Abstract
Two Drosophila myosin II point mutations (D45 and Mhc5) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc5) could stabilize (D45) or destabilize (Mhc5) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1A261T and S1G200D) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1A261T is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin·S1), and reduced ATP-induced dissociation of actin·S1), whereas S1G200D shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin·S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1A261T (dilated) and S1G200D (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1A261T weakens the affinity of S1·ADP for actin, whereas S1G200D increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up2 mutation in Drosophila.
Highlights
Myosins are a large family of actin-based molecular motor proteins, of which at least 35 different classes are known [1]
D45 is a suppressor of the TnI mutation (A116V) in the indirect flight muscle (IFM), and in Drosophila hearts, it leads to a dilated cardiomyopathy (DCM)-like phenotype in the presence of normal troponin I, whereas Mhc5 leads to RCM in the
We investigated the steady-state and transient kinetics of two transducer mutant myosin subfragment 1 (S1) fragments that 1) yield normal morphology (A261T) or hypercontraction (G200D) in the IFM [2, 8]; 2) can suppress (A261T) or enhance (G200D) the hypercontraction phenotype caused by a troponin I defect (TnI-A116V); and 3) can induce specific phenotypes in the Drosophila heart [7]
Summary
Mhc, which indicates the myosin heavy chain mutation G200D; and D45, which indicates the myosin heavy chain mutation A261T. 5 The abbreviations used are: DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; RCM, restrictive cardiomyopathy; S1, myosin subfragment 1; actin1⁄7S1, actomyosin subfragment 1; cATP, caged ATP; cADP, caged ADP; eda-deac-ATP, 3Ј-O-{N-[2-(7-diethylamino-coumarin-3carboxamido)ethyl]carbamoyl}-ATP; eda-deac-ADP, 3Ј-O-{N-[2-(7-diethylamino-coumarin-3-carboxamido)ethyl]carbamoyl}-ADP; IFI, indirect flight muscle isoform; IFM, indirect flight muscle; Tn, troponin; TnI, troponin I; EMB, embryonic myosin S1. Heart and is lethal in combination with hdp2 This suggests the likelihood of opposite effects of the two myosin mutations in regard to TnI interaction and that such differential activity may lead to the observed alternative cardiomyopathies. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1 myosin as compared with wild type, resulting in a less active myosin (S1A261T) or more active myosin (S1G200D). These observations are consistent with the induced cardiac. We report that the two mutations change the affinity of S11⁄7ADP for actin in opposite directions and that these changes can account for the differential interaction of the two mutations with the TnI held-up mutation in Drosophila
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