The role of myosin loop-4 in localizing tropomyosin on actin during muscle activation and relaxation.

Abstract

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, namely myosin loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct. In the mutant, the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366 to 372. Cryo-EM analysis of fully-decorated actin-tropomyosin filaments with either wild-type or mutant S1, yielded 3.4- to 3.6-Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum site, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the relatively high potential energy associated with wild-type tropomyosin positioning favors an effective transition to B- and C-states following release of myosin from thin filaments during relaxation.