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DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation

Nature volume 592, pages 778–783 (2021)Cite this article

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Abstract

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis1,2,3,4. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin4,5,6. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains7,8,9, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT)10,11. Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear10,12,13,14. Here we report cryo-electron microscopy structures of the human NLRP1–DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1–DPP9 interaction and accelerates degradation of the N-terminal fragment10 to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome.

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Fig. 1: Structure of the NLRP1–DPP9 complex.
Fig. 2: Detailed interfaces in the NLRP1–DPP9 ternary complex and inhibition by VbP.
Fig. 3: Functional consequences of mutations that affect assembly of the NLRP1–DPP9 ternary complex.
Fig. 4: Repression of activity of the NLRP1 CT inflammasome by formation of the ternary complex.

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Data availability

Extended protein purification protocols are available on Protocols.io at https://www.protocols.io/groups/hao-wu-lab. Raw cryo-EM data are available on EMPIAR under the accession numbers EMPIAR-10594 (NLRP1-DPP9) and EMPIAR-10595 (NLRP1–DPP9–VbP). The cryo-EM maps are available on the Electron Microscopy Data Bank (EMDB) under the accession numbers EMD-22074 (NLRP1–DPP9) and EMD-22075 (NLRP1–DPP9–VbP). The atomic coordinates are available on the Protein Data Bank (PDB) under the accession numbers 6X6A (NLRP1–DPP9) and 6X6C (NLRP1–DPP9–VbP). Pymol session files and the image analysis macro are available from https://doi.org/10.17605/OSF.IO/X7DV8. All other data can be obtained from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank T.-m. Fu of the laboratory of H.W. for initiating an apo-NLRP1 project; all members of the laboratories of H.W. and D.A.B. for helpful discussions; J. Chai for sharing their crystal structure of the FIIND of rat NLRP1 before submission; M. Su for his advice on running goCTF and CTF estimation for tilt datasets; M. Ericsson at the HMS electron microscopy facility for advice and training; R. Walsh, S. Sterling and S. Rawson at the Harvard Cryo-EM Center for Structural Biology for cryo-EM training and productive consultation; G. Skiniotis and M. Robertson for sharing their GemSpot script and for troubleshooting advice; T. Edwards and the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E; and J. Myers and the Pacific Northwest Center for Cryo-EM at Oregon Health & Science University for screening and preliminary dataset collection, under the NIH grant U24GM129547 and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. The pcDNA3.1 LIC 6A and pcDNA 3.1 LIC 6D plasmids were gifts from S. Gradia (Addgene plasmids no. 30124 and no. 30127, respectively). The pLEX_305-N-dTAG plasmid was a gift from J. Bradner and B. Nabet (Addgene plasmid no. 91797). The pLEX_307 plasmid was a gift from D. Root (Addgene plasmid no. 41392). Our research used software and computing support at SBGrid and the Extreme Science and Engineering Discovery Environment (XSEDE) Bridges at the Pittsburgh Supercomputing Center, through allocation TG-MCB190086. This work was supported by the National Institutes of Health and the Cancer Research Institute (DP1 HD087988 and R01 AI124491 to H.W., T32-GM007726 to L.R.H., the CRI Irvington Postdoctoral Fellowship to P.F., R01GM132129 to J.A.P., R01GM67945 to S.P.G., T32 GM007739-Andersen and F30 CA243444 to A.R.G. and the Memorial Sloan Kettering Cancer Center Core Grant P30 CA008748 and R01 AI137168 to D.A.B.). We apologize to authors whose work could not be cited because of space limitation.

Author information

Author notes

  1. These authors contributed equally: L. Robert Hollingsworth, Humayun Sharif, Andrew R. Griswold

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    L. Robert Hollingsworth, Humayun Sharif, Pietro Fontana, Kevin B. Dagbay & Hao Wu

  2. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    L. Robert Hollingsworth, Humayun Sharif, Pietro Fontana, Kevin B. Dagbay & Hao Wu

  3. Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA

    L. Robert Hollingsworth

  4. Tri-Institutional MD–PhD Program, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Andrew R. Griswold

  5. Pharmacology Program, Weill Cornell Graduate School of Medical Sciences and Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Andrew R. Griswold & Daniel A. Bachovchin

