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 N⁶ substituents. Introduction of this mutation, Y61G in rat myosin- Iβ, 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 N⁶-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules. Several of these N⁶-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Iβ; in doing so, these analogs locked Y61G myosin-Iβ tightly to actin. N⁶-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Iβ. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Iβ. Introduction of Y61G myosin-Iβ molecules into a cell should permit selective inhibition by N⁶-modified ADP analogs of cellular processes dependent on myosin-Iβ.