Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling.

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both "water" and "membrane" conformations of the central loop (loop-2) were determined by X-ray crystallography. The "water" conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The "membrane" conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step-the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their "water" NMR models. We found that the both toxins transform their "water" conformation of loop-2 into the "membrane" one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.

Small Amphiphilic Peptides: Activity Against a Broad Range of Drug-Resistant Bacteria and Structural Insight into Membranolytic Properties.

We report the synthesis and antibacterial activities of a series of amphiphilic membrane-active peptides composed, in part, of various nongenetically coded hydrophobic amino acids. The lead cyclic peptides, 8C and 9C, showed broad-spectrum activity against drug-resistant Gram-positive (minimum inhibitory concentration (MIC) = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria. The cytotoxicity study showed the predominant lethal action of the peptides against bacteria as compared with mammalian cells. A plasma stability study revealed approximately 2-fold higher stability of lead cyclic peptides as compared to their linear counterparts after 24 h of incubation. A calcein dye leakage experiment revealed the membranolytic effect of the cyclic peptides. Nuclear magnetic resonance spectroscopy and molecular dynamics simulation studies of the interaction of the peptides with the phospholipid bilayer provided a solid structural basis to explain the membranolytic action of the peptides with atomistic details. These results highlight the potential of newly designed amphiphilic peptides as the next generation of peptide-based antibiotics.

Stepwise Insertion of Cobra Cardiotoxin CT2 into a Lipid Bilayer Occurs as an Interplay of Protein and Membrane "Dynamic Molecular Portraits".

For many peripheral membrane-binding polypeptides(MBPs), especially ß-structural ones, the precise molecular mechanisms of membrane insertion remain unclear. In most cases, only the terminal water-soluble and membrane-bound states have been elucidated, whereas potential functionally important intermediate stages are still not understood in sufficient detail. In this study, we present one of the first successful attempts to describe step-by-step embedding of the MBP cardiotoxin 2 (CT2) from cobra Naja oxiana venom into a lipid bilayer at the atomistic level. CT2 possesses a highly conservative and rigid ß-structured three-finger fold shared by many other exogenous and endogenous proteins performing a wide variety of functions. The incorporation of CT2 into the lipid bilayer was analyzed via a 2 µs all-atom molecular dynamics (MD) simulation without restraints. This process was shown to occur over a number of distinct steps, while the geometry of initial membrane attachment drastically differs from that of the final equilibrated state. In the latter one, the hydrophobic platform ("bottom") formed by the tips of the three loops is deeply buried into the lipid bilayer. This agrees well with the NMR data obtained earlier for CT2 in detergent micelles. However, the bottom is too bulky to insert itself into the membrane at once. Instead, the gradual immersion of CT2 initiated by the loop-1 was observed. This initial binding stage was also demonstrated in a series of MD runs with varying starting orientations of the toxin with respect to the bilayer surface. Apart from the nonspecific long-range electrostatic attraction and hydrophobic match/mismatch factor, several specific lipid-binding sites were identified in CT2. They were shown to promote membrane insertion by engaging in strong interactions with lipid head groups, fine-tuning the toxin-membrane accommodation. We therefore propose that the toxin insertion relies on the interplay of nonspecific and specific interactions, which are determined by the "dynamic molecular portraits" of the two players, the protein and the membrane. The proposed model does not require protein oligomerization for membrane insertion and can be further employed to design MBPs with predetermined properties with regard to particular membrane targets.

Structure and dynamics of cardiotoxins.

Cytotoxins (or cardiotoxins; CTs) are toxins from cobra venom characterized by the three-finger (TF) fold. CTs are on average 60-residue-long peptides, possessing as many as 4 disulfide bonds. The elements of antiparallel ß-structure take origin from the hydrophobic core formed by the disulfides. The ß-strands adopt the shape of the three loops, giving the name of the fold. While neurotoxins (NTs) - also TF proteins from snake venom - exert their effect through specific interactions with protein receptors, no specific protein target has been found for CTs. Unlike NTs, CTs are amphiphilic and cytotoxic against a variety of cells, including cancer ones. Thus, the hypothesis that the activity of CTs is caused by their interactions with lipid membranes is currently central. To understand molecular basis behind variations in toxicities of CTs highly homologous in their sequences, detailed knowledge of their structure and dynamics is required. The present review summarizes experimental and computational data on the spatial organization of CTs and their dynamics in various environments (aqueous solution, membranous milieus).