Omecamtiv mecarbil and Mavacamten target the same myosin pocket despite antagonistic effects in heart contraction

Figures

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  Figure 1 –. Effect of Omecamtiv mecarbil (OM) and Mavacamten (Mava) on the motor cycle and regulation of β-cardiac myosin. (a) During its motor cycle, the myosin goes through three major structural states, associating to the actin in the pre-powerstroke (PPS) with the Lever arm up and performing the powerstroke which is responsible for heart contraction. In cardiac sarcomere, the population of available myosin is highly regulated by the presence of an inactive sequestered state that shuts down a proportion of myosin to regulate force production during contraction phases. OM and Mava have antagonistic effects on both the motor cycle and sequestered state stability. Mava is able to stabilize the sequestered state while OM destabilizes it, increasing the proportion of available heads. (b) On the active state, OM stabilizes the PPS state, increasing the number of heads available to interact with actin25, whereas Mava inhibits both the interaction with actin and the Pi release rate with an unknown mechanism.

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  Figure 2 –. Crystal structures of β-cardiac myosin complexed with Mavacamten and comparison with other PPS structures. (a) The motor domain (MD) and the S1 fragment (S1) structures complexed to Mavacamten (Mava) superimpose with a rmsd of 0.493 Å on 616 Cα. (b) Overall structure of the S1 fragment of β-cardiac myosin bound to Mava. The compound occupies a pocket between the Lever arm and the motor domain. (c) Superimposition of the OM bound and the Mava bound structures on the U50 shows that there is no difference of conformation on the actin binding interface and on the three nucleotide binding loops of the active site: Switches-1 and -2 and P-loop (spheres). (d), (e) and (f) Local conformational differences induced by the presence of OM or Mava, notably in linkers near the Transducer and in the Lever arm orientation. The Lever arm is more primed in the structures in which either of these drugs are bound, compared to the apo structure. (g), (h) and (i) Conformation of Ala767 and of the first β-strand of the Converter (Convβ1) depending on the presence of drugs.

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  Figure 3 –. Mava and OM targets the same pocket. (a) Cartoon and schematic representation of Mava binding site. (b) Cartoon and schematic LigPlot based34 representation of OM binding site. (c) and (d) Distinct shapes of the pocket where Mava and OM bind leads to different distances between the Converter and the motor domain, as shown by the distance between Y164 and N711 in the drug-bound structures. (e) Sequence alignment of class 2 myosins highlights the specificity determinants of OM and Mava. Residues important for binding both drugs are boxed in green, residues that lead to weaker affinity for skeletal myosin are colored in red, residues exclusive to OM or Mava binding are colored in deep purple and pink respectively. A star below the sequence indicates residues involved in electrostatic interactions.

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  Figure 4 –. OM and Mava stabilize the priming of the lever arm. (a) Evolution of the root mean square deviation (RMSD) of the Cα positions of the Lever arm (711-806) compared to the motor domain (MD) (1-710) for the three conditions during the time course of the simulation: Apo (blue), OM (red) and Mava (stone grey). The two extreme positions of the Apo (0 ns and 730 ns) are represented in blue and sand green, showing that a 36° swing occurred during the simulation. (b) Cartoon representation of the OM and Mava conditions which displays the extreme positions of the Lever arm. Mava allows more flexibility in Lever arm condition than OM. All the structures were aligned on the N-term subdomain for these measurements.

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  Figure 5 –. OM and Mava induce different effects on the allostery of the motor. (a) RMSD of the Cα positions of the motor domain (1-710) during the time course of the simulation for the three conditions: Apo (blue), OM (red) and Mava (grey). Following the same methodology, (b) and (c) RMSD of the Cα positions of the helix-turn-helix (HTH, 529-526) and of the HO-helix (419-445), respectively. In (d), (e) and (f), the extreme positions of the HTH (part of the actin binding interface, the HS-helix is colored differently) is represented as well as that of the connectors for Apo, OM and Mava, respectively. The Pi backdoor is found close to the salt bridge between Switch-1 R243 and Switch-2 E466. In both the Apo and the OM conditions, the communication between the Wedge and the Switch-2 remains during all the duration of the simulation through the interaction with Sw2F465. This communication stabilizes the conformation of the HTH and of the actin binding interface. In the Mava condition, the communication between the Wedge and Switch-2 is rapidly lost in the simulation by the flip of F465, leading to a drastic change in the conformation of the HTH and specifically the HS-helix (f). When Mava is…

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  Figure 6 –. Effects of Mava and OM on the β-cardiac myosin sequestered state. (a) Superimposition of the cryo-EM β-cardiac IHM structure (cartoon, colored, PDB code 8ACT 7 on the S1 structure of β-cardiac myosin complexed to OM (PDB 5N69, 25. Superimposition is done on the motor domain of the blocked head (BH) (residues 1-707). The presence of OM induces several differences in the Transducer and in the Converter (indicated in dashed lines). (b) Superimposition of the β-cardiac IHM structure on the two molecules of the asymmetric unit of PPS-S1-Mava (ribbon, in black). The superimposition was performed on the head-head interface (residues 329-447 of the BH and residues 498-518, 708-780 on the FH, rmsd 0.5 Å on 112 Cα). The structures are highly superimposable. (c) Superimposition of a molecule of the asymmetric unit of PPS-S1-Mava on the BH (residue 1-707) of the β-cardiac myosin IHM.

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  Figure 7 –. OM and Mava mechanism of action. Schematic representation of the effects of both OM and Mava on (i) folding back to form the sequestered state and (ii) Pi release. (Left) The apo condition (without drug) allows the dynamic formation of the sequestered state (Interacting-heads motif, IHM) in equilibrium with heads available for association with actin. (Center) Mava alters the actin binding interface, generating an “incompetent” PPS that slows association with the actin filament. Mava-bound heads undergo flexibility in lever arm priming, a requirement for the formation of the head/head interfaces characteristic of the cardiac sequestered state7. In the zoom (inset), the cardiac IHM blocked and free head structures are compared with the Mava-bound crystal structure (thin black lines). (Right) When OM binds (thin orange lines), the conformation of the Transducer and the orientation of the Lever arm differ from what is found in cardiac IHM. PPS-OM is thus incompatible with the sequestered state. OM bound heads can efficiently bind F-actin and promote Pi release, yet the swing of the lever arm is affected as it requires destabilization of the OM binding site26.