A Proposed Mechanism for the Initial Myosin Binding Event on the Cardiac Thin Filament: A Metadynamics Study

Abstract

The movement of tropomyosin over filamentous actin regulates the cross-bridge cycle of the thick with thin filament of cardiac muscle by blocking and revealing myosin binding sites. Tropomyosin exists in three, distinct equilibrium states with one state blocking myosin–actin interactions (blocked position) and the remaining two allowing for weak (closed position) and strong myosin binding (open position). However, experimental information illuminating how myosin binds to the thin filament and influences tropomyosin’s transition across the actin surface is lacking. Using metadynamics, we mimic the effect of a single myosin head binding by determining the work required to pull small segments of tropomyosin toward the open position in several distinct regions of the thin filament. We find differences in required work due to the influence of cardiac troponin T lead to preferential binding sites and determine the mechanism of further myosin head recruitment.

Structure and Dynamics of the Flexible Cardiac Troponin T Linker Domain in a Fully Reconstituted Thin Filament

“…Generation of computational cTF models for calcium-depleted (−Ca 2+ ) and calcium-saturated (+Ca 2+ ) thin filaments follows a similar procedure utilized previously. 47 Our previous computational cTF model 51 was refined according to recently published cryo-EM images of the cTF that demonstrated the orientation of the cTn core within the cTF thin filament and the organization of cTnT within the overlap region. 10 This previous model differs from the recent cryo-EM structures in the placement of the TnT helix within the overlap region of the thin filament and the orientation of the Tn core.…”

Free-Energy Surfaces of Two Cardiac Thin Filament Conformational Changes during Muscle Contraction

“…Structures utilized in this study were created starting from the cryo-EM structures published by Yamada, Namba, and Fujii, as previously described, , which provided structures for both Ca 2+ -saturated and -unsaturated conformations of the CTF (PDB 6KN8 and 6KN7, respectively). In the context of saturation of the CTF, a fully saturated system refers to Ca 2+ bound to site II of cTnC, while unsaturated refers to no Ca 2+ bound to site II of cTnC.…”

“…All structured fragments were aligned and the proteins were subjected to minimization until all bonds and angles were acceptable values. Any clashing of proteins was resolved by rotating dihedral angles in the unstructured regions, as previously described. , The helical pitch of Tm in the Yamada structure was corrected to agree with the protein docking study by Pavadai et al, allowing Tm to span 7 actin monomers instead of the 6 monomers in the original pdb. The structure for the cTnI C-terminal domain of the Ca 2+ -unsaturated state was also corrected to agree with the docking study by Lehman et al, which found a more energetically favorable location compared to the originally published structure.…”

“…Following this, studies by both Pavadai et al and Lehman et al have since refined certain segments in the models: Tm helical pitch and location and cTnI placement on actin, respectively. Our group has previously published results using these updated structures while also providing initial locations for unstructured segments not included in the cryo-EM maps to include all of the complete proteins of the CTF. , …”

“…Our group has previously published results using these updated structures while also providing initial locations for unstructured segments not included in the cryo-EM maps to include all of the complete proteins of the CTF. 15,16 Understanding the interactions between proteins in the cTn complex is crucial for understanding contraction at a molecular level. Previous work analyzing the opening of cTnC has shown that binding of Ca 2+ to site II creates a strain on the protein.…”