Facilitated Cross‐Bridge Interactions with Thin Filaments by Familial Hypertrophic Cardiomyopathy Mutations in α‐Tropomyosin

Abstract

Familial hypertrophic cardiomyopathy (FHC) is a disease of cardiac sarcomeres. To identify molecular mechanisms underlying FHC pathology, functional and structural differences in three FHC-related mutations in recombinant α-Tm (V95A, D175N, and E180G) were characterized using both conventional and modified in vitro motility assays and circular dichroism spectroscopy. Mutant Tm's exhibited reduced α-helical structure and increased unordered structure. When thin filaments were fully occupied by regulatory proteins, little or no motion was detected at pCa 9, and maximum speed (pCa 5) was similar for all tropomyosins. Ca2+-responsiveness of filament sliding speed was increased either by increased pCa50 (V95A), reduced cooperativity n (D175N), or both (E180G). When temperature was increased, thin filaments with E180G exhibited dysregulation at temperatures ~10°C lower, and much closer to body temperature, than WT. When HMM density was reduced, thin filaments with D175N required fewer motors to initiate sliding or achieve maximum sliding speed.

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

“…Clinically the V95A mutation causes severe symptoms and poor prognosis in patients (Karibe et al 2001), with significant increase in the Ca 2? sensitivity in thin filament (Bai et al 2013) and in vitro (Wang et al 2011). This is consistent with our finding of large mutational effects on Tpm's local flexibility and actin binding.…”

“…V95 is at the d position of the heptad repeat, so it is involved in stabilizing the Tpm coiled coil structure. As expected, the V95A mutation was found to destabilize the Tpm coiled coil structure (Wang et al 2011). Our flexibility analysis of V95A found decreased RMSF, increased PL (see Table 1), and altered local flexibility near ALA2, ALA4, and ALA5 (featuring anomalously high flexibility near ALA2, see Fig S4 in the Supporting Information).…”

“…E180 is at the e position of the heptad repeat (forming a salt bridge with R182 of another Tpm chain), so it is involved in stabilizing the Tpm coiled coil structure. As expected, the E180G mutation was shown to cause greater structural disorder (Wang et al 2011), decreased thermal stability in the C-terminal part (Kremneva et al 2004), increased local flexibility [near R133 and K233 (Ly and Lehrer 2012)], and decreased PL (Loong et al 2012a, b). Surprisingly, our flexibility analysis found lower RMSF and higher PL than the WT (see Table 1) and altered local flexibility near D137, ALA5, and E218 (see Fig S7 in the Supporting Information).…”

Ca2+-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding

“…Recombinant human α-Tm was expressed in Escherichia coli as a homodimeric fusion protein with maltose binding protein (MBP); α-Tm was purified following removal of the MBP affinity tag via thrombin cleavage as previously described [46,50,51,52,53]. After removal of the MBP tag, each of the two polypeptides in recombinant α-Tm has two extra, N-terminal amino acids (GS-); GS- is a conservative alternative to the AS-dipeptide in bacterially expressed Tm that substitutes functionally for acetylation of native Tm’s N-terminus in eukaryotic cells [54,55].…”

“…In separate experiments, ρ of ATPase-competent HMM on the flow cell surface was estimated from K-EDTA ATPase assays, assuming that the enzymatic activity of surface-adhered HMM was the same as that measured in solution [53,58,62]. …”