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  • A comparative analysis of the cleavage activity of different


    A comparative analysis of the cleavage activity of different forms of the BoNT/A endopeptidase in reduced conditions with SNAPtide and full-length SNAP-25 substrate is summarized in Fig. 5, Fig. 6. The BoNT/A LC was found to be the most active against SNAPtide, a small peptide segment of the SNAP-25 whereas the BoNT/A complex was the most active against the full-length substrate. The peptide substrate interacts with the active site of BoNT/A LC and cleavage is almost instantaneous due to its small size, although it may not be optimal. The BoNT/A complex, on the other hand, showed the highest activity with the full-length substrate. It has been observed that the BoNT/A endopeptidase (LC), as well as BoNT/A, toxin exist in a molten globule state in the reduced condition at 37 °C (Cai and Singh, 2001, Kukreja and Singh, 2005). The flexible structure of molten globule conformation in BoNT/A toxin allows the enzyme to adapt to a variety of different conditions and modify its secondary and tertiary structure to accommodate its substrate, while retaining its endopeptidase properties due to the native-like folding pattern at physiological temperatures. The above explanation seems to be fitting with the requirement of the large substrate for optimal activity. To accommodate such a large substrate not only active site should be flexible, it also requires other interacting regions of the enzyme (exosites) to have sufficient flexibility (in this case whole enzyme because the substrate encircles the whole enzyme). Also, the binding could result in structural changes leading to exposure of the active site, hence enhancing the enzymatic activity. BoNT/A complex was found to be the least active among the rest with SNAPtide. The neurotoxin associated proteins (NAPs) either obstruct the active site or alter the folding of the endopeptidase domain leading to inaccessibility of the small peptide substrate. Since SNAPtide contains only the cleavage site with some neighboring residues, there is a possibility that the lack of the most successful film other than the active site results in failure of the exposure of the active site to be as enzymatically active. In addition, an important issue related to this molecule is designing an effective antidote/inhibitor. A critical step in developing effective antidotes against any molecule requires a comprehensive knowledge of its active conformational states in solution, and the molecular basis of its function (kinetics and interaction). In general, a crystal structure can address this issue (to a certain extent). However, a crystal structure is largely a static structure, and protein molecules involve several dynamical features in solution to execute their functions. The crystal environment undermines these aspects. In the case of full-length BoNT/A LC, solubility issues and flexibility of the C-terminus have proven difficult to overcome for crystallization despite multiple efforts by researchers to solve the crystal structure. Another factor is the potency of the molecule, and in case of BoNT this factor plays a huge role. High potency means a very delicate play between flexibility, folding, and molecular functions. In other words, to achieve a high potency, the molecule needs to be flexible to adapt its structure in different environments to be more effective, stable, or able to avoid changes in its active form due to the hostile environment, thus, lasting for a longer time without losing its function. Kukreja and Singh (2005) and Kumar et al. (2014) support this observation, which strengthens the hypothesis that this molecule has a different representation in solution than what is known from the published crystal structures. The fact that different forms of the toxin respond to different sizes of the substrate strongly supports that there is a clear role of folding and flexibility in recognition and biological function of the BoNT. These two parameters need to be taken into consideration while designing an inhibitor for this molecule targeting the endopeptidase activity in different forms of the enzyme encountered under natural conditions.