BINDING OF MYOTOXIN A TO SARCOPLASMIC-RETICULUM CA2+-ATPASE - A STRUCTURAL STUDY

The interaction of myotoxin a with intact sarcoplasmic reticulum (SR) components was investigated, and two SR proteins were identified that associated with myotoxin a. One of the proteins has an apparent molecular weight similar to the Ca2+ ATPase, the major SR protein responsible for calcium loading. Ca2+-ATPase was purified, and its interaction with myotoxin a was studied. Evidence for specific binding of myotoxin a to Ca2+-ATPase was established by isolating chemically cross-linked myotoxin a-Ca2+-ATPase complexes and further proving their association with anti-myotoxin a antibodies. The binding region of myotoxin a was further delineated by cleaving the protein with cyanogen bromide (CNBr) into two fragments, a larger N-terminal fragment of 28 residues and a smaller C-terminal fragment of 14 residues. Competition experiments with I-125-myotoxin a showed that the C-terminal fragment competed better against I-125-myotoxin a than the N-terminal fragment for SR protein binding. Two overlapping peptides covering the sequence of the N-terminal fragment were synthesized to clarify the interaction of the N-terminal fragment of myotoxin a with SR proteins. A 16-residue peptide corresponding to residues 1-16 competed strongly with I-125-myotoxin a, while a second peptide (residues 13-28) did not.