Polymeric structures and dynamic properties of the bacterial actin AlfA.
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 A, 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 A. 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.
Popp
David
D
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. dpopp@imcb.a-star.edu.sg
Narita
Akihiro
A
Ghoshdastider
Umesh
U
Maeda
Kayo
K
Maéda
Yuichiro
Y
Oda
Toshiro
T
Fujisawa
Tetsuro
T
Onishi
Hirufumi
H
Ito
Kazuki
K
Robinson
Robert C
RC
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D005583:Fourier Analysis; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D055503:Protein Multimerization; D055550:Protein Stability; D053838:Scattering, Small Angle; D012965:Sodium Chloride
D000199:Actins; D001426:Bacterial Proteins; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate