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Targeted substrate loop insertion by VCP/p97 during PP1 complex disassembly

Nature Structural & Molecular Biology volume 28, pages 964–971 (2021)Cite this article

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Abstract

The AAA-ATPase VCP/p97/Cdc48 unfolds proteins by threading them through its central pore, but how substrates are recognized and inserted into the pore in diverse pathways has remained controversial. Here, we show that p97, with its adapter p37, binds an internal recognition site (IRS) within inhibitor-3 (I3) and then threads a peptide loop into its channel to strip I3 off protein phosphatase-1 (PP1). Of note, the IRS is adjacent to the prime interaction site of I3 to PP1, and IRS mutations block I3 processing both in vitro and in cells. In contrast, amino- and carboxy-terminal regions of I3 are not required, and even circularization of I3 does not prevent I3 processing. This was confirmed by an in vitro Förster resonance energy transfer assay that allowed kinetic analysis of the reaction. Thus, our data uncover how PP1 is released from its inhibitory partner for activation and demonstrate a remarkable plasticity in substrate threading by p97.

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Fig. 1: Identification of an internal recognition site in I3 for the p97–p37 ATPase complex.
Fig. 2: The IRS is required for processing of the SDS22–PP1–I3 complex and I3 unfolding.
Fig. 3: Free termini are not required for substrate unfolding.
Fig. 4: A FRET-based assay demonstrates PP1 complex disassembly driven by loop insertion.
Fig. 5: Model for I3 processing by p97 during PP1 complex disassembly.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/archive/) with the dataset identifiers PXD026696 and PXD026692. Source data are provided with this paper.

References

  1. Brautigan, D. L. Protein Ser/Thr phosphatases—the ugly ducklings of cell signalling. FEBS J. 280, 324–345 (2013).

    Article  CAS  Google Scholar 

  2. Heroes, E. et al. The PP1 binding code: a molecular-lego strategy that governs specificity. FEBS J. 280, 584–595 (2013).

    Article  CAS  Google Scholar 

  3. Cao, X., Lemaire, S. & Bollen, M. Protein phosphatase-1: life-course regulation by SDS22 and Inhibitor-3. FEBS J. https://doi.org/10.1111/febs.16029 (2021).

  4. Choy, M. S. et al. SDS22 selectively recognizes and traps metal-deficient inactive PP1. Proc. Natl Acad. Sci. USA 116, 20472–20481 (2019).

    Article  CAS  Google Scholar 

  5. Zhang, L., Qi, Z., Gao, Y. & Lee, E. Y. C. Identification of the interaction sites of Inhibitor-3 for protein phosphatase-1. Biochem. Biophys. Res. Commun. 377, 710–713 (2008).

    Article  CAS  Google Scholar 

  6. Ye, Y., Tang, W. K., Zhang, T. & Xia, D. A mighty "protein extractor" of the cell: structure and function of the p97/CDC48 ATPase. Front Mol. Biosci. 4, 39 (2017).

    Article  Google Scholar 

  7. van den Boom, J. & Meyer, H. VCP/p97-mediated unfolding as a principle in protein homeostasis and signaling. Mol. Cell 69, 182–194 (2018).

    Article  Google Scholar 

  8. Weith, M. et al. Ubiquitin-independent disassembly by a p97 AAA–ATPase complex drives PP1 holoenzyme formation. Mol. Cell 72, 766–777 e6 (2018).

    Article  CAS  Google Scholar 

  9. Kracht, M. et al. Protein phosphatase-1 complex disassembly by p97 is initiated through multivalent recognition of catalytic and regulatory subunits by the p97 SEP-domain adapters. J. Mol. Biol. 432, 6061–6074 (2020).

    Article  CAS  Google Scholar 

  10. Stolz, A., Hilt, W., Buchberger, A. & Wolf, D. H. Cdc48: a power machine in protein degradation. Trends Biochem. Sci. 36, 515–523 (2011).

    Article  CAS  Google Scholar 

  11. Olszewski, M. M., Williams, C., Dong, K. C. & Martin, A. The Cdc48 unfoldase prepares well-folded protein substrates for degradation by the 26S proteasome. Commun. Biol. 2, 29 (2019).

    Article  Google Scholar 

  12. Bodnar, N. O. & Rapoport, T. A. Molecular mechanism of substrate processing by the Cdc48 ATPase complex. Cell 169, 722–735 (2017).

