Myosin-binding protein C (MyBP-C) is a modulatory sarcomeric protein which interacts with both thick and thin filaments, and is likely to form transvere “C-links” that can activate thin filaments and resist myofilament sliding by generation of viscoelastic drag. Its significance is underlined by the number of mutations associated with hypertrophic cardiomyopathy (HCM) in the gene MYBPC3 encoding the cardiac isoform of the protein. While most of these mutations are nonsense mutations that result in reduced cMyBP-C expression, single missense mutations occur throughout all domains of the protein. It has been recently shown by our group that the middle domains play an important role in inhibiting the activating effects of the N’-terminal domains (C0-C2) and that cMyBP-C slows relaxation in cardiac myofibrils (Greenman et al., 2025; Dvornikov and Harris, 2025). Here, we utilized the same cut-and-paste technique to replace endogenous cMyBP-C with recombinant fragments having deletion of the (1) flexible 10-a.a. linker between the C4 and C5 domains (dLinker) or (2) a 28-a.a. loop in C5 domain (dLoop). We also substituted the middle domains C3-C7 with the Ig27 titin domains. Results demonstrate that deletion of the C4-C5 linker significantly increased the duration of the linear relaxation phase together with the significant reduction of the rate of fast exponential relaxation. However, the deletion of the 28-a.a. loop in C5 did not significantly affect relaxation kinetics in single cardiac myofibrils. Titin replacement also significantly slowed both phases of relaxation. We conclude that the flexible C4-C5 linker, but not the C5 loop, contributes to inhibiting effects of the N-terminal domains. We provide a model where the middle domains and the linker in particular, allow for the molecule bending, potentially causing unbind of the activating C0-C2 domains from the thin filament.
Dvornikov et al. (2026) studied this question.