An NMR Study of the Interaction of Cardiotoxin gamma from Naja nigricollis with Perdeuterated Dodecylphosphocholine Micelles

Dauplais, Marc; Neumann, Jean‐Michel; Pinkasfeld, Suzanne; Ménèz, Andre; Roumestand, Christian

doi:10.1111/j.1432-1033.1995.tb20553.x

Cited by 23 publications

(43 citation statements)

References 62 publications

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“…The intermolecular NOEs observed between LPC fatty acyl chain and CTX A3 molecule suggest further that the amino acid residues in the convex side of the loop region are involved in lipid-CTX interaction (data not shown). The results reconfirm that all three hydrophobic loops (I, II, and III) of the P-type CTXs are involved in its binding to phospholipid dispersions (18,21) and the convex side of CTX A3 molecule with hydrophobic residues is in contact with the membrane surface.…”

Section: Heparin-enhanced Ctx Binding To Lpc Micelles As Reflected Bysupporting

confidence: 65%

Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin

Sue

Chien

Huang

et al. 2002

Journal of Biological Chemistry

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It has been shown previously that the long chain fragments of heparin bind to the ␤-strand cationic belt of the three-finger cobra cardiotoxin (or cytotoxin, CTX) and hence enhance its penetration into phospholipid monolayer under physiological ionic conditions. By taking lysophosphatidylcholine (LPC) micelles as a membrane model, we have shown by 1 H NMR study that the binding of heparin-derived hexasaccharide (Hep-6) to CTX at the ␤-strand region can induce conformational changes of CTX near its membrane binding loops and promote the binding activity of CTX toward LPC. The Fourier-transform infrared spectra and NMR nuclear Overhauser effect of Hep-6⅐CTX and CTX⅐LPC complex in aqueous buffer also supplemented the aforementioned observation. Thus, the detected conformational change may presumably be the result of structural coupling between the connecting loops and its ␤-strands. This is the first documentation of results showing how the association of hydrophilic carbohydrate molecules with amphiphilic proteins can promote hydrophobic protein-lipid interaction via the stabilization of its membrane-bound form. A similar mechanism involving tripartite interactions of heparin, protein, and lipid molecules may be operative near the extracellular matrix of cell membranes.Cobra cardiotoxins (or cytotoxins, CTXs) 1 are highly basic, three-fingered polypeptides with the capability of inducing direct lytic effect on many cell types, including cardiac myocytes (1, 2). The available x-ray and NMR structures of CTXs are similar with a short antiparallel double-stranded ␤-sheet connected by loop I and with a long antiparallel triple-stranded ␤-sheet connected by loop II and III. Two distinct types of CTXs, i.e. P-and S-type, can be distinguished by the presence of Pro 30 and Ser 28 , respectively, near the tip of second finger (loop II) based on its binding activity toward neutral phospholipid bilayers and micelles (3). P-type CTXs, including the major CTX A3 from Taiwan cobra (Naja atra) venom, have been shown to induce a reversible formation of an extra outwardly rectifying conductance in bullfrog atrial myocytes (4) and also redistribute themselves between penetrating and peripheral binding states in phosphatidylcholine (PC) dispersion under favorable condition (5). Despite of the apparent perturbing effect of CTX toward phospholipid membranes, it is not clear how CTXs might act specifically toward different cell types.Recently, it was suggested that the sulfated polysaccharides such as glycosaminoglycans (GAGs), which are abundant in the extracellular matrix of most animal cells, are potential targets for CTX action (6 -8). Heparin not only binds to homologous CTXs, with different specificity, but also promotes its penetration into phospholipid monolayer under physiological ionic conditions (6). Analysis of binding of 10 CTX homologues to heparin reveals a new heparin-binding structural motif by involving several positive residues at the concave surface of the three-finger toxin (7). The positive residues flanking the...

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Section: Heparin-enhanced Ctx Binding To Lpc Micelles As Reflected Bysupporting

confidence: 65%

Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin

Sue

Chien

Huang

et al. 2002

Journal of Biological Chemistry

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“…There is also a good correlation with experimentally observed participation of residues 25, 27, 29, 31, 33, 37, 38, 42 and 53 of neurotoxins in binding with acetylcholine receptor ( [1,29], see also references in [15]), and involvement of residues 15, 21, 25 and 43 in cardiotoxin's functioning ( [30] and references therein). Recent NMR experiments also suggest the interaction of the side chains of hydrophobic residues of toxin y in the segments 5-14, 25-35, 39~,6 and 54-58 with the detergent micelles [31]. This also accords well with the predicted in the current study 'versatile' and 'functional' residues for cardiotoxins.…”

Section: Resultssupporting

confidence: 90%

Amino acid residue: is it structural or functional?

et al. 1995

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A new approach is suggested for delineating the structural and functional amino acid residues in proteins with known three-dimensional structure, basing on the involvement of residues in intramolecular hydrophobic and hydrophilic interactions and additional information about the conservativity of the residues. The approach is applied to the families of homologous neurotoxins and cardiotoxins. The results obtained concerning the role of amino acid residues in both families of toxins accord well with the similarity of their fold, but different mechanisms of action. Current approach can be used for detailed characterization of protein spatial structures, as well as for rational protein engineering.

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“…Although it lacks cardiotoxicity, CTX A5 is also called cardiotoxin because its amino acid sequence is homologous to that of CTX A3 (4,7). Fluorescence and NMR studies of CTXs in the presence of zwitterionic PC micelles indicate that L1-L3 become perturbed (7,(12)(13)(14). Previous biophysical studies suggest that association of lipids, especially negatively charged ones such as phos-phatidylglycerol (PG) and phosphatidic acid (PA), induce a significant increase in the ␤-sheet content of CTX A3 (7,13,15,16).…”

mentioning

confidence: 99%

“…Fluorescence and NMR studies of CTXs in the presence of zwitterionic PC micelles indicate that L1-L3 become perturbed (7,(12)(13)(14). Previous biophysical studies suggest that association of lipids, especially negatively charged ones such as phos-phatidylglycerol (PG) and phosphatidic acid (PA), induce a significant increase in the ␤-sheet content of CTX A3 (7,13,15,16). 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.…”

mentioning

confidence: 99%

Structural Basis of Membrane-induced Cardiotoxin A3 Oligomerization

Forouhar

Huang

Liu

et al. 2003

Journal of Biological Chemistry

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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 CTX-SDS 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.

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An NMR Study of the Interaction of Cardiotoxin gamma from Naja nigricollis with Perdeuterated Dodecylphosphocholine Micelles

Cited by 23 publications

References 62 publications

Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin

Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin

Amino acid residue: is it structural or functional?

Structural Basis of Membrane-induced Cardiotoxin A3 Oligomerization

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