Abstract
The major toxin (CTX A3) from Taiwan cobra (Naja atra) is a basic polypeptide capable of depolarizing cell membrane and causing lytic effect on many cells, including cardiomyocytes. It binds to several negatively charged ligands, such as phosphate derivatives, phospholipids and glycosaminoglycans. With unknown function, since phosphate derivatives compounds could inhibit the CTX-induced hemolysis, it is interesting to determine the binding mode to understand their structural and functional relationship. Although the complex structure of CTX A3 and dATP at acidic pH has been determined, it is not clear whether the interaction of CTX A3 and phosphate derivatives under physiological condition would be the same. Based on the NMR study and computer simulation, we found that the dATP bound to the convex side of CTX A3 at neutral pH, in sharp contrast to the concave side of CTX A2 as determined previously at low pH. The existence of two different binding modes of the dATP/CTX A3 complex at different pH was also proved by using fluorescence technique. We also found that two forms of CTX coexisted as major (M) and minor (m) form as detected in the 2D 1H NMR spectra of CTX A3. At 25℃, the m/M ratio was about 14.3%. By changing temperature, the m/M ratio decreased from 22.0% at 5℃ to 12.4% at 45℃. With addition of 2 mM tripolyphosphate at 25℃, it increased from 14.3 to 31.6%. Thermodynamic analysis suggests that the main driving force is due to the change of entropy. In addition, the chemical shift difference between two conformations indicates the mechanism of conformational transition may be derived from the tyrosine ring but the evidence corresponding to the change of molecular structure is not found yet.