title: Protofilament formation of ParM mutants.
abstract: ParM, an actin homolog, forms left-handed two-start helical filaments that segregate DNA in bacteria prior to cell division. Our recent atomic model obtained from electron microscopy (EM) reconstructions of negatively stained ParM filaments implied that two salt bridges (Glu35-Lys258 and Asp63-Arg262) may be key inter-filament contacts that stabilize the left-handed ParM helix. We made mutations of these amino acids and probed the inter-strand interface of our model experimentally by EM and X-ray fiber diffraction. We found that several mutations, such as ParM single mutants Asp258 and Asp262 and double mutant Asp258/Arg262, were incapable of forming straight filaments in aqueous buffers and appeared ragged and unstructured. However, in the presence of crowding agents, straight filaments or filament bundles formed, which allowed us to elucidate the structure of these mutant filaments. Centrifugation of filaments also resulted in a pellet of straightened filaments that could be oriented in glass capillaries and gave detailed X-ray diffraction patterns. Both EM and X-ray diffraction showed that filaments formed from these ParM mutants were not double-stranded helical filaments but single protofilaments, indicating that these residues are important for formation of the ParM helix. Our data also confirm a major prediction of crowding theory, namely that molecular crowding shifts the equilibrium of even severely impaired, unstructured cytoskeletal polymers toward their structured native and functional state. ParM is the first example of a helical actin homolog that can be induced to form protofilaments.
authors.affiliation: 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@spring8.or.jp
authors.forename: David
authors.initials: D
authors.lastname: Popp
authors.forename: Mitsusada
authors.initials: M
authors.lastname: Iwasa
authors.forename: Kayo
authors.initials: K
authors.lastname: Maeda
authors.forename: Akihiro
authors.initials: A
authors.lastname: Narita
authors.forename: Toshiro
authors.initials: T
authors.lastname: Oda
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Maéda
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000235:genetics; D001704:Biopolymers / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008854:Microscopy, Electron; D050505:Mutant Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D014961:X-Ray Diffraction
chemical_list: D000199:Actins; D001704:Biopolymers; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; C471255:ParM protein, E coli