9177-9186Crossref PubMed Scopus (30) Google Scholar). To address these issues and to gain an insight into the mechanism of CTX-induced membrane leakage and fusion processes, we co-crystallized CTX A3 with SDS and determined its three-dimensional structure under micelle conditions. The results also provide the first high resolution molecular model of CTX-lipid complex to understand how lipid-induced CTX dimerization may contribute to the CTX oligomerization required for the formation of fusion intermediate and membrane pores in the previously reported CTX-induced membrane related activities. Materials and Purification—Rhodamine B isothiocyanate, fluorescently labeled dextrans FD-4 and FD-70, and fluorescein isothiocyanate-conjugated anti-rabbit IgG were purchased from Sigma. The phospholipids of PC, phosphatidylserine (PS), PG, and PA used in this study were obtained from Avanti Polar Lipids. Because these phospholipids contain palmitoyl-oleoyl fatty acyl chains, they are named POPC, POPS, POPG and POPA, respectively. CTX A3 and CTX A5 were purified by SP-Sephadex C-25 ion exchange chromatography followed by HPLC on a reverse phase C-18 column from crude venom of N. atra (Snake's education farms, Tainan, Taiwan) previously described (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). Vesicle Preparation—Lipids were dried under vacuum overnight and then hydrated with 10 mm Tris buffer (pH 7.4) containing 150 mm NaCl. The suspension was frozen and thawed several times and was successively extruded through a polycarbonate filter with the pore size of 0.1 μm for obtaining homogeneous large unilamellar vesicles. For the pore size determination experiments, the buffer contained 2 mg/ml FD-4 and 4 mg/ml FD-70 or fluorescein isothiocyanate-conjugated IgG (17Ladokhin A.S. Selsted M.E. White S.H. Biophys J. 1997; 72: 1762-1766Abstract Full Text PDF PubMed Scopus (187) Google Scholar). Vesicles used in fluorescence-leakage experiments were formed in the presence of 10 mm Tris (pH 7.4), 75 mm NaCl, and 50 mm 6-carboxyfluorescein (6-CF). Sepharose CL-4B column was used to remove the residual fluorescent molecules outside of the vesicles, and the lipid concentration was determined by inorganic phosphate assay as described (18Lanzetta P.A. Alvarez L.J. Reinach P.S. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1794) Google Scholar). Chemical Modification of Methionine—Chemical modification of methionine residues was performed as described (19Carlsson F.H. Int. J. Biochem. 1987; 19: 915-921Crossref PubMed Scopus (8) Google Scholar) with a slight modification. Briefly, 1 mm CTX A3 in 100 mm phosphate buffer (pH 2.5) containing 6 m guanidine-HCl was reacted with 10 mm iodoacetamide at room temperature. The reaction was monitored by analytical reverse phase HPLC followed by mass spectrometry. Two single- and one double-alkylated product could typically be obtained from this reaction. Each product was further characterized by mass spectrometry after CNBr cleavage and subsequent reduction of disulfide bonds. The identified various alkylated forms of CTXs were separated and purified by reverse phase HPLC. Vesicle Leakage—Release of vesicle contents was detected by 6-CF fluorescence intensity. Although 6-CF displays low fluorescence intensity at high concentration, its intensity increases sharply at low concentrations. Vesicles containing 6-CF were incubated at a final volume of 1 ml of buffer in a 1 × 1-cm quartz cuvette. After the addition of CTX, the fluorescence intensity was monitored as a function of time for the CTX-induced vesicle leakage process. The 6-CF leakage was calculated using the following expression: leakage % = (Ft – Fi)/(Ff – Fi), where Fi is the initial fluorescence before adding proteins, Ft is the fluorescence reading at time t, and Ff is the final fluorescence determined by adding Triton 0.02% (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 excited and emitted wavelengths were 480 and 520 nm, respectively. Pore Size Determination—Fluorescein dextran/IgG-containing vesicle (1.2 mm and 50 μl) were treated either with Triton X-100, to determine the ratio of entrapped molecules, or with CTX A3, to examine differential molecule release. After 20 min, treated vesicle was applied to a 45 × 0.5-cm CL-4B column with an elution rate of 6 ml/h. The elution profile was determined by a Hitachi F1050 fluorescence spectrophotometer. Excitation and emission wavelengths were 490 and 530 nm, respectively. To estimate the fraction of released molecules, a best-fit Gaussian curve was used to determine the area of elution profiles. When the leakage fraction was relatively small and the best-fit Gaussian curve was difficult to obtain, the leakage fraction was determined by the peak height of elution profile. We assume the released fraction corresponds