  6. Department of Cell Biology, Harvard Medical School, Harvard University, Boston, MA, USA

    Julian Mintseris, Joao A. Paulo & Steven P. Gygi

  7. Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Daniel A. Bachovchin

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Contributions

L.R.H., H.S. and H.W. conceived the NLRP1–DPP9 complex study. L.R.H. designed constructs with input from H.S. L.R.H., H.S., P.F. and K.B.D. carried out preliminary expression and purification studies. L.R.H. purified the complexes. H.S. and L.R.H. made cryo-EM grids for data collection. L.R.H. screened cryo-EM grids and collected cryo-EM data. H.S. and L.R.H. analysed cryo-EM data. H.S. performed model building and refinement. L.R.H. and H.S. designed mutants for in vitro and cell-based assays. L.R.H. and A.R.G. cloned mutants for functional study. A.R.G. performed all cell-based assays, peptide mass spectrometry and chemical enrichment of protease substrates analysis under the supervision of D.A.B. J.M. and J.A.P. performed crosslinking mass spectrometry and data analysis under the supervision of S.P.G. A.R.G., L.R.H., H.S., H.W. and D.A.B. designed the experiments. L.R.H., H.S., A.R.G., H.W. and D.A.B. wrote the manuscript with input from all of the other authors.

Corresponding authors

Correspondence to Daniel A. Bachovchin or Hao Wu.

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Competing interests

H.W. is a co-founder of Ventus Therapeutics. The other authors declare no competing interests.

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Peer review information Nature thanks Edward Miao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Structural determination of the NLRP1–DPP9 complex.

a, Purification of the NLRP1–DPP9 complex by ion-exchange chromatography. The ternary complex peak is shaded in green and labelled with an arrow. b, A representative (of >1,000 images) cryo-EM micrograph. c, Representative 2D class averages. d, Workflow for the determination of the structure of the NLRP1–DPP9 complex. e, Map–map and map–model FSC curves. f, Local-resolution distribution of the final map calculated with ResMap44.

Source data

Extended Data Fig. 2 Crosslinking mass spectrometry analysis of the NLRP1–DPP9 complex.

a, Summary of BS3 crosslinking between DPP9 and NLRP1. High-confidence crosslinked peptides are displayed, and residue ranges are labelled and colour-coded by domain. Crosslinked lysine pairs are indicated in red text. Cα–Cα distances between lysine residues (in red) interpreted by the final NLRP1–DPP9 model are shown, along with the figure panels that show them in detail. All detected peptide pairs are tabulated in Source Data. b, Overview of BS3-mediated crosslinks. c–h, Close-up views highlighting crosslinked lysine pairs (red) interpreted by the final NLRP1-DPP9 model.

Source data

Extended Data Fig. 3 Sequence and structural analysis of FIIND.

a, ClustalW multiple sequence alignment between human NLRP1 (hNLRP1), mouse NLRP1 (mNLRP1, different isoforms) and rat NLRP1 (rNLRP1, different isoforms). COP, Copenhagen; ZUC, Zucker; LEW, Lewis; SD, Sprague Dawley; and CDF, Fischer. Secondary structures and residue numbers are denoted on the basis of the human FIINDA structure in the NLRP1–DPP9 ternary complex. Interface residues in the NLRP1–DPP9 complex are annotated with asterisks, and residues in the catalytic triad (H1186, E1195 and S1213) are boxed in black. b, FIINDA overview with ZU5 (blue) and UPA (light pink) subdomains. The catalytic triad residues (H1186, E1195 and S1213) are shown in sticks. c, Topology of the FIIND with secondary structures labelled. d, Superimposition of FIINDA onto the UPAB. NLRP1B must be free NLRP1 CT, because a ZU5 subdomain at site B would have clashed with ZU5 and UPA at site A and with DPP9. e, The ZU5A–UPAA–UPAB module that binds DPP9. UPAA and UPAB interact with each other in a front-to-back manner, with only a 9° rotation between them. f, Altered conformation of the UPAB N terminus that binds in the DPP9 active-site tunnel in comparison to UPAA in a complete FIINDA.

