The solution structure of alpha-cobratoxin, a neurotoxin purified from the venom of the snake Naja naja siamensis, at pH 3.2 is reported. Sequence-specific assignments of the NMR resonances was attained by a combination of a generalized main-chain-directed strategy and of the sequential method. The NMR data show the presence of a triple-stranded beta-sheet (residues 19-25, 36-41, and 52-57), a short helix, and turns. An extensive number of NOE cross peaks were identified in the NOESY NMR maps. These were applied as distance constraints in a molecular modeling protocol which includes distance geometry and dynamical simulated annealing calculations. A single family of structures is observed which fold in such a way that three major loops emerge from a globular head. The solution and crystal structures of alpha-cobratoxin are very similar. This is in clear contrast to results reported for alpha-bungarotoxin where significant differences exist.

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1H NMR has been applied to a 3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpion Androctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20-28) which is linked by two disulfide bridges to the central strand of a triple-stranded anti-parallel beta-sheet. Turns were identified at residues 15-17, 47-49 and also at residues 51-53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu37, Leu23, Val47, Tyr14, Trp45 and Tyr5. The Trp45 and Tyr5 rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.

The HIV-1 integrase is an attractive target for the therapeutics development against AIDS, as no host homologue of this protein has been identified. The integrase strand transfer inhibitors (INSTIs), including raltegravir, specifically target the second catalytic step of the integration process by binding to the DDE motif of the catalytic site and coordinating Mg(2+) ions. Recent X-ray crystallographic structures of the integrase/DNA complex from prototype foamy virus allowed to investigate the role of the different partners (integrase, DNA, Mg(2+) ions, raltegravir) in the complex stability using molecular dynamics (MD) simulations. The presence of Mg(2+) ions is found to be essential for the stability, whereas the simultaneous presence of raltegravir and Mg(2+) ions has a destabilizing influence. A homology model of HIV-1 integrase was built on the basis of the X-ray crystallographic information, and protein marker residues for the ligand binding were detected by clustering the docking poses of known HIV-1 integrase inhibitors on the model. Interestingly, we had already identified some of these residues to be involved in HIV-1 resistance mutations and in the stabilization of the catalytic site during the MD simulations. Classification of protein conformations along MD simulations, as well as of ligand docking poses, was performed by using an original learning method, based on self-organizing maps. This allows us to perform a more in-depth investigation of the free-energy basins populated by the complex in MD simulations on the one hand, and a straightforward classification of ligands according to their binding residues on the other hand.

There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme.

The 3D solution structure of α-cobratoxin, a neurotoxin purified from the Naja naja siamensis snake venom, has been determined by Nuclear Magnetic Resonance spectroscopy, in conjunction with distance geometry and restrained molecular dynamics, at pH 7.5. A total of 490 distance restraints were obtained from NOE intensities and 25 φ dihedral angle restraints deduced from J- coupling data. The generated structures are well defined with root mean square deviations from a geometrical mean structure of 0.107 ± 0.036 nm for the backbone atoms and 0.128 ±0.073 nm for the side-chain atoms (considering residues 1 to 66 minus 26 to 35). A comparison between the generated structures at pH 7.5 and the mean NMR solution structure at pH 3.2 revealed that the 3D structure of α-cobratoxin is more compact at neutral pH. This major difference is mainly due to the pH-dependant conformational variations of three residues His(18), Thr(44) and Thr(59).

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