1 H-NMR spectroscopy data, such as NOE intraprotein and (bound water)/protein contacts, 3 J coupling constants and deuterium exchange rates were used to determine the in-solution spatial structure of cytotoxin II from Naja naja oxiana snake venom (CTII). Exploiting information from two 1 H-NMR spectral components, shown to be due to cis/trans isomerization of the Val7±Pro8 peptide bond, spatial structures of CTII minor and major forms (1 : 6) were calculated using the torsion angle dynamics algorithm of the dyana program and then energy refined using the fantom program. Each form, major and minor, is represented by 20 resulting conformers, demonstrating mean backbone rmsd values of 0.51 and 0.71 A Ê , respectively. Two forms of CTII preserve the structural skeleton as three large loops, including two b-sheets with bend regions, and demonstrate structural differences at loop I, where cis/trans isomerization occurs. The CTII side-chain distribution constitutes hydrophilic and hydrophobic belts around the protein, alternating in the trend of the three main loops. Because of the V-shaped backbone, formed in participation with two bound water molecules, the tip of loop II bridges the tips of loops I and III. This ensures the continuity of the largest hydrophobic belt, formed with the residues of these tips. Comparison revealed pronounced differences in the spatial organization of the tips of the three main loops between CTII and previous structures of homologous cytotoxins (cardiotoxins) in solution.Keywords: bound water; cardiotoxin; cis/trans isomerization; NMR; protein structure.Cytotoxins, forming part of cobra snake venom, were so named because of their ability to damage cells. Because they have been observed to have a toxic effect on heart, they are often called cardiotoxins. To date cytotoxins (cardiotoxins) have been determined to attack a wide variety of cells, in particular, muscle cells, probably causing membrane depolarization [1], and nonexcitable cells, provoking their lysis ([2], and refs therein). That cardiotoxins damage cells by membrane perturbation was postulated due to their ability to interact with model membranes both directly [3±7] and after binding to oligosaccharides [8], which abound at the cell surface.The heterogeneity of the cardiotoxins was first noticed with respect to their biological properties, e.g. hemolytic and muscle contraction activity [2]. The correlation between some of the biological properties and the binding activity towards model zwitterionic membranes was noticed later [5]. Cardiotoxins also show different heparin-binding strengths [9].The three-loop b-sheet structural motif is a common feature of all determined spatial structures of cardiotoxins in crystalline form [10±12] and in solution [13±18]. Various approaches have established the role of the hydrophobic cluster at the tips of the three main loops in penetrating the membrane [19±21] or participating in heparin binding [9]. The numerous conserved, positively charged residues flanking this hydrophobic cluster are a...
