Structure of the actin-myosin complex in the presence of ATP.

Abstract

The structure of the complex between actin and myosin subfragment 1 (S1), designated the acto-S1 complex, in the presence of ATP was examined by EM. This was accomplished by using negative staining to study a complex of S1 covalently crosslinked to actin by the zero-length crosslinker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide. Two levels of S1 binding were studied, with a molar ratio of crosslinked S1 to total actin of either 20% or 50%. The lower percentage was used to observe individual S1 molecules attached to actin, while the higher percentage was used to look at the overall pattern of S1 decoration of the actin filament. In the absence of ATP, the appearances of both the 20% and 50% crosslinked filaments closely resembled the rigor apparances obtained with noncrosslinked proteins. The arrowheads observed had the conventional structure, and individual S1 molecules were elongated and curved and appeared to make an angle of 45.degree. with the thin filament. Addition of ATP to the crosslinked acto-S1 complex caused a radical change in the structure of the cross-bridges. At both 20 and 170 mM ionic strengths, individual S1 molecules appeared to be attached at variable angles which, in contrast to rigor, did not center on 45.degree.. In addition, the S1 molecules often appeared shorter and fatter than in rigor. The 50% crosslinked acto-S1 preparation no longer showed the arrowhead pattern of S1 decoration but instead appeared to be disordered with little obvious polarity. Control experiments with ADP suggest that these effects were not due simply to a weakening of the binding of S1 to actin in the presence of nucleotide but most likely were ATP-specific. The crosslinked acto-S1 complex, which hydrolyzes ATP at about the same rate as the maximal actin-activated ATPase of S1 (Vmax), is composed of a mixture of states A.cntdot.M.cntdot.ATP and A.cntdot.M.cntdot.ADP.cntdot.Pi (in which A = actin and M = myosin), with more than 50% of the crosslinked S-1 occuring in state A.cntdot.M.cntdot.ATP. Both states A.cntdot.M.cntdot.ATP and A.cntdot.M.cntdot.ADP.cntdot.Pi apparently have a very different conformation from the classic arrowhead conformation of the A.cntdot.M state.