Structural and Dynamic “Portraits” of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They?

Abstract

Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra Naja oxiana venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly C- andN-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study.

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

“…To make sure that the process of protein–lipid insertion reached an equilibrium and mode-123 corresponded to the final state of the system, we extended simulation θ 0 to 2 μs. During the entire 2 μs run, the β-structural core of the toxin molecule remained intact, while the loops were more flexible, and their dynamics was similar to that in an aqueous solution , (Figure S4).…”

“…Penetration into a membrane-like environment (detergent micelles) by means of the hydrophobic bottom has only been demonstrated experimentally for two CTs from Naja oxiana, CT1 and CT2 . Their spatial models were established by NMR spectroscopy in an aqueous solution , as well as in zwitterionic dodecylphosphocholine (DPC) micelles. , CT2 retains its 3D structure in both environments, water and membrane-like to a significant extent . The hypothesis suggesting that the three-finger binding mode, which CT2 demonstrated interacting with micelles, would also be a valid scenario for planar lipid bilayers requires verification.…”

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

“…Among the spatial structures of CTs the quality of the NMR-determined models is usually inferior to that of X-ray ones [7]. One of the reasons for this is slow conformational equilibrium between cis-and trans-isomers of the X-Pro8 peptide bond, which occurs in aqueous solution but not in the crystal [39,40]. This equilibrium results in an increase in the number of cross-peaks in NMR spectra of the toxins in aqueous solution.…”

“…This equilibrium results in an increase in the number of cross-peaks in NMR spectra of the toxins in aqueous solution. Normal mode analysis [7], MD study [39], and NMR investigation [41] of the dynamics of CTs revealed that the residues involved in beta-sheet formation, are more rigid compared to those that are not involved in backbone hydrogen bonding. The latter are localized in the tips of loops (residues 7-11, 16-18, 28-31, and 46-48).…”

“…To check reproducibility of the simulations, three independent runs were performed for each toxin/lipid system listed in Table 3. In these starts, the orientations of the toxin molecules with respect the membrane surface were identical, but the distance from the center of mass of the toxin molecule to the center of the lipid bilayer was varied in the range 37-45 Å. a see Table 1 for the amino acid sequences of the toxins; b solution conformations of the major form of CT1No [22,39] or CT2Nk [37]; and c conformation of CT1No in detergent micelles [22].…”