Engineering of the myosin-ibeta nucleotide-binding pocket to create selective sensitivity to N(6)-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-Ibeta, 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-Ibeta; in doing so, these analogs locked Y61G myosin-Ibeta tightly to actin. N(6)-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Ibeta. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Ibeta. Introduction of Y61G myosin-Ibeta molecules into a cell should permit selective inhibition by N(6)-modified ADP analogs of cellular processes dependent on myosin-Ibeta.
Gillespie
P G
PG
Department of Physiology, The Johns Hopkins University, Baltimore, Maryland 21205, USA. gillespp@ohsu.edu
Gillespie
S K
SK
Mercer
J A
JA
Shah
K
K
Shokat
K M
KM
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004023:Dictyostelium; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009068:Movement; D009154:Mutation; D020125:Mutation, Missense; D009218:Myosins / Q000037:antagonists & inhibitors* / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D051381:Rats
D000199:Actins; D007527:Isoenzymes; C400525:N(6)-(2-methylbutyl)adenosine diphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins