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β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

  1. Adam Langea,d,g,3
  1. aDepartment of NMR-Based Structural Biology and
  2. eElectron Microscopy Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
  3. bProkaryotic Cell Biology Group, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany;
  4. cFaculty of Biology and
  5. fLOEWE Center for Synthetic Microbiology, Philipps-Universität, 35043 Marburg, Germany;
  6. dDepartment of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; and
  7. gInstitut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany

  1. Edited by Robert Tycko, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board December 9, 2014 (received for review September 24, 2014)

Significance

Bactofilins are a new class of cytoskeletal proteins that are involved in key cellular processes. For instance, in the human pathogen Helicobacter pylori, they are responsible for maintaining its characteristic helical cell shape, a feature required for cells to efficiently colonize the gastric mucus. So far the atomic structure of bactofilin filaments has remained elusive, as the large bactofilin assemblies are not amenable to standard methods for 3D structure determination. Here, we have applied a combination of solid-state NMR and electron microscopy and discovered that bactofilins adopt a β-helical architecture, which has not been observed before for other cytoskeletal filaments. Interestingly, however, the structure bears similarities to that of the fungal prion protein HET-s.

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.

Footnotes

  • ↵1S.V., L.L., and C.S. contributed equally to this work.

  • ↵2Present address: Université de Bordeaux, Chimie et Biologie des Membranes et des Nano-objets (CBMN) (UMR 548), Institut Européen de Chimie et de Biologie, 33607 Pessac, France.

  • ↵3To whom correspondence may be addressed. Email: alange{at}fmp-berlin.de or thanbichler{at}uni-marburg.de.

  • Author contributions: M.T. and A.L. designed research; S.V., L.L., C.S., and D.R. performed research; S.V., L.L., C.S., B.H., D.R., J.K., M.T., and A.L. analyzed data; and S.V., L.L., C.S., M.T., and A.L. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission. R.T. is a guest editor invited by the Editorial Board.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1418450112/-/DCSupplemental.

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