Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human α‐tropomyosin

Abstract

Mutations in the protein a-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca 2+ . However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg 2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.Keywords: circular dichroism; differential scanning calorimetry; Pichia pastoris; tropomyosin.Tropomyosins (Tms) are a family of highly conserved proteins found in most eukaryotic cells. The striated muscle isoform is an a-helical protein, which forms a parallel coiled-coil dimer twisted around the long axis of the actin filament. Each polypeptide chain has 284 amino acid residues, and each dimer binds to seven actin monomers and one troponin (Tn) complex (TnC, TnI and TnT). In striated muscle cells the Tm polymerizes in a head-to-tail fashion, and together with the troponin complex, regulates the Ca 2+ sensitivity of the actomyosin Mg 2+ ATPase complex [1]. The Tm amino acid sequence shows a sevenresidue pattern (a to g) repeated throughout the entire sequence. Positions a and d, on the same side of the helices, are usually occupied by apolar amino acids that allow hydrophobic interactions between chains. Positions e and g are often occupied by charged residues, and therefore contribute to the stabilization of the parallel coiled-coil structure by ionic interactions with residues at positions e¢ and g¢ of the other helix. Positions b, c and f are occupied by polar or ionic residues and they interact with solvent or other proteins [1]. In addition to the heptapeptide repeat, there are seven consecutive repetitions of approximately 40 residues each in the entire length of the chain, which correspond to the actin binding sites [2].Recom...

The Effect of Mutations in α-Tropomyosin (E40K and E54K) That Cause Familial Dilated Cardiomyopathy on the Regulatory Mechanism of Cardiac Muscle Thin Filaments

“…Consistent with this is the absence of any reduction in V max , cooperative unit size, K T , or K b . These aspects of the E54K mutation, in fact, resemble the results reported for HCM mutations, which also decrease actin-tropomyosin binding affinity (6,9,10) and increase Ca 2ϩ affinity for troponin C; however, other properties of the E54K mutation are similar to other DCM mutations, notably the reduced Ca 2ϩ sensitivity (EC 50 increased by an average 167% (8,11)). …”

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation

“…Since K70 is at the g position of the heptad repeat (forming a salt-bridge with E75 on the other Tpm chain), it is involved in stabilizing the Tpm coiled coil structure. Indeed, the K70T mutation was found to severely destabilize the Tpm coiled coil leading to decreased thermal stability (Heller et al 2003;Hilario et al 2004). Our flexibility analysis found increased RMSF and PL than the WT (see Table 1) and altered local flexibility near ALA1, ALA3, ALA5, and E218 (see Fig S3 in the Supporting Information).…”

Tropomyosin Flexural Rigidity and Single Ca2+ Regulatory Unit Dynamics: Implications for Cooperative Regulation of Cardiac Muscle Contraction and Cardiomyocyte Hypertrophy

“…A number of FHC-related mutations have been found in human cardiac α-tropomyosin (αTm), along with many other mutations primarily in cardiac cytoskeletal proteins (Bing et al, 2000; Fatkin and Graham, 2002; Roberts, 2002; Towbin and Bowles, 2002; Takeda, 2003; Wolska and Wieczorek, 2003). FHC-related αTm mutants have been linked to decreased thermal stability (Hilario et al, 2004; Kremneva et al, 2004; Wang et al, 2011) and a lower binding affinity for actin (Bing et al, 1997; Kremneva et al, 2004) compared to WT. In vitro studies with mutant αTm using myofibrillar ATPase activity, motility assays, and isometric force generation show significantly enhanced Ca 2+ -sensitivity and/or reduced cooperativity (Bing et al, 2000; Chang et al, 2005; Bai et al, 2011; Mathur et al, 2011; Wang et al, 2011).…”