Extended Data Fig. 4 Structural determination of the NLRP1–DPP9 complex with VbP.

a, Purification of the NLRP1–DPP9 complex in the presence of VbP by ion-exchange chromatography. The ternary complex peak is shaded in green and labelled with an arrow. b, A representative (of >1,000 images) cryo-EM micrograph. c, Representative 2D class averages. d, Workflow for the determination of the structure of the NLRP1–DPP9–VbP complex. e, Map–map and map–model FSC curves. f, Local-resolution distribution of the final map calculated with ResMap44.

Source data

Extended Data Fig. 5 VbP interactions in the DPP9 active site and comparison to a DPP substrate and NLRP1.

a, Schematic of covalent linkage between the S730 of DPP9 and VbP. b, Fit of VbP into the cryo-EM density. VbP is shown in stick, with carbon atoms in light brown. The charged amino group of VbP interacts with the DPP9 EE-loop (which also coordinates a substrate N terminus), and the carbonyl oxygen of VbP interacts with R133 of the R helix. The covalent linkage of VbP with S730 (the catalytic serine) is displayed. c, Structural alignment of the VbP-bound DPP9 model (green) and the crystal structure of bacterial DPP4 bound to the substrate Ile-Pro (PDB code 5YP3) (orange)25. VbP assumes a pose that is notable similar to a model substrate. d, NLRP1 CT−DPP9 complex, in which DPP9 is coloured by Cα–Cα distances between NLRP1-bound and VbP-bound structures, as indicated. A distance scale bar is shown. VbP is displayed in sticks to mark the active site, with carbon atoms in green, oxygen atoms in red, nitrogen atoms in blue and boron atoms in orange. UPA of NLRP1 CT is shown in magenta.

Extended Data Fig. 6 Lack of cleavage of intact NLRP1 CT, but the cleavage of its isolated N-terminal peptide, by DPP9.

a, N-terminal sequencing of the purified NLRP1–DPP9 complex, showing that the NLRP1 CT is not cleaved by the co-expressed DPP9. b, Chemical enrichment of protease substrates assay, showing that DPP9 does not cleave NLRP1 CT. In brief, wild-type NLRP1 expressed in DPP8 DPP9 double-knockout HEK293T cells was incubated with PBS or recombinant DPP9 before labelling with a 2PCA–biotin probe (which selectively biotinylates free N termini, except for those with a proline in the second position), followed by capture of biotinylated proteins. The inputs and the eluents were analysed by immunoblots using anti-NLRP1 CT (full-length NLRP1 and NLRP1 CT), anti-GAPDH and anti-streptavidin (biotinylated proteins) antibodies. DPP9 treatment did not increase the biotinylation of NLRP1 CT, as would be expected after the removal of the N-terminal Ser-Pro dipeptide. c, Evidence of cleavage of the isolated 15-residue N-terminal peptide in NLRP1 CT by recombinant DPP9 from mass spectrometry analysis. d, Inhibition of DPP9 catalytic activity against Ala-Pro-AMC by the isolated NLRP1 CT peptide. e, Schematic illustrates the ability of DPP9 to cleave an isolated UPA N-terminal peptide, but not dipeptides from an intact NLRP1 CT. f, Comparison of the binding modes of the UPAB N-terminal peptide in the NLRP1–DPP9 complex and the Ile-Pro dipeptide in an acyl-enzyme intermediate25. g, Theoretical dipeptide cleavage does not dampen activity of the NLRP1 inflammasome by LDH release or inflammasome signalling. n = 3 independent biological replicates. Data are mean ± s.e.m. Anti-Flag (NLRP1 CT), anti-GSDMD and anti-GAPDH antibodies were used in the immunoblots. p30, GSDMD N-terminal fragment from caspase-1 cleavage. h, Theoretical dipeptide cleavage does not dampen activity of the NLRP1 inflammasome by formation of ASC specks. n = 10 quantified fields of view. Data are mean ± s.e.m. Right, representative superimposed images of nuclei (blue), RFP (red) and GFP–ASC (green). All data are representative of >2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired two-sided t-test. Exact P values are provided in Source Data.

