C.J. Miller, E. Bobkova, P. Cheung, *P.A. Rubenstein, and E. Reisler
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, CA 90095 and *the Department of Biochemistry, College of Medicine, University of Iowa, Iowa City, IA 52242
The results of recent studies on three weak binding sites for myosin on actin, the N-terminus, D24/D25, and E99/E100, and on their functional role in the myosin cross-bridge cycle shed light on the weak actomyosin interactions. Yeast actin mutants of these sites, with the acidic residues either replaced or deleted, had identical sliding properties under diverse conditions of in vitro motility assays. In these assays, the N-terminal mutants DNEQ and DDSE, as well as the D24A/D25A and E99A/E100A yeast actins, showed the same dependence of their sliding velocities on the density of HMM on the cover slip surface, generated the same amount of force against external load (~ 70% of that obtained with wild type actin), required methylcellulose to overcome the weak binding deficiency, and failed to move at the ionic strength of 150 mM. These results suggest that myosin binding to all three weak binding sites is required for cross-bridge transition from the weakly to strongly bound states under physiological ionic strength conditions. In addition, these results and the equilibrium binding data suggest that the orientational freedom of S1 on actin in the weakly bound states results from structural flexibility in S1 and not from alternate modes of acto-S1 docking. The strong binding of S1 and the in vitro motility of actin were found to be influenced considerably by the intermolecular interactions of loop 38-52 in subdomain 2 of actin. In agreement with previous studies, subtilisin cleavage of G-actin between M47 and G48 decreased ~ tenfold both the strong and weak binding of S1 to F-actin, did not change the Vmax value of acto-S1 ATPase, and reduced the in vitro motility of actin. The sliding velocity of cleaved actin showed a strong dependence on the density of HMM on the cover slip surface suggesting that the reduced motility of cleaved actin is related to its lower affinity to myosin. This interpretation was confirmed by experiments with cleaved actin-MgADP-BeFx-. BeFx- increased the stability of cleaved actin filaments, had no effect on the Vmax and Km values of acto-S1 ATPase, increased four-fold the strong binding affinity of S1 to cleaved actin, and increased also the sliding velocity of the cleaved actin. These results provide evidence for the importance of actin filament structure to actomyosin binding and force generation by myosin.