Human Beta-Cardiac Myosin Cardiomyopathy Mutations R712l And E497d Disrupt A Key Salt-Bridge In The Coupling Domain

Mutations in the human beta-cardiac myosin gene (MYH7) are responsible for a large number of hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Omecamtiv mecarbil (OM) is a small molecule drug that induces allosteric changes in beta-cardiac myosin activity and is in clinical trials for treatment of systolic heart failure. OM binds in a narrow cleft adjacent to the SH1 helix, relay helix, and converter domain of beta-cardiac myosin and interacts with residues involved in linking changes in the nucleotide pocket to rotation of the lever arm, known as the ‘coupling region’. Cardiomyopathy mutations R712L and E497D disrupt a salt bridge between R712 and E497 that is centered in the OM binding pocket and plays a role in coupling the relay helix to a small beta-sheet of the converter domain. In the present study, we have characterized the effect of the mutations and OM on the unloaded shortening velocity of R712L and E497D beta-cardiac HMM mutants. The R712L mutation causes a 5-fold slowing of motility, and increases the IC50 of OM binding to ∼30 μM from 0.1 μM for the WT cHMM. OM also restores the velocity of R712L cHMM to 75% of WT cHMM velocity. On the other hand, the E497D mutation has little effect on the velocity, but increases the IC50 of OM to 4.5 μM. OM inhibits the velocity of E497D cHMM but only by 2.5-fold compared to over 25-fold for WT cHMM. In conclusion, the dramatic effect on motility (R712L) and impact on the IC50 of OM (R712L and E497D) suggest a central role of this buried salt bridge in coupling the nucleotide state to rotation of the converter domain.