Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V

Figures

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  Figure 1. Schematic diagram showing the domain organization of MyBPC and its C2 domain. (A) MyBPC consists of eight IgI-like domains (blue), three FnIII-like domains (orange), and one unique M domain. Binding partners are indicated above the respective subdomains. The positions of the MYBPC3Δ25bp/D389V mutation are indicated. PA: proline–alanine-rich region. (B) Alignment of MyBPC C2 protein sequences. Residue D389 is highly conserved across different mammalian isoforms. Rn: Rattus norvegicus, Mm: Mus musculus, Hs: Homo sapiens, Bt: Bos taurus. Alignment was created using T-Coffee [29] and Jalview [30]. (C) Cartoon representation of the MyBPC C2 domain (PDB: 5k6p [31]) with relevant residues D389, K395, and A417 displayed as spheres. (D) Domain topology scheme of the C2 domain as generated by EMBL-EBI PDBsum (https://bio.tools/pdbsum_generate; last accessed on 1 November 2021). A1–A4 and B1–B6 represent strands of the β-sheets A and B.

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  Figure 2. Free energy landscapes of MyBPC C2 wt (A) and D389V (B) domains generated using 100 ns of CORE-MD II simulations. The effect of the D389V mutation on the conformational space in the vicinity of the mutation is explored. We define the interatomic distances between D389V Cγ/V389 Cβ–K395 Nζ and D389V Cγ/V389 Cβ–A417 Cα as principal coordinates of the FEL. The scale of the color bar is given in units of kBT and represents the free energy ΔF (see Equation (5), Section 4.3). (1) to (4) indicate conformational states of C2 wt, and (3′) and (4′) indicate those of C2 D389V. (C) Free energy differences ΔΔF between C2 wt and C2 D389V (see Equation (6), Section 4.3). Negative values indicate an abundance of wt, and positive values indicate an abundance of D389V states. (D) Sample conformer extracted from State (4) in (A). C2 wt is depicted with the interacting residues D389, K395, and A417. D1 represents the x-axis and D2 represents the y-axis of the distances plotted in figures (A) to (C) for all conformations adapted over the course of the simulation.

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  Figure 3. Kinetic network representation derived from the clustered major conformations of C2 wt and D389V. Representative conformations of the States (1) to (4) identified in Figure 2 are shown for C2 wt (black), and (3′) and (4′) are shown for D389V (blue). Backbone Cα is shown as a tube representation, and the side chains of residues D389, V389, K395, and A417 are shown as ball–and–stick models with CPK coloring. The opaque underlay displays 40 conformers of each state. The numbers with circular background denote different states. Their size corresponds to the relative occupancy of each state over the course of the simulation, which is shown below the respective conformers. Arrows and percentages reflect probabilities of transition between two states.

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  Figure 4. Mean smallest distance matrices for MyBPC C2 wt (A) and D389V (B). The time-averaged minimum distance of each residue to every other residue is plotted in heat maps with white indicating the smallest distance and black indicating the largest distance ranging from 0 to 1.5 nm. This map reveals differences in the global conformational space of C2 wt and D389V. Dotted boxes indicate the β4–5–6 interstrand contacts within the C-terminus of the domain. Dashed boxes indicate the β1–6 interstrand contacts of C2 wt that are destabilized in C2 D389V (for topology see Figure 1D). (C,D) Sample structures of C2 wt (grey) and D389V (blue) to illustrate the differences in C-terminal conformation regarding the dotted boxes in (A,B). Residues 430 to 450 are marked in red.

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  Figure 5. High-speed cosedimentation experiments of MyBPC C0–C2 with F-actin and β-cardiac myosin. (A) Actin (0–40 µM) and MyBPC C0–C2 (5 µM) were cosedimented and the fraction of C0–C2 in the pellet was determined. A quadratic fit yields the dissociation constants Kd for wt of 3.2 ± 0.9 µM and D389V of 2.8 ± 0.6 µM. (B) MyBPC C0–C2 (0–30 µM) was cosedimented with 2.5 µM of β-cardiac myosin. The amount of C0–C2 bound to synthetic thick filaments was determined using a protein standard range of C0–C2. A quadratic fit yields Kd values for wt of 12.7 ± 4.7 µM and D389V of 14.8 ± 7.7 µM. Data points indicate mean ± SD from at least n = 3 experiments.

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  Figure 6. Constant velocity steered molecular dynamics (cvSMD) simulation of MyBPC C2 domain unfolding. The SMD atom P452Cα was pulled at a velocity of 0.01 Å/ps and the spring constant was set to 7 kcal/mol/Å. (A) Sample traces for an unfolding simulation of each wt and D389V MyBPC C2 domain. The force acting on the SMD atom is plotted against the time of the simulation. Data were smoothened using Savitzky–Golay filter to compensate for the noise created by the spring acting on the fixed atom. (B) Sample structures of time points 0, 1400, and 2400 ps in the unfolding experiments displayed in (A). C2 wt and C2 D389V are shown in grey and blue, respectively, with residue 389 as a CPK model. Residues A372–K377 and T445–P452 of the parallel β1–6 strands are marked in red. (C) Average of n = 10 independent unfolding simulations of each wt and D389V MyBPC C2 domain. The force acting on the SMD atom is plotted against the time of the simulation. Data were smoothened using Savitzky–Golay filter to compensate for the noise created by the spring acting on the fixed atom. (D) The time point of the major unfolding event is plotted for each individual unfolding experiment. The major unfolding event is…

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  Figure 7. Proposed disease mechanisms as a result of age-related alternative splicing. All carriers in this model are heterozygous for one of the variants and produce wt protein from at least one allele at all times. Young and middle-aged MYBPC3Δ25bp carriers predominantly produce wt mRNA from both alleles, resulting in mild or absent symptoms. Incorporation of MyBPCD389V into the contractile zone of sarcomeres in young and middle-aged MYBPC3Δ25bp/D389V carriers leads to myocardial remodeling mediated by a poison polypeptide mechanism. As patients age, the alternatively spliced transcript on the allele carrying the intron deletion predominates in both MYBPC3Δ25bp and MYBPC3Δ25bp/D389V carriers. We assume that the truncated forms of the protein are efficiently eliminated by the ubiquitin–proteasome system in cardiomyocytes with smaller fractions of MyBPCC10mut and MyBPCD389V/C10mut mislocalizing to the Z-disc. As a result, C-zone haploinsufficiency is predicted to become the dominant disease mechanism in elderly MYBPC3Δ25bp and MYBPC3Δ25bp/D389V carriers.