to Ii = Ioi(1 – exp(–Ri)) where Ii, Ioi, and Ri were the released amount, total amount, and intrinsic leakage factor of different marker i, respectively. The selectivity was defined by the ratio of intrinsic leakage factor of co-encapsulated markers,Selectivity=RFD−4RFD−70=ln(1−IFD−4/IOFD−4)ln(1−IFD−70/IOFD−70) where IFD and IOFD were estimated by elution profiles after treatment with different concentrations of CTX A3 and Triton 0.8%, respectively. Crystallography—Crystals of CTX A3 in complex with SDS, belonging to P21212 space group with cell parameters of a = 74.90 Å, b = 76.20 Å, and c = 47.78 Å, were grown by the hanging drop method. One μl of the protein solution (10 mg/ml) was mixed with 1 μl of reservoir solution containing 100 mm sodium acetate (pH 4.6), 20% polyethylene glycol 400, 3% glycerol, and 24 mm SDS, which is well above its critical micelle concentration (7–10 mm). Crystals were flash-frozen in liquid nitrogen followed by cryo-data collection on an R-Axis IV imaging plate mounted on a Rigaku RU 300-rotating anode and subsequent data processing using DENZO (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38231) Google Scholar). The structure was solved by the molecular replacement method using the crystal structure of CTXγ (accession code 1TGX) as the search model. Using AMoRe (21Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar), the initial solution containing three molecules in the asymmetric unit had an R-factor of 40%. After manually adjusting the position of L2, which significantly differed from that of the model (1TGX), as revealed by omit map of residues 26–34, and the addition of SDS and water molecules, the R-factor dropped to about 30%. Because the L2 of each molecule in the asymmetric unit adopts a different conformation, non-crystallographic symmetry was not imposed during refinement, for which CNS (22Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16909) Google Scholar) and XtalView (23McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2015) Google Scholar) were used. The current model with R-factor and R-free values of 22 and 27.75% from 30- to 1.9-Å resolution (Table I) was obtained after several iterative cycles of CNS refinement. PROCHECK (24Laskowski R.A. MacArthur M.W. Moss D.S. Thorton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) showed that 85.6% of the residues are in the most favored region with the remaining residues in the additional favored region. Coordinates have been deposited in the Protein Data Bank under accession code 1h0j.Table IData collection and refinement statisticsCollectionResolution (Å)1.9No. of total reflections93,866No. of unique reflections21,036Overall completeness (outermost) (%)94.4 (96.3)Overall RmergeaRmerge(I) = Σh Σi|Ii — I| Σh ΣiI, where I is the mean intensity of the i observations of reflection h. (outermost) (%)4.6 (26.6)RefinementResolution limits (Å)30.0-1.9Rfactor (%)22.01Rfree (%)27.75No. protein atoms1,395No. water molecules285No. of ligand atoms170r.m.s.d. bond length (Å)0.005r.m.s.d. bond angles (degree)1.28a Rmerge(I) = Σh Σi|Ii — I| Σh ΣiI, where I is the mean intensity of the i observations of reflection h. Open table in a new tab Fluorescence Labeling—CTX (0.2 mm) was mixed with rhodamine B isothiocyanate (0.4 mm) in the presence of 100 mm phosphate buffer (pH 7.4) containing 6 m guanidine-HCl. The reaction mixture was incubated at room temperature for 12 h, and the resulting fluorescence-conjugated CTX was purified by HPLC. Single fluorescent probe-conjugated CTX was further identified using electrospray ionization mass spectrometry (Quatro Ultima, MicroMass). For characterization of the conjugated position on CTX, the sample was dissolved in 100% trifluoroacetic acid at 40 °C for 20 min to obtain conjugated amino acids of CTX and the subsequent molecular weight verified by mass spectrometry. Concentrations of fluorescence-labeled and unlabeled CTX were determined using extinction coefficients of [epsis ]558 = 105,000 m–1cm–1 for rhodamine B-conjugated CTX A3, [epsis ]276 = 4185 m–1cm–1 for CTX A3, and [epsis ]276 = 2813 m–1cm–1 for CTX A5. For each experiment, only N-terminal fluorescence-conjugated CTXs were used. Attenuated Total Reflection Fourier Transform Infrared (ATR FTIR) Experiments—ATR-FTIR spectra were collected at ambient temperature using a Bomem DA 8.3 FTIR system with a liquid nitrogen-cooled MCT detector. The internal reflection element was a zinc-selenium ATR plate (50 × 5 × 2 mm, Harrick, Ossining, NY) with an aperture angle of 45°. The ATR plates were washed with alcohol and deionized water and cleaned by plasma cleaner (Harrick) for the generation of clean and damp surface. CTX (20 μg), dissolved in D2O solution in the absence and presence of lipids (40 μg), was dried on the surface of the ATR plate and sealed in a D2O-saturated sample holder. The spectra (200 scans) were recorded at a spectral resolution of 2 cm–1 with triangular apodization. Fourier self-deconvolution was calculated, with the optimal parameter of 14–1 cm for the half-width of undeconvolution band and 2.2 for the resolution enhancement factor K, as previously described (25Lin Y.H. Huang W.N. Lee S.C. Wu W. Int. J. Biol. Macromol. 