Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA |
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Authors: | David Popp Akihiro Narita Umesh Ghoshdastider Yuichiro Maéda Toshiro Oda Tetsuro Fujisawa Kazuki Ito |
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Institution: | 1 ERATO “Actin Filament Dynamics” Project, Japan Science and Technology Corporation, c/o RIKEN Harima Institute at Spring 8, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan 2 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos 138673, Singapore 3 Structural Biology Research Center and Division of Biological Sciences, Nagoya University Graduate School of Science, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan 4 RIKEN Harima Institute at Spring 8, Kouto, Sayo, Hyogo 679-5148, Japan 5 Department of Biomolecular Science, Graduate School of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan |
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Abstract: | AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 Å, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 Å. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5′-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes. |
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Keywords: | EM electron microscopy TIRF total internal reflection fluorescence 3-D three-dimensional GTP guanosine 5&prime -triphosphate 2-D two-dimensional EDTA ethylenediaminetetraacetic acid Pi inorganic phosphate HP-SAXS high-pressure small angle X-ray scattering |
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