CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3,4,5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.
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The crystal structures have been deposited into the Protein Data Bank database with the accession codes 7QDA, 8B0R and 8B0U. The SAXS data and models have been deposited into the Small Angle Scattering Biological Data Bank with the accession codes SASDQM4, SASDQN4, SASDQP4 and SASDQQ4. The following Protein Data Bank entries were used in this study: 2H27, 3K1J, 4ME7, 4IZJ, 4LUP, 5ZX2, 5CR2, 6VM6, 6SCE and 7RWK. Source data are provided with this paper.
No custom code was used in this work.
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The synchrotron MX data were collected at beamline P13, operated by staff at EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). We thank G. Bourenkov and I. Bento for the assistance in using the beamline; V. Siksnys for helpful discussions; S. Shirran and S. Synowsky for the MS analysis; N. Brenner for technical assistance; and M. Drag and J. Grzymska for discussions and an initial peptide screen. M.G. and J.L.S.-B. are funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy–EXC2151–390873048. M.F.W. acknowledges a European Research Council Advanced Grant (grant number 101018608) and the China Scholarship Council (reference 202008420207 to H.C.). B.E.B. acknowledges EPR equipment funding by BBSRC(BB/R013780/1 and BB/T017740/1). G.H. is grateful for funding by the Deutsche Forschungsgemeinschaft (grant number HA6805/6-1).
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Purification and Structure of CalpL.
a, Gelfiltration chromatography (Superdex 200 16/60) of CalpL. Inset: SDS-PAGE analysis of the fractions indicated by the blue bar in the chromatogram. The experiment was performed multiple times (n > 3 biological replicates). b, TM-prediction by the TMHMM 2.0 server28 vs experimental structure. c, Representative electron density of the SeMet CalpL crystal structure. The structural model is drawn in ball-and-stick representation. Selected residues are labeled. The black mesh is a 2mFo-DFc electron density map contoured at 1.0 σ. d, Topology diagram of CalpL. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 CalpL in comparison to structurally related proteins.
a, CalpL is drawn as a cartoon model color-coded as in Fig. 1. The Lon protease from T. onnorineus (PDB-ID: 3K1J, DALI Z-score: 12.8)76 is shown as a white cartoon model. b, Table listing proteins with similar domain structures17,23,27,29,30,76,77,78. c, Surface electrostatics of the Lon protease active site region. The catalytic dyad is marked. The grey line marks the likely substrate binding site. d, Superposition of CalpL active site with the acyl-enzyme intermediate of yellowfin asciitis virus protease. CalpL is in sticks representation and color-coded as in Fig. 1. Chain D of structure 4IZJ27 (residues 630–640) was superimposed on the corresponding residues of CalpL (150-160) leading to an r. m. s. d. of 0.314 Å. Of 4IZJ, only the acyl-enzyme intermediate is shown in sticks mode. Selected residues and the positions of the P1-P3 sites are indicated. e, Superposition of CalpL (color scheme as in Fig. 1) with the Cap4 protein (white, PDB-ID: 6VM6)23. f, Superposition of the CalpL SAVED domain (color scheme as in Fig. 1) with the Can1 protein (white, PDB-ID: 6SCE)17. g, Superposition of the CalpL SAVED domain (color scheme as in Fig. 1) with the CARF domains of the Cap5 protein (white, PDB-ID: 7RWK)29.
Extended Data Fig. 3 The CalpL/cA4 complex.
a, Close-up of cA4 (green) bound to the SAVED domain of CalpL. The blue mesh is a 2mFo-DFc electron density map contoured at 1.0 σ. b, Superposition of CalpL apo (white) onto the cA4 complex structure (color coded as in Fig. 1). c, Structural alignment of the SAVED domains of CALP/cA4 and Cap4/cA3 (white).
Extended Data Fig. 4 CalpT is a MazF homolog and the target of the CalpL protease.
a, b, A superposition of the predicted CalpT structure (compare Fig. 2B) with one monomer of the MazF/ssRNA complex (purple/orange) (PDB-IDs: 5CR2)79. The AlphaFold233 prediction confidence is mapped onto the CalpT structure (pLDDT48, predicted local distance difference test). b, A superposition of the predicted CalpT structure (compare Fig. 2b) with one monomer of the MazE/F complex (PDB-IDs: 4ME7)35. The AlphaFold233 prediction confidence is mapped onto the CalpT structure (pLDDT48, predicted local distance difference test). c, Gel filtration chromatography (Superdex 75 16/60) of CalpT The experiment was performed multiple times (n > 3 biological replicates). According to the MALS data in Fig. 3, isolated CalpT behaves as a monomer. Inset: SDS-PAGE analysis of the fractions indicated by the blue bar. d, Peptide fingerprints of cleavage bands. The indicated gel-bands were cut from the gel and submitted for identification at the Mass spectrometry and proteomics facility at the University of St Andrews (Fife, UK, https://mass-spec.wp.st-andrews.ac.uk). Red letters indicate peptides that were identified in the respective sample. The experiment was performed once. e, Mutational analysis of potential CalpL cleavage sites in CalpT. The experiment was performed multiple times (n > 3 technical replicates). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 Characterization of the CalpL/T complex.
