Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus
Introduction
Crotamine is a polypeptide present in the venom of the South American rattlesnake Crotalus durissus terrificus. First isolated by Goncalves and Vieira (1950), crotamine contains a single polypeptide chain of 42 amino acid residues (Laure, 1975). There are six cysteines, which are all involved in disulfide-bonds (Kawano et al., 1982). The globular conformation of crotamine is very stable in solution (Hampe et al., 1978).
Crotamine is part of a family of small basic peptides present in rattlesnake venoms, which are non-enzymatic and have myonecrotic activity. These myotoxins show high amino acid sequence homology (Radis-Baptista et al., 1999). They also show similar action mechanisms during the envenomation (Fletcher et al., 1996), where they reduce the membrane potential and increase the influx of ions through the membrane, thus modifying conductance by a Na+ channel-mediated mechanism and releasing Ca2+ from the heavy fraction of the sarcoplasmatic reticulum (Ownby, 1998).
The myotoxins has been extensively studied, including structural studies by SAXS (Beltran et al., 1990), laser-Raman spectroscopy (Kawano et al., 1982), and 1H NMR (Endo et al., 1989, Nicastro et al., 2003). In these previous studies, the cysteine pairing pattern could not be unambiguously determined by SAX (Beltran et al., 1990) or by the NMR structure determination, which was therefore based on ad hoc disulfide connectivities (Nicastro et al., 2003). In this study, the NMR structure determination of crotamine was repeated, using the highest available field strength for improved NMR sensitivity and resolution. Furthermore, fully automatic interpretation of the 2D [1H,1H]-NOESY data was obtained with the software package ATNOS/CANDID/DYANA (Herrmann et al., 2002a,b; Güntert et al., 1997), which also yielded an automatic determination of the disulfide covalent bond connectivities as a result of the NOE-derived distance information network. The computational approach used here for identification of the unknown disulfide bond connectivities based entirely on [1H,1H]-NOESY distance constraints, will be generally applicable for studies of globular proteins with unknown cysteine pairings, including other polypeptide toxins. A previously determined NMR structure of crotamine (Nicastro et al., 2003) could thus be confirmed and refined.
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Purification of crotamine
Crotalus durissus terrificus venom was extracted from snakes maintained at the FMRP serpentarium of São Paulo University, and dried under vacuum. Six hundred milligrams of crude venom were dissolved in 5 ml of 0.25 M ammonium formate buffer at pH 3.5, and the bulk of crotoxin, the major venom component, was eliminated by low speed centrifugation as a heavy precipitate that formed upon slow addition of 20 ml of cold water to the solution. Dropwise addition of Tris-base solution was then used to
Resonance assignment
The sequential assignment of crotamine was based on homonuclear 1H NMR spectroscopy (Wüthrich, 1986), using 2D 2QF-[1H,1H]-COSY (Rance et al., 1983), 2D [1H,1H]-TOCSY (Griesinger et al., 1996) and 2D [1H,1H]-NOESY (Wider et al., 1984) spectra. Except for Lys2 and Gln3, all expected backbone amide proton resonances were identified and assigned. Fig. 1 contains a survey of the data used for the backbone assignments. All the α-protons and 90% of the carbon-bound side chain protons were assigned.
Comparison of crotamine with related small disulfide-rich proteins
Crotamine and myotoxin a, the most extensively studied members of the myotoxin family, show differences in only three sequence positions (Fig. 4(a)) and have similar biological activities. The present study on crotamine further implies that they have the same disulfide bond patterns, i.e. Cys4/Cys36, Cys11/Cys30 and Cys18/Cys37, which was determined for myotoxin a by biochemical methods (Fox et al., 1979).
Structural similarities are also readily apparent when comparing crotamine with the
Acknowledgements
We thank L.H.A. Pedrosa and C.J. Laure for a generous supply of Crotalus durissus terrificus venom, and F. Fiorito for help in performing the structure calculations. Financial support was obtained from Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES-PDEE (process # 1243-012), FAPESP (SMOLBNet, 01/07532-0), CNPq (300851-98-7), and the Schweizerischer Nationalfonds (projects 31.49047.96 and 3100A0-105570/1). The use of the computational facilities of the Center of
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