Abstract
We have identified pentabromopseudilin (PBP) as a potent inhibitor of myosin-dependent processes such as isometric tension development and unloaded shortening velocity. PBP-induced reductions in the rate constants for ATP binding, ATP hydrolysis and ADP dissociation extend the time required per myosin ATPase cycle in the absence and presence of actin. Additionally, coupling between the actin and nucleotide binding sites is reduced in the presence of the inhibitor. The selectivity of PBP differs from that observed with other myosin inhibitors. To elucidate the binding mode of PBP, we crystallized the Dictyostelium myosin-2 motor domain in the presence of Mg2+-ADP–meta-vanadate and PBP. The electron density for PBP is unambiguous and shows PBP to bind at a previously unknown allosteric site near the tip of the 50-kDa domain, at a distance of 16 Å from the nucleotide binding site and 7.5 Å away from the blebbistatin binding pocket.
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Acknowledgements
We thank S. Fujita-Becker (Max-Planck-Institute for Medical Research, Heidelberg), B. Westermann (University of Bayreuth), T. Scholz, A. Schweda (Hannover Medical School) A. Malnasi-Csizmadia and M. Kovacs (Eötvös Lorand University, Budapest), and C.R. Bagshaw (University of Leicester) for reagents; M.A. Geeves for discussions; C. Thiel, C. Wassmann, Y. Henker and L. Litz for excellent technical assistance. We are grateful to the staff of beamline EMBL X11 at the DORIS storage ring of DESY for their support.
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R.F. performed crystallization, structure determination and molecular modeling; M.B. performed the initial kinetic screening of the pseudilin derivatives and the yeast assays; G.T. supervised and codesigned the protein preparation, kinetic and in vitro motility assays, and performed the transient kinetic analysis with F.K.H. and M.H.T.; F.K.H. performed in vitro motility assays and steady-state analysis; M.H.T. performed transient kinetic experiments; P.B. crystallized the protein inhibitor complexes; B.B. designed and performed single-fiber experiments; R.M. synthesized PBP; H.-J.K. designed and supervised compound synthesis; H.O.G. designed and supervised the initial kinetic screening of PBP and the yeast assays; D.J.M. supervised kinetic analysis, crystallization and data collection, molecular modeling, in vitro motility analysis, advised on the design of the experiments, and wrote the manuscript with G.T., R.F., H.O.G., B.B. and H.-J.K.
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Fedorov, R., Böhl, M., Tsiavaliaris, G. et al. The mechanism of pentabromopseudilin inhibition of myosin motor activity. Nat Struct Mol Biol 16, 80–88 (2009). https://doi.org/10.1038/nsmb.1542
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DOI: https://doi.org/10.1038/nsmb.1542
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