Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus

Abstract

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 °C, using standard homonuclear ¹H NMR spectroscopy at 900 MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4–Cys36, Cys11–Cys30 and Cys18–Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an α-helix with residues 1–7 and a two-stranded anti-parallel β-sheet with residues 9–13 and 34–38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the β-defensin fold, although the two proteins have low sequence homology, and display different biological activities.

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.