Motions and structural variability within toxins: Implication for their use as scaffolds for protein engineering

Abstract

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short ␣/␤ scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short ␣/␤ scorpion fold is highly compact, whereas the "three-finger" fold is a ␤ structure presenting large flexible loops. First, the crystal structure of the snake toxin ␣ was solved at 1.8-Å resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin ␣ and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin ␣. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.

The three-fingered protein domain of the human genome

“…Vol. 65,2008 Research Article short neurotoxin from N. nigricollis (P01246, 1IQ9) [77] is shown in Table 2S. Both tables illustrate that the fragments have moderate conservation with the Cterminal segment reaching the highest conservation level and the lowest divergence between its AA composition and that derived from the human genomic database.…”

Conformational Exchange Is Critical for the Productivity of an Oxidative Folding Intermediate with Buried Free Cysteines

“…Thus, our results strongly suggest that the lacking disulfide of Tox61 intermediate C is 42-53 (des [42][43][44][45][46][47][48][49][50][51][52][53]), as found in Tox62 intermediate C.…”

“…In the case of Tox62, such intermediates were already observed by Ruoppolo et al 26 and were named C and D. 26 They contain all but one native disulfide. The lacking disulfide is 43-54 (des [43][44][45][46][47][48][49][50][51]) in the case of intermediate C and 17-41 (des ) in the case of intermediate D.…”

“…The crystal structure of Tox62 was compared to the crystal structure of Tox61 solved at 1.8 Å resolution. 27 A 3D alignment was performed using the PyMOL software 28 and an RMSD of 1 Å was obtained on 459 heavy atoms (Fig. S7).…”

“…44 Density fitting and refinement were carried out with the aid of electron density maps (omit σ Aweighted 2F o − F c and F o − F c ) calculated and displayed using the XtalView suite of programs. 45 Refinement was carried out with REFMAC 46 and phenix.refine.…”