title: Reconstitution of the Escherichia coli cell division ZipA-FtsZ complexes in nanodiscs as revealed by electron microscopy.
abstract: ZipA is an element of the bacterial division ring complex that provides an anchor to the membrane to FtsZ, a GTPase ancestor of tubulin. In vitro reconstitution and characterization of these interactions is challenged by the difficulty to integrate a physiological membrane environment. Here a single copy of the full-length ZipA protein from Escherichia coli incorporated into phospholipid bilayer nanodiscs (Nd-ZipA) has been visualized using negative-staining electron microscopy (EM). The EM images reveal the presence of discs, mostly organized in two distinct populations of 11 and 13nm in diameter. The globular FtsZ-binding C-terminal domain of ZipA (ZBD) was not visible in 3D reconstructions of Nd-ZipA or 2D averages, suggesting that this domain is separated from the membrane by the large flexible domain connecting the N-terminal trans-membrane region to the ZBD. We tested if Nd-ZipA were appropriate models for the in vitro reconstitution of ZipA-FtsZ interactions. First we observed that the ZBD region of ZipA was accessible for the interaction with other proteins in the context of the nanodisc, as revealed by its recognition by specific antibodies. In addition, Nd-ZipA attached to carbon coated EM grids, but not empty nanodiscs, were able to capture FtsZ filaments without inducing significant filament bundling, consistent with a model in which FtsZ filaments are loosely attached to the cell-membrane. These observations are compatible with the plastic nature of the ZipA-FtsZ complexes formed at the membrane, evidenced in the moderate binding affinity of Nd-ZipA to FtsZ oligomers and polymers recently measured.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas-CSIC, Ramiro de Maetzu 9, 28040 Madrid, Spain.
authors.forename: Víctor M
authors.initials: VM
authors.lastname: Hernández-Rocamora
authors.forename: Concepción
authors.initials: C
authors.lastname: García-Montañés
authors.forename: Germán
authors.initials: G
authors.lastname: Rivas
authors.forename: Oscar
authors.initials: O
authors.lastname: Llorca
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002455:Cell Division; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D004926:Escherichia coli / Q000737:chemistry* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional; D007106:Immune Sera / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008854:Microscopy, Electron; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C071525:FtsZ protein, Bacteria; D007106:Immune Sera; D008051:Lipid Bilayers; D011994:Recombinant Proteins; C103795:ZipA protein, E coli