Source data

Extended Data Fig. 7 Mutational analysis of the interactions in the NLRP1–DPP9 ternary complex.

a, Disorder-to-order transition of several DPP9 surface loops from the isolated DPP9 crystal structure (PDB code 6EOQ)24 to the NLRP1-bound DPP9 cryo-EM structure. b, Genomic confirmation of DPP8 knockout generated in DPP9-knockout HEK293T cells that stably express Cas920, to create DPP8 DPP9 double-knockout HEK293T cells. The single-guide RNA (sgRNA) sequence is highlighted. c, Immunoblots of the input lysates for the Flag co-immunoprecipitation with wild-type or mutant DPP9 and wild-type NLRP1–Flag, related to Fig. 2h. Anti-DPP9, anti-NLRP1 (full-length NLRP1 and NLRP1 CT) and anti-GAPDH antibodies were used in the immunoblots. d, Cleavage rate of a model DPP9 substrate, Gly-Pro-AMC, by wild-type DPP9 and its structure-guided mutants. Only the catalytically dead mutant DPP9(S730A) disrupts catalytic activity and sensitivity to VbP. n = 3 technical replicates. Data are mean ± s.e.m. e, Immunoblots of the input lysates for the Flag co-immunoprecipitation with wild-type or mutant NLRP1–Flag and wild-type DPP9, related to Fig. 2i. Anti-DPP9, anti-NLRP1 (full-length NLRP1 and NLRP1 CT) and anti-GAPDH antibodies were used in the immunoblots. All data are representative of >2 independent experiments.

Extended Data Fig. 8 The ZU5 domain and DPP9 sterically hinder UPA polymerization.

a, Modelling of a FIIND polymer using the observed UPAA–UPAB relationship. Adjacent ZU5 molecules would clash, which suggests that UPA polymerization cannot occur in complete FIIND. b, Modelled recruitment of free UPA adjacent to UPAA and UPAB in the ternary complex with DPP9. The additional UPA subdomain next to FIINDA clashes with the ZU5 subdomain, and the additional UPA next to UPAB clashes with both DPP9 monomers in the complex, which suggests that DPP9 inhibits UPA oligomerization. c, A modelled UPA oligomer on the basis of the near front-to-back interaction in the NLRP1–DPP9 ternary complex. In the model, the N-terminal tails of free UPAs are shown in the UPAA (pink) or UPAB (magenta) conformation in complex with DPP9 but, in reality, this conformation is likely to be different. d, Modelling of a UPA oligomer formed on one side of a NLRP1 FIINDA–NLRP1 CTB complex. The NLRP1 FIINDA−NLRP1 CTB binary complex can polymerize with freed NLRP1 CT. In a–d, DPP9 is coloured in green, and NLRP1 domains are coloured as indicated.

Extended Data Fig. 9 VbP displaces NLRP1 from DPP9 in vitro and in cells.

a, Schematic of the on-bead displacement experiment. The ternary complex is expressed in HEK293T cells, which are then lysed and incubated with Flag beads. Once bound, beads are split equally and washed with compounds or DMSO. The remainder of the protein is eluted off of the beads. MeBS, bestatin methyl ester. b, Two structurally distinct DPP9 inhibitors (VbP and 8J) displace DPP9 from NLRP1(S1213A) by the on-bead displacement assay. Anti-Flag (NLRP1(S1213A)), anti-MYC (NLRP1 CT) and anti-V5 (DPP9) antibodies were used in the immunoblots. Representative of two independent experiments. c, Schematic of the dTAG experiment. FKBP12 with the F36V mutation (dTAG) is fused to the N terminus of NLRP1. The dTAG13 ligand recruits an E3 ligase to FKBP12(F36V), leading to its ubiquitination and N-terminal degradation of the fusion protein. NLRP1 CT (UPA–CARD) that results from FIIND autoprocessing are released to assemble the inflammasome. d, NLRP1(FIIND/S1213A) expression in reconstituted HEK293T inflammasome system rescues GSDMD cleavage resulting from dTAG13-induced NLRP1 degradation. VbP prevents GSDMD rescue without inducing additional NLRP1 degradation. Anti-HA (dTAG–NLRP1, and dTAG–NLRP1 N-terminal fragment), anti-Flag (NLRP1(FIIND/S1213A)), anti-GSDMD and anti-GAPDH antibodies were used in the immunoblots. p30, GSDMD N-terminal fragment from caspase-1 cleavage. Representative of two independent experiments.

Extended Data Table 1 Cryo-EM data collection and refinement statistics of NLRP1–DPP9 structures
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Hollingsworth, L.R., Sharif, H., Griswold, A.R. et al. DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation. Nature 592, 778–783 (2021). https://doi.org/10.1038/s41586-021-03350-4

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