Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA

• Published in: Journal of molecular biology 2010-04, Vol.397 (4), p.1031-1041
• Main Authors: Popp, David, Narita, Akihiro, Ghoshdastider, Umesh, Maeda, Kayo, Maéda, Yuichiro, Oda, Toshiro, Fujisawa, Tetsuro, Onishi, Hirufumi, Ito, Kazuki, Robinson, Robert C.
• Format: Article
• Language: English
• Subjects: Actin; Actin Cytoskeleton - metabolism; actin homolog; Actins - chemistry; Actins - metabolism; Actins - ultrastructure; Adenosine Triphosphate - metabolism; AlfA; ATP; Bacillus subtilis; Bacillus subtilis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacterial Proteins - ultrastructure; Cytoplasm; DNA; electron microscopy; Elongation; Filaments; Fluorescence; Fourier Analysis; Guanosine; Guanosine Triphosphate - metabolism; helical symmetry; Hydrogen-Ion Concentration; Kinetics; Microscopy, Electron, Transmission; Models, Molecular; Monomers; Nets; pH effects; Phosphate; Polymerization; Protein Multimerization; Protein Stability; Salts; Scattering, Small Angle; Sodium Chloride; treadmilling; X-ray scattering
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2010.02.010

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.