Engineering of the myosin-Iβ nucleotide-binding pocket to create selective sensitivity to N6-modified ADP analogs

Distinguishing the cellular functions carried out by enzymes of highly similar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large myosin superfamily, we designed a mutation in myosin's nucleotide-binding pocket that permits binding of adenine nucleotides modified with bulky N-6 substituents. Introduction of this mutation, Y61G in rat myosin-I beta, did not alter the enzyme's affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate. We also synthesized several N-6-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules, Several of these N-6-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-I beta; in doing so, these analogs locked Y61G myosin-I beta tightly to actin, N-6-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-I beta. Furthermore, at small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-I beta. Introduction of Y61G myosin-I beta molecules into a cell should permit selective inhibition by N-6-modified ADP analogs of cellular processes dependent on myosin-I beta.