    Article  CAS  Google Scholar 

  13. Blythe, E. E., Olson, K. C., Chau, V. & Deshaies, R. J. Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP*NPLOC4*UFD1L is enhanced by a mutation that causes multisystem proteinopathy. Proc. Natl Acad. Sci. USA 114, E4380–E4388 (2017).

    Article  CAS  Google Scholar 

  14. Meyer, H. H., Wang, Y. & Warren, G. Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4. EMBO J. 21, 5645–5652 (2002).

    Article  CAS  Google Scholar 

  15. Twomey, E. C. et al. Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding. Science 365, eaax1033 (2019).

    Article  CAS  Google Scholar 

  16. Cooney, I. et al. Structure of the Cdc48 segregase in the act of unfolding an authentic substrate. Science 365, 502–505 (2019).

    Article  CAS  Google Scholar 

  17. Pan, M. et al. Mechanistic insight into substrate processing and allosteric inhibition of human p97. Nat. Struct. Mol. Biol. 28, 614–625 (2021).

    Article  CAS  Google Scholar 

  18. Prakash, S., Tian, L., Ratliff, K. S., Lehotzky, R. E. & Matouschek, A. An unstructured initiation site is required for efficient proteasome-mediated degradation. Nat. Struct. Mol. Biol. 11, 830–837 (2004).

    Article  CAS  Google Scholar 

  19. Antos, J. M. et al. A straight path to circular proteins. J. Biol. Chem. 284, 16028–16036 (2009).

    Article  CAS  Google Scholar 

  20. Weber-Ban, E. U., Reid, B. G., Miranker, A. D. & Horwich, A. L. Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401, 90–93 (1999).

    Article  CAS  Google Scholar 

  21. Choi, B., Rempala, G. A. & Kim, J. K. Beyond the Michaelis–Menten equation: accurate and efficient estimation of enzyme kinetic parameters. Sci. Rep. 7, 17018 (2017).

    Article  Google Scholar 

  22. Avellaneda, M. J. et al. Processive extrusion of polypeptide loops by a Hsp100 disaggregase. Nature 578, 317–320 (2020).

    Article  CAS  Google Scholar 

  23. Kraut, D. A. & Matouschek, A. Proteasomal degradation from internal sites favors partial proteolysis via remote domain stabilization. ACS Chem. Biol. 6, 1087–1095 (2011).

    Article  CAS  Google Scholar 

  24. Burton, R. E., Siddiqui, S. M., Kim, Y. I., Baker, T. A. & Sauer, R. T. Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine. EMBO J. 20, 3092–3100 (2001).

    Article  CAS  Google Scholar 

  25. Han, H. et al. Structure of Vps4 with circular peptides and implications for translocation of two polypeptide chains by AAA+ ATPases. eLife 8, e44071 (2019).

    Article  CAS  Google Scholar 

  26. Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl Acad. Sci. USA 99, 11020–11024 (2002).

    Article  CAS  Google Scholar 

  27. Selo, I., Negroni, L., Creminon, C., Grassi, J. & Wal, J. M. Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays. J. Immunol. Methods 199, 127–138 (1996).

    Article  CAS  Google Scholar 

  28. Pan, D., Brockmeyer, A., Mueller, F., Musacchio, A. & Bange, T. Simplified protocol for cross-linking mass spectrometry using the MS-cleavable cross-linker DSBU with efficient cross-link identification. Anal. Chem. 90, 10990–10999 (2018).

    Article  CAS  Google Scholar 

  29. Gotze, M. et al. Automated assignment of MS/MS cleavable cross-links in protein 3D-structure analysis. J. Am. Soc. Mass. Spectrom. 26, 83–97 (2015).

    Article  Google Scholar 

  30. Kress, E. et al. The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A. J. Cell Biol. 201, 559–575 (2013).

    Article  CAS  Google Scholar 

  31. Eiteneuer, A. et al. Inhibitor-3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore-bound protein phosphatase-1. EMBO J. 33, 2704–2720 (2014).

    Article  CAS  Google Scholar 

  32. Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).

    Article  CAS  Google Scholar 

  33. Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G. J. Jalview Version 2 – a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  Google Scholar 

  34. Loetscher, P., Pratt, G. & Rechsteiner, M. The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. Support for the pest hypothesis. J. Biol. Chem. 266, 11213–11220 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The work was supported by Deutsche Forschungsgemeinschaft (DFG) grant Me1626/3-2 to H. Meyer, by SFB1093 projects B2, A2 and B5 to H. Meyer, M.K. and A.M., respectively, a GRK1739 project to H. Meyer and by INST 20876/322-1 FUGG to M.K.

