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

Mark J Heller,Mahta Nili,Earl Homsher,Larry S Tobacman

doi:10.1074/jbc.m303408200

Mark J Heller, Mahta Nili + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m303408200

Copy DOI

Abstract

The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of alpha-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 degrees C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 degrees C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0-1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca2+ binding sites of thin filament in two settings: Ca2+-dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.

Highlights

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
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
A new approach to understanding tropomyosin has been provided by the discovery that tropomyosin missense mutations can cause the autosomal dominant disorder familial hypertrophic cardiomyopathy (HCM)

Summary

The abbreviations used are

Troponin; HCM, hypertrophic cardiomyopathy; MOPS, 4-morpholinepropanesulfonic acid. In examining cardiomyopathic mutations A63V and K70T [26, 27], located instead in the N-terminal domain of tropomyosin, the current report combines with the earlier evidence to imply that a characteristic result of the cardiomyopathic tropomyosin mutations is to decrease thermal stability, which presumably reports an increased tropomyosin flexibility. Despite these findings, the cooperativity of thin filament activation, either by myosin or by Ca2ϩ, was not altered by the mutations. The data suggest that the flexibility of the tropomyosin N-domain influences Ca2ϩ sensitivity, This paper is available on line at http://www.jbc.org but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION