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Newly folded substrates inside the molecular cage of the HtrA chaperone DegQ

Nature Structural & Molecular Biology volume 19, pages 152–157 (2012)Cite this article

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Abstract

The HtrA protein family combines chaperone and protease activities and is essential for protein quality control in many organisms. Whereas the mechanisms underlying the proteolytic function of HtrA proteins are well characterized, their chaperone activity remains poorly understood. Here we describe cryo-EM structures of Escherichia coli DegQ in its 12- and 24-mer states in complex with model substrates, providing a structural model of HtrA chaperone action. Up to six lysozyme substrates bind inside the DegQ 12-mer cage and are visualized in a close-to-native state. An asymmetric reconstruction reveals the binding of a well-ordered lysozyme to four DegQ protomers. DegQ PDZ domains are located adjacent to substrate density and their presence is required for chaperone activity. The substrate-interacting regions appear conserved in 12- and 24-mer cages, suggesting a common mechanism of chaperone function.

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Figure 1: The DegQ 24-mer cryo-EM map reveals a potential β-casein binding site.
Figure 2: DegQ 12-mer complexes with a peptide and lysozyme.
Figure 3: Five or six folded lysozymes are bound to the DegQ 12-mer.
Figure 4: Lysozyme–DegQ interaction.
Figure 5: Chaperone activity of DegQ.

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Acknowledgements

We thank D. Clare, N. Lukoyanova and E. Orlova for advice on EM data collection and processing; L. Wang, D. Houldershaw and R. Westlake for computing and EM support; and T. Daviter for help with fluorescence spectroscopy. This work was supported by Wellcome Trust (079605 and 089050) and European Science Foundation (BB/F010281/1) grants to H.R.S.; by ERA-Net NEURON, FWF I 235-B09, to F.C. and T.C. and by Institute of Structural and Molecular Biology Wellcome Trust studentships to J.Y. and K.T. The Research Institute of Molecular Pathology is funded by Boehringer Ingelheim.

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  1. Hélène Malet & Justyna Sawa

    Present address: Present addresses: Unit of Virus Host-Cell Interactions, Joint International Unit 3265, Grenoble Cedex 9, France (H.M.); Max F. Perutz Laboratories, University of Vienna, Vienna, Austria (J.S).,

Authors and Affiliations

  1. Institute of Structural and Molecular Biology, Crystallography, Birkbeck College, London, UK

    Hélène Malet & Helen R Saibil

  2. Research Institute of Molecular Pathology, Vienna, Austria

    Flavia Canellas, Justyna Sawa & Tim Clausen

  3. Division of Biosciences, Institute of Structural and Molecular Biology, University College London, London, UK

    Jun Yan & Konstantinos Thalassinos

  4. Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany

    Michael Ehrmann

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Contributions

EM data collection and processing, fitting of atomic coordinates into EM maps and tryptophan fluorescence were carried out by H.M. under the supervision of H.R.S. Protein purification and complex formation were done by F.C., J.S. and H.M. MS experiments were conducted by J.Y. under the supervision of K.T. The refolding assays were carried out by J.S. and F.C. under the supervision of T.C. and M.E. H.R.S. and T.C. supervised the project. H.M., H.R.S. and T.C. wrote the manuscript.

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Correspondence to Helen R Saibil.

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Supplementary Figures 1–7, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 6872 kb)

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Malet, H., Canellas, F., Sawa, J. et al. Newly folded substrates inside the molecular cage of the HtrA chaperone DegQ. Nat Struct Mol Biol 19, 152–157 (2012). https://doi.org/10.1038/nsmb.2210

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