A Failure to Communicate: Myosin Residues Involved in Hypertrophic Cardiomyopathy Affect Inter-Domain Interaction [Molecular Bases of Disease]

October 7th, 2015 by Kronert, W. A., Melkani, G. C., Melkani, A., Bernstein, S. I.

Our molecular modeling studies suggest a charge-dependent interaction between residues E497 in the relay domain and R712 in the converter domain of human β-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (~80%) as well as in basal (~67%) and actin-activated (~84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ~73% of wild type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. While sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Further, the same inter-domain interaction is disrupted when modeling human β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease.
  • Posted in Journal of Biological Chemistry, Publications
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Mapping Interactions Between Myosin Relay and Converter Domains that Power Muscle Function [Protein Structure and Folding]

March 13th, 2014 by Kronert, W. A., Melkani, G. C., Melkani, A., Bernstein, S. I.

Intra-molecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (I508, N509 and D511) in communicating with converter domain residue R759. We find that the N509K relay mutation suppresses defects in myosin ATPase, in vitro motility, myofibril stability and muscle function associated with the R759E converter mutation. Through molecular modeling we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disables motor function and myofibril assembly, suggesting productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.