Cardiomyopathic Tropomyosin Mutations That Increase Thin Filament Ca2+ Sensitivity and Tropomyosin N-domain Flexibility

Abstract

The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of ␣-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10°C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10°C, respectively, indicating destabilization of the Ndomain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and Cdomain fold with a cooperative free energy of 1.0 -1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca 2؉ binding sites of thin filament in two settings: Ca 2؉ -dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca 2؉ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca 2؉ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca 2؉ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca 2؉ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.Contraction of cardiac and skeletal muscle is controlled by the reversible binding of calcium to the N-domain of TnC, 1 which is the regulatory subunit of troponin (for reviews, see Refs. 1 and 2). Troponin and tropomyosin bestow Ca 2ϩ dependence on the productive interactions of actin and myosin: rapid ATPase activity, generation of force, and generation of movement. Three-dimensional, helical reconstructions of thin filament electron micrographs imply three positions of tropomyosin on the actin surface (3). These data, supported by x-ray diffraction of thin filaments (4), indicate that a major component of regulation consists of tropomyosin sterically interfering with myosin binding to actin. In the absence of Ca 2ϩ , much of the myosin-binding site on actin is obscured by tropomyosin. Calcium binding to troponin causes tropomyosin to shift position, exposing much of the myosin-binding site. Strong actinmyosin binding requires a further repositioning of tropomyosin. These findings do not imply that steric interference fully explains regulation. For example, addition of troponin and tropomyosin to bare actin filaments increases actin-myosin affinity, force production, and sliding speed in the presence of Ca 2ϩ (5-7). Nevertheless, as was first proposed 30 years ago (8), most recent reports (albeit not all (e.g. Refs. 9 and 10)) point to the shifting position of tropomyosin ...

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