Author information

Author notes

  1. Dongqing Pan

    Present address: Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan

  2. These authors contributed equally: Johannes van den Boom, Anja F. Kueck.

Authors and Affiliations

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

    Johannes van den Boom, Anja F. Kueck, Bojana Kravic, Helen Müschenborn, Maike Giesing, Andrea Musacchio & Hemmo Meyer

  2. Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Dongqing Pan & Andrea Musacchio

  3. Analytics Core Facility, Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany

    Farnusch Kaschani & Markus Kaiser

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  1. Johannes van den Boom
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Contributions

J.v.d.B. and A.F.K. performed and analyzed all in vitro assays and protein purifications. B.K., H. Müschenborn and M.G. helped with cells and performed proliferation assays. D.P. and A.M. performed XL-MS and analyzed XL-MS data. F.K. and M.K. did the MS analysis on photo-crosslinks. H. Meyer conceived and supervised the project, and wrote the manuscript.

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Correspondence to Hemmo Meyer.

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Peer review information Nature Structural & Molecular Biology thanks Claudio Iacobucci and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Anke Sparmann and Florian Ullrich were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Effect of IRS mutation and p97 inhibition on I3 distribution (related to Fig. 2).

(a) Coomassie-stained SDS-gels of purified proteins used in the in vitro assays. Eos fusion proteins were UV-irradiated to convert from the green to the red Eos version leading to fragmentation as indicated. (b) Co-immunoprecipitation confirms interaction of I3 with PP1 and SDS22 in the SDS22–PP1–I3 (SPI) complex peak fractions. Myc-tagged I3WT or the PP1 binding-deficient I3RAXA mutant were transiently over-expressed in HEK293 cells. Lysates were separated by size exclusion chromatography and SPI complex peak fractions (corresponding to fractions 4 and 5 in Fig. 2d) subjected to anti-myc coimmunoprecipitation, followed by Western blot analysis as indicated. Asterisk indicates the light chain of the anti-myc antibody. (c) Size exclusion chromatography of lysates from HEK293 cells expressing myc-tagged I3WT treated with p97 inhibitor NMS-873 (10 µM) or DMSO for 2 h, followed by Western blot analysis as indicated. Note the shift in the distribution from free I3 to the PP1-bound form (SPI complex) upon NMS-873 treatment. (d) Quantification of signals for endogenous I3 in (c) as indicated.

Source data

Extended Data Fig. 2 Unfolding of DHFR fusions and effect of GroEL trapping mutant (related to Fig. 3).

(a) Unfolding reactions with Eos–DHFRI3 with or without methotrexate (Mtx). Reactions without the p37 adapter served as control as indicated. SDS22–PP1–Eos-DHFRI3 (35 nM) or variants thereof were mixed with p97 (175 nM hexamer) and p37 (500 nM), and the reaction started by addition of ATP (2 mM). Red Eos fluorescence was recorded. Mean ± SD, n=3 independent experiments. (b) Inhibition of DHFR activity in SDS22–PP1–Eos-DHFRI3 by Mtx. Conversion of dihydrofolate to tetrahydrofolate was followed spectrometrically by measuring the absorbance of NADPH at 340 nm, at indicated Mtx concentrations. Mean ± range, n=2 independent experiments. (c) Unfolding reactions of circular SPEosI3 with indicated concentrations of GroELD87K mutant that prevents refolding of green Eos. Green Eos fluorescence was recorded. Mean ± SD, n=3 independent experiments.

Source data

Extended Data Fig. 3 Controls and titrations of the FRET-based unfolding assay (related to Fig. 4).

