Letter | Published:

SbsB structure and lattice reconstruction unveil Ca2+ triggered S-layer assembly

Nature volume 487, pages 119122 (05 July 2012) | Download Citation

Abstract

S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria1,2,3. The nanoscale repeat structure of the S-layer lattices and their self-assembly from S-layer proteins (SLPs) have sparked interest in their use as patterning and display scaffolds for a range of nano-biotechnological applications4,5,6,7. Despite their biological abundance and the technological interest in them, structural information about SLPs is limited to truncated and assembly-negative proteins8,9,10. Here we report the X-ray structure of the SbsB SLP of Geobacillus stearothermophilus PV72/p2 by the use of nanobody-aided crystallization. SbsB consists of a seven-domain protein, formed by an amino-terminal cell-wall attachment domain and six consecutive immunoglobulin-like domains, that organize into a ϕ-shaped disk-like monomeric crystallization unit stabilized by interdomain Ca2+ ion coordination. A Ca2+-dependent switch to the condensed SbsB quaternary structure pre-positions intermolecular contact zones and renders the protein competent for S-layer assembly. On the basis of crystal packing, chemical crosslinking data and cryo-electron microscopy projections, we present a model for the molecular organization of this SLP into a porous protein sheet inside the S-layer. The SbsB lattice represents a previously undescribed structural model for protein assemblies and may advance our understanding of SLP physiology and self-assembly, as well as the rational design of engineered higher-order structures for biotechnology4,5,6,7.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for SbsB32–920:NbKB6 are deposited in the Protein Data Bank under accession code 4AQ1.

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Acknowledgements

We thank staff at beamlines Proxima-1 and SWING (Soleil), beamline BM30A (European Synchrotron Radiation Facility) and beamline X33 (Deutsches Elektronen-Synchrotron) for technical assistance during data collection. We thank G. Waksman for suggestions on the manuscript, and D. Levy for advice on cryo-EM grid preparation and data collection. This work was supported by VIB (Vlaams Institute voor Biotechnologie) grant PRJ9 to H.R., the Fonds Wetenschappelijk Onderzoek-Vlaanderen through an Odysseus grant to H.R., a postdoctoral fellowship to A.P.G. and research grant FWO551 to J.S. R.F. is supported by the Centre National de la Recherche Scientifique (CNRS) and the Institut Pasteur. G.P.A. is supported by the CNRS. E.P. received support from Interuniversity Attraction Poles grant P6/19. D.P. was funded by Biotechnology and Biological Sciences Research Council grant BB/E010466/1, and S.H. is grateful for support from University College London. We acknowledge Professor Emeritus U. Sleytr for his lifelong devotion to S-layer protein research and G. stearothermophilus SbsB in particular.

Author information

Affiliations

  1. Structural and Molecular Microbiology, VIB Department of Structural Biology, VIB, Pleinlaan 2, 1050 Brussels, Belgium

    • Ekaterina Baranova
    • , Nani Van Gerven
    •  & Han Remaut
  2. Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

    • Ekaterina Baranova
    • , Abel Garcia-Pino
    • , Nani Van Gerven
    • , Els Pardon
    • , Jan Steyaert
    •  & Han Remaut
  3. Unité G5, Biologie structurale de la sécrétion bactérienne, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Rémi Fronzes
  4. Unité de recherche mixte 3538, Centre national de la recherche scientifique, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Rémi Fronzes
    •  & Gérard Péhau-Arnaudet
  5. Department of Chemistry, Institute of Structural and Molecular Biology, University College London, London WC1H 0AJ, UK

    • David Papapostolou
    •  & Stefan Howorka

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Contributions

K.B. produced and crystallized SbsB:NbKB6 complexes, collected and analysed diffraction and SAXS data and determined their structures. A.P.G. analysed SAXS data and collected SAXS, isothermal titration calorimetry and circular dichroism data. N.V.G. performed mutagenesis and crosslinking experiments. R.F. grew S-layers in vitro and analysed cryo-EM data. G.P.A. prepared cryo-EM grids and collected cryo-EM data. E.P. and J.S. produced SbsB-binding nanobodies. D.P. performed crosslinking experiments. S.H. produced SbsB constructs, supervised D.P. and wrote the manuscript. H.R. supervised the work, collected diffraction data, solved and analysed the structures and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Han Remaut.

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DOI

https://doi.org/10.1038/nature11155

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