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Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions

Nature Structural & Molecular Biology volume 18, pages 150–158 (2011)Cite this article

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Abstract

The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain–binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered CH1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.

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Figure 1: Kinetics and thermodynamics for the interaction of BiP with the CH1 domain and the CH1-derived HTFPAVL peptide.
Figure 2: Labeling positions within BiP for spFRET measurements.
Figure 3: Single-pair FRET analysis of BiP 166/518.
Figure 4: Single-pair FRET analysis of BiP 518/636.
Figure 5: BiP and substrate binding by ERdJ3 and its influence on BiP conformation.
Figure 6: Influence of ERdJ3 on substrate binding by BiP.
Figure 7: Model for the ERdJ3-regulated chaperone cycle of BiP.

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Acknowledgements

We thank H. Krause for performing mass spectrometry experiments and V. Kudryavtsev for valuable discussions regarding analysis of the spFRET data. Funding of M.M. and M.J.F. by the Studienstiftung des deutschen Volkes, of M.H. by the International Doctorate Program NanoBioTechnology (IDK-NBT), of D.B. by the International Graduate School of Science and Engineering (IGSSE) and of J.B. by the SFB 749, the Fonds der chemischen Industrie and the Bayerische Forschungsstiftung is gratefully acknowledged. D.C.L. wishes to thank the Deutsche Forschungsgemeinschaft (SFB 749), the Center for Nano Science, the Ludwig-Maximilians-Universität Munich (LMUInnovativ BioImaging Network) and the Nanosystems Initative Munich (NIM) for financial support. The authors thank L. Hendershot for discussions and helpful comments on the manuscript.

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Author notes

  1. Matthias J Feige

    Present address: Present address: Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.,

  2. Moritz Marcinowski and Matthias Höller: These authors contributed equally to this work.

Authors and Affiliations

  1. Department Chemie, Technische Universität München, Garching, Germany

    Moritz Marcinowski, Matthias J Feige, Danae Baerend & Johannes Buchner

  2. Department Chemie and Center for Nano Science, Ludwig-Maximilians-Universität München, München, Germany

    Matthias Höller & Don C Lamb

  3. Center for Integrated Protein Science, Munich, Germany

    Moritz Marcinowski, Matthias Höller, Matthias J Feige, Danae Baerend, Don C Lamb & Johannes Buchner

  4. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Don C Lamb

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Contributions

M.M., M.H., M.J.F., D.C.L. and J.B. designed the study and wrote the paper. M.M., M.J.F. and D.B. performed ensemble experiments and M.H. performed single molecule experiments. M.M., M.H., M.J.F. and D.C.L. analyzed data.

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Correspondence to Don C Lamb or Johannes Buchner.

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Marcinowski, M., Höller, M., Feige, M. et al. Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions. Nat Struct Mol Biol 18, 150–158 (2011). https://doi.org/10.1038/nsmb.1970

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