To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.

Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure-activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Nav channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structurefunction homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of antiinfective or therapeutic peptides that selectively target microbial or abnormal host cells.channel ͉ defensin ͉ host defense ͉ toxin

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ␤-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the ␤-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ␤-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.

Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo-and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most relevant difference between the NMR structure ensembles of apo-and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligandbinding site. These comprise ␣-helix II, the subsequent linker to ␤-strand B, the hairpin turn between ␤-strands C and D, and the ␤E-␤F turn. The internal backbone dynamics, obtained from 15 N relaxation data (T 1 , T 2 , and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the ␤C-␤D and ␤E-␤F turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment.Vitamin A derivatives play important roles in a variety of biological processes including vision, cell growth, cell differentiation, and morphogenesis (1). Plasma transport of retinol to target cells and intracellular transport for either storage or metabolic conversion are performed by binding proteins that belong to the calycin superfamily. The cytosolic carriers are members of the intracellular lipid-binding protein (i-LBP) 1 family, characterized by molecular masses of around 15 kDa. Their structure consists of a 10-stranded ␤-barrel, formed by two orthogonal ␤-sheets, and two short ␣-helices (2). The two best known intracellular carriers of retinol are cellular retinolbinding protein type I (CRBP), widely distributed in various tissues (3, 4), and cellular retinol-binding protein type II (CRBP-II), present in the enterocytes of the small intestine and in neonatal hepatocytes (5, 6). The structures of rat apo-and holoCRBP-II have been solved both in the crystal (7) and in solution (8, 9), whereas the only structure of CRBP available to date was that of the holoprotein in the crystal (10). More recently, two other retinol carriers have been identified as follows: murine CRBP-III, expressed primarily in heart, muscle, and adipose tissue (11); and human CRBP-III, most abundant in liver and kidney, whose structure in the retinol-free form has been solved by x-ray crystallography (12).In the cell, the poorly water-soluble retinol is stored within membranes as a retinyl-ester derivative of long-cha...

Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross-linked by three disulfide bridges and characterized by a mild toxicity (LD 50 ¼ 820 lg per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage-sensitive Na + channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N-terminal a-helix and a small antiparallel triple-stranded b-sheet arranged in an ab 1 b 2 b 3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na + channels similar to the one reported for crotamine, exhibit an a/b fold, though with a b 1 ab 2 b 3 topology.In addition, as the antibacterial b-defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human b-defensins shows a similar fold and a comparable net positive potential surface.To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na + channel.Keywords: b-defensin; myotoxin; NMR; scorpion toxin; structure.Despite the fact that Na + channels are affected by a large variety of toxins from arthropods, coelenterates, microorganisms, fish and plants, they are seldom the targets of toxins from snake venom [1]. One exception is crotamine (Crt), a protein of 42 amino acids present in the venom of Crotalus durissus terrificus [2,3] and characterized by a wide spectrum of biological activities. This toxin, in fact, has been known for a long time to be able to induce membrane depolarization dependent muscle contractions by increasing the Na + permeability of skeletal muscle membranes [4-9] and to affect, in an allosteric fashion, the action of other Na + channel neurotoxins (i.e. tetrodotoxin, veratridine, batrachotoxin and grayanotoxin) [4,5,[7][8][9][10]. In addition, while loose patch-clamp recording of macroscopic sodium currents in frog skeletal muscle has revealed that Crt inhibits the inactivation of the Na + channel in a fashion similar to that of scorpion a-toxins [11], other experiments have shown that, at low doses, it has an analgesic activity involving both central and peripheral mechanisms [12]. Moreover, Crt actively interacts with lipid membranes being able to form vacuoles and exhibiting myonecrotic activity [13,14].Crt belongs to a family of small basic rattlesnake venom myotoxins that includes myotoxin a [15], peptide C [16], myotoxin I and II [17] and the CAM toxin [18]. They exhibit high primary sequence identity (Fig. 1) and, in addition, they are antigenically related [19]. However, when compared to the other members of the family, Crt exhibits a reduced toxicity (intraperitoneal injection LD 50 ¼ 820 lg per ...