Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tendd to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.
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