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Abstract
Mutations in 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 remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (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) 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 subfragment 1 for binding to F-actin and effectively inhibit actomyosin 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 Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.
Keywords: actin; cryo-EM; muscle regulation; myosin binding protein C; tropomyosin.
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Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Effects of C0 and C1…
Fig. 1.
Effects of C0 and C1 on TF-dependent myosin-S1 ATPase activity. Data in the…
Fig. 1. Effects of C0 and C1 on TF-dependent myosin-S1 ATPase activity. Data in the presence of C0 was fit by a simple inhibition equation V(C0) = Vo/(1+ ([C0]/KiC0)), where Ki C0 = 12.1 ± 1 μM, whereas data in the presence of C1 shows cooperative activation and inhibition that yields a best fit using V(C1) = V1/(1 + (Km/[C1])n + ([C1]/Ki)n) + Vo equation, where V1 = 3.2 ± 0.3⋅s−1, Km = 8.4 ± 1 µM, Ki = 15.2 ± 3 µM, and n = 2.1 ± 0.3 and Vo = 0.6 ± 0.1⋅s−1. Final protein concentrations used were 0.65 µM S1 and 5 µM TFs. Domain structure of the NTD of cMyBP-C is shown in the Inset.
Fig. 2.
The C0 Ig-like domain of…
Fig. 2.
The C0 Ig-like domain of cMyBP-C binds to three distinct sites on the…
Fig. 2. The C0 Ig-like domain of cMyBP-C binds to three distinct sites on the surface of actin filaments and does not interfere with the closed position of the Tm on F-actin. Reconstructions are shown as gray transparent surfaces. The frequency of each structural mode is denoted on the left of each map. Actin molecules are shown as tan ribbons, Tm is shown as red ribbons, and the NMR structure of the C0 Ig domain (PDB: 2K1M) is shown in magenta (C0-F1 mode), blue (C0-F2 mode), or cyan (C0-S mode). Actin subdomains are numbered in black. (A–C) Three-dimensional reconstructions of structural classes of TF segments that have Tm stripped off, but are uniformly decorated with C0. C0 binds to the front of F-actin in two structural modes indicated as C0-F1 (A) and C0-F2 (B). C0 also binds to the side of F-actin, denoted here as C0-S mode (C). (D) Three-dimensional reconstruction of TF segments uniformly occupied with Tm, but not with the C0 domain, shows Tm (red ribbons) in the closed position (21). (E and F) Three-dimensional reconstructions of TF segments uniformly occupied with Tm (red ribbons) and possessing C0 domain bound in either C0-F1 (E) or C0-F2 (F) modes. C0 in both structural modes is distal from the Tm in the closed position (blue arrows). (G–I) Projected position of the S1 myosin head on the surface of F-actin (solid yellow) (24) clashes with C0 in either C0-F1 (G) or C0-F2 (H) modes (red arrows) but has no steric hindrance with C0 in the C0-S mode (I, green arrows).
Fig. 3.
The C1 Ig-like domain of…
Fig. 3.
The C1 Ig-like domain of cMyBP-C displaces Tm from its closed position on…
Fig. 3. The C1 Ig-like domain of cMyBP-C displaces Tm from its closed position on F-actin when bound in the front mode and traps Tm in its open state when bound to TF in the side mode. Reconstructions are shown as gray transparent surfaces. The frequency of each structural mode is denoted on the left of each map. Actin molecules are shown as tan ribbons, Tm in the open state is shown as green ribbons, whereas Tm in the closed state is shown as red ribbons and red surface. The crystal structure of the C1 Ig-like domain (PDB: 2V6H) is shown in blue (C1-F1 mode), cyan (C1-F2 mode), magenta (C1-F3 mode), or purple (C1-S mode). Actin subdomains are numbered in black in A. All complexes were formed at low Ca2+ levels (pCa > 8). (A–C) Three-dimensional reconstructions of structural classes of TF segments that have Tm stripped off, but are uniformly decorated with C1. C1 binds to the front of F-actin in three structural modes indicated as C1-F1 (A), C1-F2 (B), and C1-F3 (C). (D) Three-dimensional reconstruction of TF segments uniformly occupied with Tm, but not with the C1, show Tm (green ribbons) in the open position on the surface of F-actin (24). (E and F) Three-dimensional reconstructions of TF segments uniformly occupied with Tm (green ribbons) and possessing C1 bound in either C1-F1 (E) or side (F) modes. Importantly, in the side mode (C1-S + Tm) C1 makes a prominent contact with Tm (F, red arrows). (G) Both open (green ribbons) and closed (red ribbons and red surface) positions of Tm on F-actin are shown. (H and I) Wheres C1 bound in C1-F1 (H) or C1-F2 (I) modes does not clash with the Tm in the closed state (H and I, green arrows), it collides with Tm when bound in C1-F3 mode (J, blue arrow). (K–N) In all three front modes, C1 interferes with the actomyosin interaction (K–M, red arrows), but is not projected to have steric clashes with myosin heads when bound in side mode (N, cyan arrows).
Fig. 4.
Effects of C0 and C1…
Fig. 4.
Effects of C0 and C1 on passive and Ca 2+ -activated force in…
Fig. 4. Effects of C0 and C1 on passive and Ca2+-activated force in permeabilized myocytes. (A) C0 (gray) and C1 (black) (100 μM) did not affect passive force in the absence of Ca2+ (pCa 9.0). Passive force at pCa 9.0 was expressed as a fraction of total force (PT, where PT = total maximal active and passive force measured at pCa 4.5). (B) Relative Ca2+-activated force measured in solutions of low Ca2+ (pCa 5.8) and maximal activating Ca2+ (pCa 4.5). Active forces (P) are expressed normalized to maximal active force at pCa 4.5 (P0, where P0 = PT – P9.0) under control conditions in the absence of C0 or C1 (open bar). C0 had no effect on force at either pCa 5.8 or pCa 4.5, whereas C1 increased force measured at pCa 5.8. (C) Summary tension–pCa relationships before and after addition of 100 μM C0 or C1. C1 significantly increased myofilament Ca2+ sensitivity of tension [pCa50 values before and after C1 were 5.58 ± 0.033 and 5.89 ± 0.072 (n = 5), respectively]. (Inset) C0 had no effect on myofilament Ca2+ sensitivity of tension [pCa50 values before and after C0 were 5.57 ± 0.027 and 5.54 ± 0.030 (n = 5), respectively]. See this image and copyright information in PMC
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