Cryo-EM Reveals How Cardiomyopathy Therapeutic Drugs Modulate the Myosin Motors of the Heart

Figures

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  Fig 1.. Cryo-EM density maps of the human β-cardiac myosin IHM under three experimental conditions. (A–C) M2β HMM with mava (A), without drug (B), and with OM (C). Each condition reveals two major IHM conformational states: a docked state (left images, shades of blue), where the S2 region bends and interacts with the FH, and an undocked state (right images, shades of green), where S2 is displaced from the FH and stays straight. 3D classification analysis (percentages shown for docked and undocked structures, see Tables S2 and S3 with statistical analysis) quantify the relative populations of the two states, showing that Mava favoring the docked and OM the undocked conformation. FSC global resolution is shown at the bottom left of each map. (D–F) Density maps fitted with atomic models focusing on the drug-binding sites and surrounding residues within the blocked and free myosin heads. (D, E) Mava (Magenta) in the docked and undocked IHM conformation binds to a conserved allosteric pocket, stabilizing (left, FH; right, BH) interdomain interactions. (F) OM binding site in the undocked IHM conformation showing OM (Purple) binding in both heads in the same allosteric binding pocket as Mava.…

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  Fig 2.. Displacement of S2 and the free head between bent and straight S2 conformations for the three experimental conditions. (A–C) Structural alignment of docked and undocked IHM conformations from WT+Mava (A), WT (B), and WT+OM (C) datasets. Within each panel, the docked (blue shades) and undocked (green shades) conformations are superimposed by aligning the BH to highlight the angular displacements of the FH (arrows to the left) and S2 (arrows to the right). Arrows indicate the direction and magnitude of structural shifts as S2 moves from docked to undocked position. These shifts reflect destabilization of the BH-FH interface as S2 undocks from the FH in each of the different conditions. While Mava favors minimal movement compared to WT, OM promotes a larger structural rearrangement (Table S4). Adjacent to each alignment, the corresponding undocked conformation is displayed and color-coded by per-residue RMSD relative to the docked state, mapped from low (blue) to high (orange) deviation, further showcasing the structural deviation in the S2 and FH regions. The bottom (yellow) region of the RMSD map, below the more strongly deviated orange region, represents the fulcrum of the…

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  Fig 3.. Cryo-EM density maps and atomic models of the WT+Mava (A,B) docked and (C,D) undocked IHM conformations. (A,C) Zoom in views show the interaction between Loop 2 (pink) of the BH and FH with different (lower and upper, respectively) regions of S2. (B,D) Zoomed-in panels of S2 – mesa interfaces showing S2 (from FH) interacting with positively charged residues from BH mesa, forming a triad of electrostatic interactions that strengthens the IHM configuration. Red-dotted rectangles show unique electrostatic interactions R904-E536 and D896-R663, possible only in the docked and undocked conformations respectively due to different positions of S2. (E, F) Electrostatic surface representation of WT+Mava docked IHM structure, highlighting charge complementarity regions between the motor domains and coiled-coil S2 region. Key interaction zones between Loop 2 of BH and Ring 1 of S2, and between Loop 2 of FH and Ring 2 of S2 (lower and upper rectangles respectively), showing well-aligned positive (blue) and negative (red) surface potentials. (G) Detail of FH Loop 2 – S2 Ring 2 interface, showing putative interacting charged residues, positive on Loop 2 and negative in Ring 2 of S2. (H) BH Loop…

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  Fig 4.. Comparative conformational dynamics and BH–FH interface interactions across WT, WT+Mava, and WT+OM. (A) Overlap of docked and undocked conformations of WT+Mava with their cryo-EM maps and models, showing angular displacements in the FH and S2 regions, and a 180° rotated view shows the BH–FH interface, indicated by the dotted box. (B) Quantitative comparison of FH and S2 angular shifts and buried surface area at the BH–FH interface. (C–E) BH–FH head-head interaction interfaces in docked conformations of WT+Mava, WT, and WT+OM, showing hydrogen-bonding interactions that stabilize the interface, with decreasing number of interactions from Mava to OM. (F–H) Corresponding views of undocked conformations reveal loss or rearrangement of these hydrogen bonds, with WT+OM showing the most distinct destabilization. To distinguish the BH and FH interacting residues, the BH is shown semi-transparent. Together, the comparisons illustrate how drug binding affects the BH–FH interface hydrogen bonding network and differentially modulates IHM stability across the variants.

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  Fig 5.. Lever arm angular deviations in docked and undocked IHM states upon drug binding. (A, B) Structural overlays of the three docked conformations (WT, WT+Mava, WT+OM) aligned on the blocked head (A) or the free head (B). Lever arm angular deviations for WT+Mava and WT+OM are measured relative to WT, which reveals drug-induced shifts in lever arm positioning (Table S5). (C-D) Structural overlays of the three undocked conformations (WT, WT+Mava, WT+OM) aligned on the blocked head (C) or free head (D). The small triangle depicts the fulcrum of lever arm deviation. Angular measurements demonstrate the differential effects of Mava and OM on lever arm orientation in both docked and undocked conformations.

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  Fig 6.. Proposed molecular mechanism for the allosteric control of IHM stability by Mava and OM (A). Schematic overview of thick and thin filament interactions illustrating how WT, HCM, DCM, and drug treatment shift the equilibrium between the sequestered IHM and open myosin states. In the normal WT heart (middle), both IHM and open states coexist in an optimal equilibrium. HCM mutations and OM shift the balance toward open myosin states, increasing actin-binding capability. DCM mutations and Mava shift the equilibrium toward increased IHM population, favoring energy conserving states. (B). Structural interpretation of IHM stability based on cryo-EM analysis. The docked state is maintained by three major interfaces: S2–FH, BH–FH, and S2–BH (yellow stars). Dotted-line ellipses and solid arrows highlight the transitions of the lever arm and S2 between docked and undocked conformations. Mava promotes lever arm shifting toward the motor domain, restricting lever arm flexibility and strengthening electrostatic interactions across the three interfaces, thereby stabilizing the bent S2 and docked IHM. In contrast, OM induces major leaver arm reorientation, weakening these interfaces and…