Effects of Two Familial Hypertrophic Cardiomyopathy-Causing Mutations on α-Tropomyosin Structure and Function

Abstract

Missense mutations in alpha-tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled tropomyosins differed in their response to myosin S1 binding to the actin-tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.

Functional Consequences of Hypertrophic and Dilated Cardiomyopathy-causing Mutations in α-Tropomyosin

“…The widely studied HCM-causing Tm mutation, glutamic acid 180 to glycine (E180G), was chosen as a control for the new method based on in vitro and in vivo data reported by several other groups. In vitro studies showed that this mutation causes a significant decrease in actin binding affinity when measured by co-sedimentation assays (31)(32)(33). E180G also caused a loss in the stability of the Tm structure when thermal denaturation was measured by circular dichroism and differential scanning calorimetry (32,33).…”

“…In vitro studies showed that this mutation causes a significant decrease in actin binding affinity when measured by co-sedimentation assays (31)(32)(33). E180G also caused a loss in the stability of the Tm structure when thermal denaturation was measured by circular dichroism and differential scanning calorimetry (32,33). In addition, E180G-Tm caused a decrease in thin filament motility when measured by in vitro motility assays (31,34).…”

“…A better explanation for the loss of inhibition is that the HCM mutants do not bind actin as well. It is already known for E180G that this mutation causes large decreases in actin binding affinity of as much as 3-fold (31)(32)(33)37). When Tm does not bind well to actin, more myosin may bind and form strong crossbridges (1), the cycling of which may contribute to a shift in the Ca 2ϩ sensitivity of ATPase activity.…”

Mutations of the Light Meromyosin Domain of the β-Myosin Heavy Chain Rod in Hypertrophic Cardiomyopathy

“…Missense mutations in the myosin rod have previously been shown to cause a significant dominant phenotype in both nematode 27,28 and Drosophila 29 ; in these models, dysfunction is caused, at least in part, by aberrant thick filament assembly and, in the case of Drosophila, by possible altered interaction with flightin, 29 a myosin-binding protein in indirect flight muscle. Disruption of ␣-helical coiled-coil structures has also been associated with human disease: altered stability of the ␣-helical coiled-coil has been demonstrated for the Asp175Asn and Glu180Gly HCM mutations in ␣-tropomyosin, 30 and a missense mutation in the desmin rod domain causes autosomal dominant distal myopathy through a dominant-negative effect on filament formation. 31 It is possible that some of the reported MyBP-C mutations may also act by affecting thick filament assembly/ stability, although it is equally likely that they act by disrupting the modulating effect of MyBP-C on contractility.…”

Mechanistic Heterogeneity in Contractile Properties of α-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies

“…Type Tm-To test for functional differences between WT and mutant Tms, we first examined their binding affinity with actin (40,41). With the exception of E62Q and I172T, the mutations altered the K d values (Fig.…”