a, Single cycle kinetics SPR data of the CalpL/T interaction. The interaction is very strong but cannot be satisfyingly fitted with a 1:1 binding model. The experiment was performed twice (n = 2 technical replicates). b, As a), but an artificial construct of an unspecific VHH fused to CalpT was used as analyte in this experiment. The interaction is very similar to the CalpL/T interaction. The experiment was performed twice (n = 2 technical replicates). c, Schematics of two artificial constructs containing the CalpL cleavage site. d, CalpL cleaves an artificial construct of an unspecific VHH fused to CalpT10 but not a construct of two VHHs fused by the CalpL cleavage site. The experiment was performed once. e, SEC-MALS traces (solid lines: UV280, dashed lines: MWMALS) of proteolysis reactions with different combinations of CalpL S152A, CalpT, and cA4. The schematic indicates the molecular species behind the individual peaks. The experiments were performed twice with slightly different buffer conditions (n = 2 technical replicates). f, Binding of CalpL wt to the indicated CalpT mutants in the absence of cA4. The schematic indicates the position of the mutant in the CalpL/T complex. The experiments were performed once. g, Representative electron density of the CalpL/T10 crystal structure. Selected residues are labeled. The black mesh is a 2mFo-DFc electron density map contoured at 1.0 σ. h, SEC-SAXS experiment of the CalpL/T10 complex. The experiment was performed once. Thirty sample intensity frames and sixty buffer intensity frames were collected and averaged. For each data set and angular point the errors were computed following the Poisson statistics. The data points represent the average intensity difference (sample-buffer) and the error bars represent the standard deviation. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6 cA4 induced oligomerization of CalpL studied by DLS and SAXS.
a, Dynamic light scattering experiments (six timeseries, each series marked by a dashed circle, single data points are shown) at different protein concentrations and in the absence (t = 0: light grey to t = 60 min: dark grey) and presence (t = 0: cyan to t = 60 min: violet) of cA4 reveal a cA4-dependent oligomerization of CalpL. The experiment was performed twice (n = 2 technical replicates) b, SAXS experiments at different concentrations. The experiments were performed once. For each experiment, thirty sample intensity frames and sixty buffer intensity frames were collected and averaged. For each data set and angular point the errors were computed following the Poisson statistics. The data points represent the average intensity difference (sample-buffer) and the error bars represent the standard deviation. c, Ab initio/rigid-body model of a CalpL dimer created with DAMMIF and SASREFMX by a global fit of a monomer-dimer mixture to the different concentrations (red lines). The crystal structure of the CalpL monomer is shown on the same scale.
Extended Data Fig. 7 Probing the RNase activity of the activated toxin and checking for cA4 induced dimerization of CalpT with pulsed EPR.
a, Fluorescence image of the denaturing PAGE to determine ribonuclease activity of the reactions in b) against six fluorescently labelled RNA substrates (listed in c)). No cleavage was observed after 30 min incubation with RNAs at 60 °C. The experiment was performed three times (n = 3 biological replicates) b, SDS-PAGE analysis of cA4-induced cleavage of CalpT (33 kDa) by CalpL. Cleavage is complete after 60 min at 60 °C. The experiment was performed three times (n = 3 biological replicates) c, Sequences of the RNA substrates d, left: MazF was incubated with a single stranded RNA library containing 10 random bases. Illumina sequencing was used to check for sequences that were cleaved by MazF. Compared to a control reaction without MazF, sequences containing the known MazF target site (ACA) were depleted. right: same experiment but with CalpL/T ± cA4 instead of MazF. No off-diagonal sequences and hence no ssRNase activity were observed. The experiment was performed two times (n = 2 biological replicates). e, Oligonucleotides for the experiments in d) f, AlphaFold2 dimer models of CalpT23. g, Best model (pLDDT48, predicted local distance difference test) including MTSSL spin label80. h, X-band cw-EPR spectrum of of CalpL/T E119R1. The amount of free label (sharp spikes) is ~10%. The labelling efficiency determined as ~100%. i, PELDOR time traces of CalpL/T E119R1 in the presence (red) and absence (black) of cA4. j, Consensus distributions and corresponding uncertainty bands. Colored bars indicate reliability ranges (green: shape reliable; yellow: mean and width reliable; orange: mean reliable; red: no quantification possible). Predicted distance calculated with mtsslWizard80. The EPR experiment was performed twice (n = 2 technical replicates). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 8 AlphaFold2 predictions of CalpS.
a, Prediction of CalpS alone. The protein is shown as cartoon and colored according to the prediction confidence (pLDDT48, predicted local distance difference test) b, Prediction of the CalpT/S complex. c, Superposition of CalpS with 4LUP81 and 2H2782 identify the DNA binding regions of CalpS. d, Model of CalpS in the context of a RNAP/ECF σ-factor/promotor complex (PDB: 5ZX283, grey, yellow, green) from M. tuberculosis. Note that the linker region between the σ2 and σ4 subunits of CalpS has been cut to allow the superposition of the σ2 and σ4 domains onto those of 5ZX2. The linker is long enough to bind to the RNAP in a similar way as the σ-factor in the 5ZX2 structure (yellow).
Supplementary Fig. 1
Uncropped and unedited versions of all gels used in this study.
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Rouillon, C., Schneberger, N., Chi, H. et al. Antiviral signalling by a cyclic nucleotide activated CRISPR protease. Nature 614, 168–174 (2023). https://doi.org/10.1038/s41586-022-05571-7
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