2000; 27: 171-176Crossref PubMed Scopus (14) Google Scholar). Fluorescence Homotransfer—The steady-state fluorescence spectra for the determination of CTX oligomerization upon binding to anionic lipids were obtained on an SLM-4800 fluorescence spectrometer with excitation and emission wavelengths set at 550 and 580 nm, respectively. Fluorescence-labeled and unlabeled CTX were mixed in an appropriate molar ratio, as shown in Fig. 2, in the presence of 10 mm Tris buffer (pH 7.4) containing 150 mm NaCl. The final concentration of both proteins was maintained at 0.1 μm. Upon the addition of anionic lipids vesicles (30 μm) to fluorescence-labeled CTX, the fluorescence intensity spontaneously decreased as a result of the fluorescence energy homotransfer (self-quenching) during the oligomerization process. The effect of self-quenching became less if the intrinsic CTX was added to dilute the fluorescence-labeled CTX (26Runnels L.W. Scarlata S.F. Biophys. J. 1995; 69: 1569-1583Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 27Sharpe J.C. Landon E. J. Membr. Biol. 1999; 171: 209-221Crossref PubMed Scopus (65) Google Scholar). All experiments were performed at 25 °C. Anionic Lipid-induced Oligomerization and Membrane Pore Formation of CTX A3—Although P-type CTXs are known to bind to micelles of zwitterionic lipid or membrane vesicles of sphingomyelin, their interaction with PC membranes at liquid crystalline state causes no detectable lytic effect (green line, Fig. 1B). However, introduction of acidic lipids such as PS into the model membrane leads to a significant CTX A3-induced leakage of 6-CF fluorescence probe (red and black lines, Fig. 1B). In contrast, despite stronger interaction of CTX A5 than CTX A3 with the PC membranes, there is no detectable CTX A5-induced leakage even in vesicles with 100% PS (blue line, Fig. 1B). That similar CTX-induced vesicle leakage can also be observed for vesicles formed by PG, PA, or sulfatide suggests electrostatic interactions between anionic lipids and cationic CTX A3 play a role in the CTX-induced leakage of negatively charged membranes. Concentration-dependent study of the effect of CTX A3-induced leakage further reveals a bimolecular interaction of CTX A3 might be involved since the initial leakage rate of the process depends on the square of the CTX A3 concentration (Fig. 1C and the inset). CTX A3-induced membrane leakage may stem from formation of a toxin pore and/or its direct lytic action on membranes; both mechanisms require membrane-induced toxin oligomerization. To investigate whether the CTX A3 and CTX A5 molecules oligomerize in the presence of anionic lipid membranes, fluorescence energy transfer experiments were performed in the presence of rhodamine-labeled CTX A3 (Rh-CTX A3), or CTX A5 (Rh-CTX A5). If Rh-CTX is a monomer near the membrane surface, dilution of the Rh-CTX with the intrinsic CTX molecules could cause no change in the fluorescence intensity (black dashed line in Fig. 2). Conversely, if Rh-CTX exists as either dimer or oligomer upon dilution of Rh-CTX with intrinsic CTX, the efficiency of fluorescence energy transfer among Rh-CTXs would decrease. Theoretical consideration of the quantitative effect of fluorescence energy transfer suggests a linear decrease for the CTX dimer and an even faster decrease for the CTX oligomer (Fig. 2A). Quantitative analysis of the result indicates that although CTX A3 forms oligomer (>dimer) in the presence of negatively charged lipids (green line in Fig. 2A), CTX A5 forms only dimer (Fig. 2B). This in turn suggests an interactive relationship between oligomerization and membrane leakage in the case of CTX A3. To see whether membrane pore formation of CTX A3 indeed occurs near the anionic lipid membrane surface, we ask whether the CTX-induced leakage of the co-entrapped fluorescence probe exhibits selectivity toward molecules with different sizes. Although Triton-treated PS vesicles allow the complete leakage of the fluorescently labeled dextran probe of both FD-70 (Mr = 50.7 kDa) and FD-4 (Mr = 4.4 kDa) (green line in Fig. 3A), CTX A3-treated PS vesicles retain more of FD-70. FD-70 has been suggested to be a prolate ellipsoid (28Bohrer M.P. Deen W.M. Robertson C.R. Troy J.L. Brenner B.M. J. Gen. Physiol. 