title: β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR.
abstract: Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.
authors.affiliation: Department of NMR-Based Structural Biology and.
authors.forename: Suresh
authors.initials: S
authors.lastname: Vasa
authors.affiliation: Prokaryotic Cell Biology Group, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany; Faculty of Biology and.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of NMR-Based Structural Biology and Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; and.
authors.forename: Chaowei
authors.initials: C
authors.lastname: Shi
authors.affiliation: Department of NMR-Based Structural Biology and.
authors.forename: Birgit
authors.initials: B
authors.lastname: Habenstein
authors.affiliation: Electron Microscopy Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
authors.forename: Dietmar
authors.initials: D
authors.lastname: Riedel
authors.affiliation: Prokaryotic Cell Biology Group, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany; Faculty of Biology and.
authors.forename: Juliane
authors.initials: J
authors.lastname: Kühn
authors.affiliation: Prokaryotic Cell Biology Group, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany; Faculty of Biology and LOEWE Center for Synthetic Microbiology, Philipps-Universität, 35043 Marburg, Germany; alange@fmp-berlin.de thanbichler@uni-marburg.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Thanbichler
authors.affiliation: Department of NMR-Based Structural Biology and Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; and Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany alange@fmp-berlin.de thanbichler@uni-marburg.de.
authors.forename: Adam
authors.initials: A
authors.lastname: Lange
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D016935:Caulobacter crescentus / Q000737:chemistry* / Q000235:genetics; D017124:Conserved Sequence; D003599:Cytoskeleton / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D055032:Electron Microscope Tomography; D057927:Hydrophobic and Hydrophilic Interactions; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins
keywords: cytoskeleton; filaments; protein structure; solid-state NMR