Botulinum Endopeptidase: SAXS Experiments and MD Simulations Reveal Extended Solution Structures That Account for Its Biochemical Properties.

Development of antidotes against botulism requires understanding of the enzymatically active conformations of Botulinum neurotoxin serotype angstrom (BoNT/A) light chain (LCA). We performed small angle X-ray scattering (SAXS) to characterize the solution structures of truncated light chain (tLCA). The 34-37 angstrom radius of gyration of tLCA was 1.5-times greater than the averaged 22-23-angstrom radius from the crystal structures. The bimodal distribution of interatomic distances P(r) indicated the two-domain tLCA structure with 129-133 angstrom size, and Kratky plots indicated the tLCA partial unfolding in the 25-37 degrees C temperature range. To interpret these data, we employed molecular dynamics simulations and machine learning. Excellent agreement between experimental and theoretical P(r) profiles helped to resolve conformational subpopulations of tLCA in solution. Partial unfolding of the C-terminal portion of tLCA (residues 339-425) results in formation of extended conformations with the larger globular domain (residues 2-298) and the smaller unstructured domain (339-425). The catalytic domain, buried 20 A-deep inside the crystal structure, becomes accessible in extended solution conformations (8-9 angstrom deep). The C- and N-termini containing different functional sequence motifs are maximally separated in the extended conformations. Our results offer physical insights into the molecular basis of BoNT/A function and stress the importance of reversible unfolding-refolding transitions and hydrophobic interactions.