The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy

Abstract

Point mutations targeting muscle thin filament proteins are the cause of a number of cardiomyopathies. In many cases, biological effects of the mutations are well-documented, whereas their structural and mechanical impact on filament assembly and regulatory function is lacking. In order to elucidate molecular defects leading to cardiac dysfunction, we have examined the structural mechanics of two tropomyosin mutants, E180G and D175N, which are associated with hypertrophic cardiomyopathy (HCM). Tropomyosin is an α–helical coiled-coil dimer which polymerizes end-to-end to create an elongated superhelix that wraps around F-actin filaments of muscle and non-muscle cells, thus modulating the binding of other actin-binding proteins. Here, we study how flexibility changes in the E180G and D175N mutants might affect tropomyosin binding and regulatory motion on F-actin. Electron microscopy and Molecular Dynamics simulations show that E180G and D175N mutations cause an increase in bending flexibility of tropomyosin both locally and globally. This excess flexibility is likely to increase accessibility of the myosin-binding sites on F-actin, thus destabilizing the low-Ca2+ relaxed-state of cardiac muscle. The resulting imbalance in the on-off switching mechanism of the mutants will shift the regulatory equilibrium towards Ca2+-activation of cardiac muscle, as is observed in affected muscle, accompanied by enhanced systolic activity, diastolic dysfunction, and cardiac compensations associated with HCM and heart failure.

Energy landscapes reveal the myopathic effects of tropomyosin mutations

“…Hence, mutations associated with charge changes at these sites (e.g. at residues 70, 91, 168, 175, 180) may increase local coiled-coil flexibility, and thus modify tropomyosin position on actin filaments [32, 33]. This could amplify effects noted above that weaken actin-tropomyosin interaction and thereby exacerbate aberrant responses to troponin and myosin.…”

Thin Filament Structure and the Steric Blocking Model

“…Li et al found that the tropomyosin coiled-coil is rather stiff with a persistence length about 12 times its own length (108,(134)(135)(136)(137)199). Thus, local troponin-and myosininduced shifts of semirigid tropomyosin on the actin filament can be expected to be propagated along the tropomyosin cable with limited decrement, producing expected filament cooperative effects.…”

“…Therefore each successive actin subunit along F-actin is matched to a tropomyosin pseudorepeat (138). Here, roughly 30 electrostatic interactions define the weak actin-tropomyosin electrostatic interactions (7,126,137,138,170). Thus, the charged electrostatic track along actin that attracts tropomyosin does not necessarily define a truly specific axial or azimuthal position for tropomyosin on the track.…”

Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

“…Another approach for measuring the bending stiffness of actin filaments is based on the visualization of the filaments subjected to the Brownian bending and data analysis with the WLC theory (9,11,(23)(24)(25)(26)(27). The major parameter in the WLC theory is the persistence length, L p , which is determined by the bending stiffness K and absolute temperature T: L p ¼ K/k B T, where k B is the Boltzmann constant.…”

“…However, these hypotheses were not tested directly. It was shown theoretically (9,10) and experimentally (9,11) that the bending stiffness of a Tpm molecule may be an important parameter in the activation of thin filaments, particularly in the propagation of the mechanical wave of activation along the filament.…”