Abstract
The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001.
Highlights
Mammalian cells typically produce in excess of 10,000 different proteins displaying enzymatic activities
We provide evidence indicating that EMD 57033 binds to an allosteric pocket in the myosin motor domain near the base of the lever arm
Our results show that EMD 57033 is a member of a new class of pharmacological chaperones that stabilize, enhance the activity, and correct stress-induced misfolding of their target protein
Summary
Mammalian cells typically produce in excess of 10,000 different proteins displaying enzymatic activities. To obtain more detailed information about the kinetics of the EMD 57033-mediated refolding reaction, we performed three sets of experiments recording the time and concentration dependence of EMD 57033-mediated increases in the fraction of myosin molecules that are able to undergo nucleotide induced conformational changes, hydrolyze ATP, and produce movement in the in vitro motility assay. The kobs values derived from the hyperbolae determined for EMD 57033-mediated changes in ATPase activity and motility (Figure 4F,G) and from changes in intrinsic protein fluorescence (data not shown) indicate a linear dependence between the progress of the refolding reaction and the concentration of EMD 57033. Hyperthermic stress-induced ANP expression in controls, while the presence of 10 μM EMD 57033 fully suppressed the expression of ANP during hyperthermic stress (Figure 5G)
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