The Dilated Cardiomyopathy E525K Mutation Stabilizes the Cardiac Myosin Interacting-Heads Motif While Activating the Isolated Motor Domain

The E525K mutation in beta-cardiac myosin is a frequent cause of inherited cardiomyopathies. This mutation impacts both thick filament regulation and motor activity, but the structural reasons for these alterations have been unknown until now. Researchers utilized cryo-EM and molecular dynamics simulations to understand how the E525K mutation impacts the conformational state of cardiac myosin, both in its autoinhibited interacting-heads motif and isolated myosin head states.

In the autoinhibited IHM, the E525K-mutated myosin consistently adopted a single S2 conformation, unlike the wild-type myosin which showed conformational diversity. Analyzing the structural and electrostatic properties of the mutation, they discovered that E525K boosted positive charge density at the blocked-head (BH) - S2 interface, enhancing interactions with negatively charged residues within S2.

Through 3D variability analysis and molecular dynamics simulations of the E525K IHM, they found reduced S2 mobility, consistent with the strengthened BH - S2 interactions, and decreased conformational flexibility, indicating a structural mechanism for stabilizing the IHM state. To examine the isolated myosin head, they determined cryo-EM structures of both wild-type and E525K subfragment-1 (S1).

The mutation triggered local conformational changes in the activation-loop and Loop 3 region, the SH3-like domain, and the essential light chain of S1, revealing structural alterations that support the enhanced motor activity previously observed in isolated E525K myosin motors. These findings demonstrate that the E525K mutation stabilizes the autoinhibited IHM by electrostatically stabilizing the BH - S2 interface, while simultaneously inducing structural changes consistent with the activation of the isolated motor domain.

This study provides structural insights into how a single DCM mutation differentially regulates the structure and function of cardiac myosin S1 and IHM.