THE CONFORMATIONAL STATE OF THE ACTO-S-1 COMPLEX

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We have proposed that during the actomyosin ATPase cycle, the myosin cross-bridge alternates between two major conformations, which differ markedly in their strength of binding to actin and in their overall structure. In one conformation, which occurs in the absence of ATP, myosin binds very tightly to actin at a 45 degree angle. In the second conformation, which occurs only transiently when ATP or ADP.Pi is bound to myosin, myosin binds weakly to actin at an angle postulated to be 90 degrees. We have now obtained structural evidence for the two different conformational states by using cross-linked actin.S-1. Both by negative staining and by freeze etching techniques, electron micrographs of cross-linked actin.S-1 shows that the overall structure of the cross-linked actin.S-1 is very disordered in the presence of ATP, with individual cross-linked S-1 molecules attaching to actin at variable angles centering on 90 degrees. In the absence of ATP, the cross-linked actin.S-1 shows the typical arrowhead appearance, characteristic of the 45 degrees conformation. The structure of the cross-linked complex was also examined in the presence of the ATP analog, AMP-PNP. In contrast to ATP, the structure of cross-linked actin.S-1 appears very rigor-like in the presence of AMP-PNP suggesting that the structural change induced by ATP is specific. In addition to these structural studies, we have also obtained biochemical evidence that cross-linked actin.S-1 can exist in two different conformations by studying the effect of troponin-tropomyosin on the cross-linked filament. We found that the conformation of cross-linked actin.S-1 depends on the nucleotide bound to the S-1. On the other hand, cross linked actin.pPDM.S-1 and cross-linked actin.NEM.S-1 always remain in the 90 degrees weak-binding and 45 degrees strong-binding conformations, respectively, regardless of the nucleotide bound to S-1. This is in agreement with our previous studies which show that, irrespective of the nucleotide bound to S-1, pPDM.S-1 and NEM.S-1 form with actin stable analogs of the 90 degrees and 45 degrees conformations, respectively.