The interaction between the regulatory light chain domains on two heads iscritical for regulation of smooth muscle myosin

Recent findings have suggested that the interaction between the two heads i s critical for phosphorylation-dependent regulation of smooth muscle myosin . We hypothesized that the interaction between the two regulatory light cha ins on two heads of myosin dictates the regulation of myosin motor function . To evaluate this notion, we engineered and characterized smooth muscle he avy meromyosin (HMM), which is composed of one entire HMM heavy chain and o ne motor domain truncated heavy chain containing the S2 rod and regulatory light chain (RLC) binding site, as well as the bound RLC (SMDHMM). SMDHMM w as inactive for both actin-translocating activity and actin-activated ATPas e activity in the dephosphorylated state, demonstrating that the interactio n 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 mu scle myosin motor activity. When phosphorylated, SMDHMM was activated for b oth actin-translocating activity and actin-activated ATPase activity; howev er, these activities were lower than those of double-headed HMM, implying p artial release of inhibition by phosphorylation in SMDHMM and/or cooperativ ity between the two heads of smooth muscle myosin. The present results indi cate that the RLC domain is critical for phosphorylation-dependent regulati on of smooth muscle myosin motor activity. On the other hand, similar to do uble-headed HMM, SMDHMM showed both "folded" and "extended" conformations, and the ratio of those conformations is dependent on ionic strength, sugges ting that the RLC domain is sufficient to regulate the conformational trans ition in myosin.