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

Peter G. Gillespie, Susan K.H. Gillespie, John A. Mercer, Kavita Shah, Kevan M. Shokat

Research output: Contribution to journalArticlepeer-review

58 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)31373-31381
Number of pages9
JournalJournal of Biological Chemistry
Issue number44
StatePublished - Oct 29 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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