Engineering of the myosin-I beta nucleotide-binding pocket to create selective sensitivity to N-6-modified ADP analogs

Distinguishing the cellular functions carried out by enzymes of highly simi lar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large my osin superfamily, we designed a mutation in myosin's nucleotide-binding poc ket that permits binding of adenine nucleotides modified with bulky N-6 sub stituents. Introduction of this mutation, Y61G in rat myosin-I beta, did no t alter the enzyme's affinity for ATP or actin and actually increased its A TPase 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 mor e than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-t ype myosin-I beta; in doing so, these analogs locked Y61G myosin-I beta tig htly to actin, N-6-(2-methylbutyl) ADP abolished actin filament motility me diated by Y61G, but not wild-type, myosin-I beta. Furthermore, at small fra ction 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 be ta.