Determination of the critical residues responsible for cardiac myosin binding protein C's interactions

Abstract

Despite early demonstrations of myosin binding protein C's (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure-function relationships is needed to fully understand cMyBP-C's ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-C's N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-C's C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for the formation and/or maintenance of normal cMyBP-C sarcomeric distribution.

Fig. 1

Detecting functional peptide-peptide interactions between cMyBP-C and actin or cMyBP-C and S2-MyHC. (A) Shown are the N-terminal fragments derived from cMyBP-C that were used for the Y2H assays. (B) Y2H screen using cMyBP-C fragments subcloned into pGBKT7 as bait and full-length mouse α-cardiac actin subcloned into pGADT7 as prey. (C) Y2H screen using cMyBP-C fragments as bait and the S2-myosin fragment as prey identifies the m domain as being competent and sufficient to interact with S2-MyHC.

Fig. 2

Identifying essential residues for protein-protein interactions. (A) Sequence alignment identifying possible homologies underlying an actin binding site. Residues K190, K193, K195 were mutated to alanines. (B) cMyBP-C’s N-terminal fragments with the C1 domain mutation used in the Y2H assays. (C) Y2H experiments showing growth/no growth on restrictive media. (D) ablation of the actin binding domain did not interfere with cMyBP-C/S2-MyHC binding.

Fig. 3

Critical residues in the m domain for actin or S2-MyHC binding. (A) The m domain mutant constructs used for the Y2H assays. The residues in red were chosen for mutagenesis. (B) Y2H experiments showing mut-4 (R279A and R280A), mut-5 (R279A, R280A and T281A) and mut-8 (R304A and R305A) are not competent to mediate actin binding. (C) Mut-1 (R266A, R270A and R271A), mut-5 (R279A, R280A and T281A), mut-6 (K298A, K299A and R300A) and mut-7 (K298A, K299A) are not competent to mediate interaction with S2-MyHC. (D, E) cMyBP-C interactions with F-actin and S2-MyHC are mutually exclusive. (D) Cells were grown on SD/-His/-Leu/-Trp/X-α-gal plates: growth indicates interaction of the m domain with actin or S2-MyHC. (E) Cells plated on SD/-His/-Leu/-Met/-Trp/X-α-gal plates cannot grow, indicating that the third protein (either actin or S2-MyHC, depending upon the construct) significantly inhibited interaction between the m domain and S2-MyHC, or actin, respectively.

Fig. 4

siRNA-mediated knock down of endogenous cMyBP-C. (A) Western blot analysis shows that siRNA targeted to cMyBP-C effectively decreases endogenous cMyBP-C levels. n=4, *P<0.05 (B) Immunofluorescent staining shows silencing of endogenous cMyBP-C (green). Cardiomyocytes are counterstained with a TnI antibody (red).

Fig. 5

Mutant cMyBP-C expression in cardiomyocytes. (A) C1-mutated (K190A, K193A and K195A) cMyBP-C (mut-cMyBP-C) proteins in NRCs. Western blot analysis showing endogenous cMyBP-C knockdown using cMyBP-C siRNA and replaced with either normal (wt-cMyBP-C) or the mutated cMyBP-C (mut-cMyBP-C). Constructs contained a myc-tag for quantitation. Representative protein blots are shown illustrating the decreased levels of endogenous cMyBP-C and replacement to normal levels using adenovirus-driven expression of the normal or mutated cMyBP-C. Quantitation of total cMyBP-C levels (top panel, right) shows restoration of normal levels. (n=4/treatment). (B) Immunofluorescent staining with NRCs counterstained with a TnI antibody (red).