C0 and C1 N-Terminal Ig-Domains of Myosin Binding Protein-C Exert Different Effects on thin Filament Activation

Abstract

Mutations in the genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac Myosin Binding Protein-C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g. C0, C1, M and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remain unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we employed an integral structure-function approach using cryo-electron microscopy, kinetics and force measurements to reveal how the first two Ig-domains of cMyPB-C (e.g. C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) from the closed to the open structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin S1 for binding to F-actin and effectively inhibit acto-myosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ may modulate the interaction of cMyBP-C with the TF in the sarcomere.