Formation of the myosin.ADP.gallium fluoride complex and its solution structure by small-angle synchrotron X-ray scattering.

Abstract

In the presence of MgADP, a novel phosphate analogue of gallium fluoride (GaFn) forms a ternary complex with the myosin subfragment-1 (S-1), in the same way that has been previously reported with aluminum fluoride (AlF4-), beryllium fluoride (BeFn), scandium fluoride (ScFn), and vanadate (Vi), and this complex formation may mimic different states along the ATPase kinetic pathway. This novel complex has been characterized and compared with other complexes to ascertain whether it forms a transition-state analogue of myosin ATPase. The complex formed quickly, although several times slower than the BeFn complex. The half-life of the myosin.ADP.GaFn complex was about 50 h at 4 degreesC. The formation of the myosin.ADP.GaFn complex was accompanied by an increase in tryptophane fluorescence, similar to that observed upon the addition of ATP, but slightly lower than that of the M**.ADP.Pi complex. Upon addition of GaFn to acto-myosin.ADP, acto-myosin did not dissociate, and the S-1.ADP.GaFn complex was scarcely decomposed by actin, like the AlF4- and ScFn complexes but unlike the BeFn and Vi complexes. The conformations at the localized region of SH1, SH2, and RLR, which are very accessible to the binding of ATP, were studied by fluorescent labeling and chemical modification, and the results suggested that these conformations are very similar to that of the M**.ADP.Pi state. Small-angle X-ray solution scattering showed that the radius of gyration value decreases by about 3 A when S-1 forms an S-1.ADP.GaFn complex, suggesting that the shape of the complex becomes compact or rounded in shape, similar to that in the presence of ATP or complexes with other phosphate analogues, and thus mimics the myosin**.ADP.Pi state closely. The overall results may indicate that the complex mimics a somewhat different transient state from that of other complexes but has a similar global conformation along the ATPase kinetic pathway.