Myosin SH1 Thiol as a Sensor of Rotation of the C-terminal Region of the Motor Domain, the “Converter”

Abstract

Actin-myosin interaction in smooth muscle is regulated by the phosphorylation of myosin regulatory light chains (RLCs), which is mediated by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). Therefore, contraction and regulation are controlled by the intracellular Ca2+ concentration. Previously we found that chemical modification of a reactive thiol SH1 of smooth muscle myosin leads to a complete loss of Ca2+-regulation of the contractile system. Here we investigate why SH1 modification can functionally mimic the phosphorylation of RLCs, even though the position of SH1 is far away from the neck region, where phosphorylation sites reside. SH1 locates near the converter, which rotates by ∼70° upon the transition from the “nucleotide-free” to “pre-power stroke” state. The modification rate of SH1 with a thiol reagent, IAEDANS, was dramatically inhibited by the formation of 10S myosin. Comparison between myosin structures in the pre-power stroke state and the nucleotide-free state explained why SH1 is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the converter rotation. Modeling of the myosin structure in the pre-power stroke state, in which SH1 was selectively modified with IAEDANS, revealed that this bulky probe buried in a deep cleft of myosin becomes an obstacle when the converter rotates toward its position in the pre-power stroke state. This result suggests that SH1-modified myosin cannot assume 10S myosin formation, because of an incomplete rotation of the converter in the pre-power stroke state. We propose that the loss of the phosphorylation-dependent regulation of the actin-activated ATPase activity of smooth muscle myosin by SH1 modification is due to the modification-induced inhibition of the head-head interaction proposed by Wendt et al. [J. Cell Biol. 147, 1385-1390 (1999)].