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Recent findings have suggested that the interaction between the two heads is critical for phosphorylation-dependent regulation of smooth muscle myosin. We hypothesized that the interaction between the two regulatory light chains on two heads of myosin dictates the regulation of myosin motor function. To evaluate this notion, we engineered and characterized smooth muscle heavy meromyosin (HMM), which is composed of one entire HMM heavy chain and one motor domain truncated heavy chain containing the S2 rod and regulatory light chain (RLC) binding site, as well as the bound RLC (SMDHMM). SMDHMM was inactive for both actin-translocating activity and actin-activated ATPase activity in the dephosphorylated state, demonstrating that the interaction between the two RLC domains on the two heads and/or a motor domain and a RLC domain in a distinct head is sufficient for the inhibition of smooth muscle myosin motor activity. When phosphorylated, SMDHMM was activated for both actin-translocating activity and actin-activated ATPase activity; however, these activities were lower than those of double-headed HMM, implying partial release of inhibition by phosphorylation in SMDHMM and/or cooperativity between the two heads of smooth muscle myosin. The present results indicate that the RLC domain is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. On the other hand, similar to double-headed HMM, SMDHMM showed both "folded" and "extended" conformations, and the ratio of those conformations is dependent on ionic strength, suggesting that the RLC domain is sufficient to regulate the conformational transition in myosin.
Li et al. (2000) studied this question.