1997; 74: 583-593Crossref Scopus (130) Google Scholar). Based on the short axis of dextran, it is estimated that the lower limit of the pore size is ∼20 Å (17Ladokhin A.S. Selsted M.E. White S.H. Biophys J. 1997; 72: 1762-1766Abstract Full Text PDF PubMed Scopus (187) Google Scholar). The selectivity value of CTX-induced pore was determined to be ∼1.8 ± 0.3, which was smaller than that of melittin-treated in POPC vesicles. This result implies that the size or lifetime of the CTX-induced pore is larger or longer than that of the melittin-induced pore. Because all co-entrapped IgG remained within the CTX A3-treated vesicles (Fig. 3B), the result further suggests an upper limit for the size of the pore is ∼100 Å, which corresponds to the diameter of an IgG molecule (29Sarma V.R. Silverton E.W. Davies D.R. Terry W.D. J. Biol. Chem. 1971; 246: 3753-3759Abstract Full Text PDF PubMed Google Scholar). Based on the aforementioned result, we conclude that the anionic lipid may induce the oligomerization of CTX A3 near the membrane surface and formation of a pore with a size ranging between 20 and 100 Å. Because the interaction of CTX with other anionic lipid such as cardiolipin also induces the formation of well defined membrane particles, presumably inverted micelles, as its fusion intermediates, it is desirable to obtain a high resolution structure to understand the mechanism responsible for the CTX action on phospholipid membranes. We consequently co-crystallized CTX A3 with anionic lipid of SDS and determined its three-dimensional structure at 1.9-Å resolution. Overall Structure of CTX A3—There are three crystallographically unrelated CTX A3 molecules in an asymmetric unit; each molecule forms a dimer with its closest neighbor from an adjacent asymmetric unit. The crystal structure of CTX A3 contains five β-sheets comprising residues 2–4 (β1), 11–13 (β2), 20–26 (β3), 35–39 (β4), and 49–54 (β5). The three functional loops are formed by residues 4–11 (L1), 26–35 (L2), and 39–49 (L3) (Fig. 1A). The Cα structural overlay of the three molecules in the asymmetric unit (Fig. 4A) reveals that the most significant structural variation between monomers occurs at the tip of L2. Two monomeric (blue and magenta in Fig. 4A) L2s adopt similar conformations, with each bound to an SDS head group, whereas the L2 of the third CTX A3 molecule (green in Fig. 4A) has a conformation that closely resembles that of the molecule in solution determined by 1H NMR (yellow in Fig. 4A) (11Sue S.C. Jarrell H.C. Brisson J.R. Wu W. Biochemistry. 2001; 40: 12782-12794Crossref PubMed Scopus (20) Google Scholar, 30Bhaskaran R. Huang C.C. Chang D.K. Yu C. J. Mol. Biol. 1994; 235: 1291-1301Crossref PubMed Scopus (71) Google Scholar). This suggests that L2 undergoes a local and specific conformational change in the presence of SDS. CTX A3 contains only two acidic residues, Asp-40 and Asp-57, of which the side chain of Asp-40 is exposed to the solvent, and the OD1 of Asp-57 hydrogen bond with the NH of Lys-2. The latter observation is consistent with fluorescence data generated from a study into the effect of the mutation of Asp-57 to Asn on the unfolding process of CTXs (31Lo C.C. Hsu J.H. Shen Y.C. Chiang C.M. Wu W. Fann W. Tsao P.H. Biophys. J. 1998; 75: 2382-2388Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Interactions of a CTX A3 Monomer with SDS Molecules—In the current structure, 10 SDS molecules were found to interact with 3 CTX A3 monomers in the asymmetric unit. Although any of the positively charged amino acids distributed throughout the slightly curved surface of CTX A3 can potentially interact with the SDS sulfate head groups, those residing on or near the three loops are found to be involved in the CTX A3-SDS interactions (Figs. 4B and 5A). Interestingly, the toxin backbone of 2 amino acid triads, Lys-5-Leu-6–Val-7 and Thr-29–Pro-30–Lys-31, from the respective L1 and L2 are also important contributors in CTX A3-SDS interactions, as each adopts a conformation that wraps around the SDS sulfate moiety (see SDS 2 and 4 in Fig. 4B). Consistent with previous biochemical and NMR studies in aqueous solution, the mode of the CTX A3-SDS interaction observed in the crystal implies that the three loops, L1-L3, initiate the CTX A3-membrane interaction. However, the current structure provides a molecular model on how negatively charged lipids may interact with the positively charged amino acids flanking the three hydrophobic loops that enhance the CTX-membrane interactions. Although all the three loops interact with SDS, they differ in the intensity of their interaction, with L2 providing the" @default.
• W2055158765 created "2016-06-24" @default.
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• W2055158765 date "2003-06-01" @default.
• W2055158765 modified "2023-10-02" @default.
• W2055158765 title "Structural Basis of Membrane-induced Cardiotoxin A3 Oligomerization" @default.
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