TWO CLASSES OF MYOSIN INHIBITORS, BLEBBISTATIN AND MAVACAMTEN, STABILIZE β-CARDIAC MYOSIN IN DIFFERENT STRUCTURAL AND FUNCTIONAL STATES

ABSTRACTIn addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a newly-discovered low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional and the super-relaxed states are widely assumed to correspond respectively to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin. Disruption of the SRX population in different heart diseases, such as hypertrophic cardiomyopathy, results in unwarranted muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examine the structure-function relationships of two myosin ATPase inhibitors, mavacamten, and blebbistatin, and found that binding of mavacamten to myosin at a site different than blebbistatin populates myosin into the SRX state. Blebbistatin, and para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps non-functional, ‘ultra-relaxed’ state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations that diverge significantly from the hypothetical open and closed states and furthermore, mavacamten treatment causes a greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and blebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing SRX states.