Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation

Matthew H. Doran, Michael J. Rynkiewicz, Elumalai Pavadai, Skylar M.L. Bodt, David Rasicci, Jeffrey R. Moore, Christopher M. Yengo, Esther Bullitt, William Lehman

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically 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, where the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366–372. Cryo-EM analysis of fully decorated actin-tropomyosin filaments with wild-type and mutant S1, yielded 3.4–3.6 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 when filaments were decorated with either mutant or wild-type S1. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of filaments containing 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, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the high potential energy associated with tropomyosin positioning in wild-type filaments favors an effective transition to B-and C-states following release of myosin from the thin filaments during relaxation.

Original languageEnglish (US)
Article numbere202213274
JournalJournal of General Physiology
Volume155
Issue number2
DOIs
StatePublished - Feb 6 2023

All Science Journal Classification (ASJC) codes

  • Physiology

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