(a) Coomassie-stained SDS-gel of purified Clover fusion protein complexes used in the in vitro assays. (b) Left panel: Coomassie gel of purified NIPP1 variants used in the in vitro assays. Note the shift to a higher molecular weight after TAMRA conjugation. Right panel: TAMRA fluorescence image of the same gel. (c) FRET time course measurements of disassembly reactions containing p97 (160 nM), p37 (480 nM), and NIPP1TAMRA (640 nM) after addition of ATP (2 mM) with indicated concentrations of SDS22–PP1Clover–I3. Shown is the normalized acceptor fluorescence (TAMRA). Δp37, p37 was omitted. (d) FRET time course measurements of disassembly reactions containing p97 (160 nM), SDS22–PP1Clover–I3 (160 nM), and NIPP1TAMRA (480 nM) at indicated time points after addition of ATP (2 mM) with indicated concentrations of p37. Shown are normalized ratios of acceptor and donor fluorescence. Δp37, p37 was omitted. (e) FRET time course measurements of disassembly reactions containing p97 (160 nM), p37 (480 nM), and SDS22–PP1Clover–I3 (160 nM) with indicated concentrations of NIPP1TAMRA. Shown is the normalized donor fluorescence (Clover). Δp37, p37 was omitted. (f) Co-IP analysis of the disassembly reaction under FRET assay conditions (160 nM p97, 160 nM SDS22–PP1Clover–I3, 480 nM NIPP1TAMRA and 480 nM p37). PP1Clover was immuno-isolated at indicated time points and associated proteins detected by Western blot analysis. Note association of NIPP1TAMRA over time. (g) Bayesian approach-based analysis of progress curves to determine kinetic parameters. Upper panel: FRET input data of SDS22–PP1Clover–I3 disassembly with low and high substrate concentrations for computation of kinetic parameters at 30 °C. p97 and SDS22–PP1Clover–I3 concentrations as indicated, p37 (480 nM), NIPP1TAMRA (low concentration, 480 nM; high concentration, 9 µM). Lower panel: Output of kinetics analysis. All 100000 calculated combinations of KM and kcat are plotted as a point density function in a 2D-kernel plot. Geometric means (solid lines) and geometric SD (dashed lines) of KM and kcat are depicted. (h) Cartoon depiction of linear and circular I3 cleavage by TEV protease, resulting in linear proteins. (i) Re-linearization control reaction of circular I3 by TEV protease. Note that circular I3 shifts back to a slower migrating band after TEV-mediated linearization. Coomassie-stained gel as indicated.

Source data

Supplementary information

Reporting Summary

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Supplementary Tables 1–4

Supplementary Table 1: Related to Fig. 1a. Data of nonredundant crosslinks in SDS22–PP1–I3 crosslinked with DSBU. All identified crosslinks with identified residues, peptides, scores, false discovery rates (FDRs) and scan numbers are listed in compact (left) and detailed view (right). The list has been reduced to contain each crosslink only once (non-redundant dataset). Supplementary Table 2: Related to Fig. 1c. Data of nonredundant crosslinks in p97–p37–SDS22–PP1–I3 crosslinked with DSBU. All identified crosslinks with identified residues, peptides, scores, FDRs and scan numbers are listed in compact (left) and detailed view (right). The list has been reduced to contain each crosslink only once (nonredundant dataset). Supplementary Table 3: Related to Fig. 1e. Data of UV-induced crosslinks of p97314pBpA (top) or p97592pBpA (bottom) with p37 and SDS22–PP1–I3. All identified crosslinks with identified residues, peptides, scores, and scan numbers with FDR < 5% are listed. Supplementary Table 4: Related to Fig. 3. Identified peptides covering the circularization site (red) in EosI3 subjected to control (linear) or sortase (circular) reactions detected by mass spectrometry. Samples were analyzed in triplicates. Runs where indicated peptides were identified are labeled in green. High confidence of identification by indicated search engines marked in green.

Source data

Source Data Fig. 1

Uncropped western blots and Coomassie gels.

Source Data Fig. 1

Numerical values of the graph.

Source Data Fig. 2

Uncropped western blots.

Source Data Fig. 2

Numerical values of graphs.

Source Data Fig. 3

Uncropped western blots and Coomassie gels.

Source Data Fig. 3

Numerical values of graphs.

Source Data Fig. 4

Uncropped western blots and fluorescence scans.

Source Data Fig. 4

Numerical values of graphs.

Source Data Extended Data Fig. 1

Uncropped western blots and Coomassie gels.

Source Data Extended Data Fig. 1

Numerical values of the graph.

Source Data Extended Data Fig. 2

Numerical values of graphs.

Source Data Extended Data Fig. 3

Uncropped Western blots and fluorescence scans.

Source Data Extended Data Fig. 3

Numerical values of graphs.

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van den Boom, J., Kueck, A.F., Kravic, B. et al. Targeted substrate loop insertion by VCP/p97 during PP1 complex disassembly. Nat Struct Mol Biol 28, 964–971 (2021). https://doi.org/10.1038/s41594-021-00684-5

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