Effect of phosphate analogs and divalent cations on the conformation of myosin si at two specific sites

The interaction of myosiii with actin coupled to hydrolysis of ATI1 is the molecular basis of contraction. Si. a myosin segment, is responsible for thr ATPase activity and interaction with actin. The intermediate steps of th> ATP hydrolysis are accompanied by conformational changes, which détermine the affinity of each intermediate to actin. Phosphate analogs (PAs) vanadatc (Vi), aluminum fluoride (A1F4), and beryllium fluoride (BeFx) which form stable complexes with SI and ADP, mimic the structures of the intermediates of the ATP hydrolysis. We studied the effect of PAs on the SI structure at two sites by measuring the rate of modification of Cys707 and LysHU with coumarin maleimide and trinitrobenzene sulfonate, respectively. By comparing the reaction rates obtained in the presence of PAs plus ADP to those of ATP hydrolysis intermediates we found that the structure of Sl-ADP BeF\ mimics that of M-ADP-Pi while the conformations of SI MgADP-AlF4 and SI MgADP-Vi are similar to that of M-ADP-Pi at both sites. Since diva lent cations are essential for the formation of the stable Si-ADP- PA complexes we also measured the rates of modification at the two sites in the presence of Mn, Co and Ca and compared them to the rates obtained in the presence of Mg. The cations significantly affected the rates of modification at both sitc-s. However, also in the presence of Mil, Co and Ca the struct ures of the S l metal ADP-PA complexes mimic the structures of the ATP hydrolysis intermediates similarly as found with Mg.