Functional and structural properties of cardiotoxin isomers produced by blocking negatively charged groups

In this study, we investigated the functional roles of Asp40, Asp57, and C-terminal Asn60 in Naja atra cardiotoxin 3 (CTX3) structure and function by modifying these three carboxyl groups with semicarbazide. The conjugation of the carboxyl groups with semicarbazide produced two conformational isomers whose gross and fine structures were different from those of CTX3. The blocking of the carboxyl groups increased the structural flexibility of CTX3 in response to trifluoroethanol-induced effect. Despite presenting modest to no effect on decreasing the induction of permeability in zwitterionic phospholipid vesicles, the carboxyl group-modified CTX3 showed a marked reduction in its permeabilizing effect on anionic phospholipid vesicles in comparison to that of the native protein. Compared with native CTX3, carboxyl group-modified CTX3 exhibited lower activity in inducing membrane leakage in U937 cells. The CD spectra of lipid-bound toxins and the color transition of poly-diacetylene/lipid assay showed that the membrane interaction mode of CTX3 was distinctly changed by the modification in the carboxyl groups. Given that the selective modification of Asp40 does not cause the confor-mational isomerization of CTX3, our data indicate that the carboxyl groups in Asp57 and Asn60 are essential in maintaining the structural topology of CTX3. Furthermore, modification of carboxyl groups changes the inter-dependence between the infrastructure and the global conformation of CTX3 in modulating membrane per-meabilizing activity.

Automated summary

This study investigated the role of carboxyl groups in maintaining the structure and function of Naja atra cardiotoxin 3 (CTX3). Modification of the carboxyl groups resulted in changes in the gross and fine structures of CTX3. The modified CTX3 showed reduced permeabilizing effect on anionic phospholipid vesicles and lower activity in inducing membrane leakage in U937 cells. The membrane interaction mode of CTX3 was also altered by the modification of carboxyl groups.