Dynamic characterization of the water binding loop in the P-type cardiotoxin: Implication for the role of the bound water molecule

Recent studies of cobra P-type cardiotoxins (CTXs) have shown that the wate r-binding loop (loop II) plays a crucial role in toxin binding to biologica l membranes and in their cytotoxicity. To understand the role of bound wate r in the loop, the structure and dynamics of the major P-type CTX from Taiw an cobra, CTX A3, were determined by a comprehensive NMR analysis involving H-1 NOESY/ ROESY, C-13{H-1} NOE/T-1 relaxation, and O-17 triple-quantum fi ltered NMR. A single water molecule. was found to be tightly hydrogen bonde d to the NH of Met26 with a correlation time (5-7 ns) approaching the isotr opic, tumbling time (3.8-4.5 ns) of the CTX A3 molecule. Surprisingly, desp ite the relatively long residence time (ca. 5 ns to 100 mus), the bound wat er molecule of CTX A3 is located within a dynamic (order parameter S-2 simi lar to 0.7) and solvent accessible loop. Comparison among several P-type CT Xs suggests that proline. residues in the consensus sequence of MxAxPxVPV s hould play an important role in the formation of the water binding loop. It is proposed that the exchange rate of the bound water may play a role in r egulating the lipid binding mode of amphiphilic CTX molecules near membrane surfaces.