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| Title | Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation. |
|---|---|
| Journal, issue, pages | J Gen Physiol, Vol. 155, Issue 2, Year 2023 |
| Publish date | Feb 6, 2023 |
 Authors | Matthew H Doran / Michael J Rynkiewicz / Elumalai Pavadai / Skylar M L Bodt / David Rasicci / Jeffrey R Moore / Christopher M Yengo / Esther Bullitt / William Lehman /  |
| PubMed 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 ...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. |
 External links | J Gen Physiol / PubMed:36459134 / PubMed Central |
| Methods | EM (helical sym.) |
| Resolution | 3.4 - 3.6 Å |
| Structure data | EMDB-28083, PDB-8efi: EMDB-28270, PDB-8enc: |
| Chemicals |  ChemComp-ADP:  ChemComp-MG: |
| Source |
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 Keywords |  MOTOR PROTEIN / actin / tropomyosin / myosin / cardiac |
