Abstract
Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR–Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR–Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy–AcrIF24–promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.
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Data availability
Cryo-EM reconstructions of Csy–AcrIF24, Csy–AcrIF24 dimer, Csy–AcrIF24–DNA dimer, Csy–AcrIF24–Promoter DNA and Csy–AcrIF24–Promoter DNA dimer have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-25660, EMD-25661, EMD-25662, EMD-25789 and EMD-25788, respectively. Coordinates for atomic models of Csy–AcrIF24, Csy–AcrIF24 dimer, Csy–AcrIF24–DNA dimer, Csy–AcrIF24–Promoter DNA and Csy–AcrIF24–Promoter DNA dimer have been deposited in the Protein Data Bank under accession numbers 7T3J, 7T3K, 7T3L, 7TAX and 7TAW, respectively. Structures of Csy alone and Csy–AcrIF4 are available in the Protein Data Bank under accession numbers 6B45 and 7JZW, respectively. Source data are provided with this paper.
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Acknowledgements
We thank T. Klose and J. Sun for help with cryo-EM; S. Wilson for computation; R. V. Stahelin for providing access to the GE Amersham Imager 600 system; A. R. Davidson for sharing plasmids; J. Hopkins for assistance with the SAXS and MALS experiments and analyses; and other beamline staff of the BioCAT beamline at the Advanced Photon Source, Argonne National Laboratory, for their help with data collection. This work was supported by National Institutes of Health (NIH) grant R01GM138675 and a Core Pilot grant from the Indiana Clinical and Translational Sciences Institute to L.C. C.G. is supported by a grant from the NIH (T32GM132024). The BioCAT resources are supported by grant P30 GM138395 from the National Institute of General Medical Sciences (NIGMS) of the NIH. The use of the Pilatus3 1M detector was provided by grant 1S10OD018090 from the NIGMS. This work made use of the Purdue Cryo-EM Facility and resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by the Argonne National Laboratory under contract number DE-AC02-06CH11357.
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L.C. supervised the study. I.A.M. prepared samples, with help from C.G. I.A.M. performed the biochemical assays. I.A.M., C.G. and L.C. collected and processed cryo-EM data. I.A.M. and N.N. performed SAXS experiments and analyzed the data. L.C. and I.A.M. built the structural models. J.B.-D. identified the promoter sequence and elements. I.A.M. and L.C. prepared the manuscript, with input from C.G., N.N. and J.B.-D.
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J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics and a scientific advisory board member and co-founder of Acrigen Biosciences. The Bondy-Denomy laboratory receives research support from Felix Biotechnology. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 AcrIF23 and AcrIF24 inhibit in vitro DNA cleavage by Csy and Cas2/3.
(a) Cartoon diagram of the acrIF23-acrIF24 operon found in a P. aeruginosa prophage. No aca gene downstream of the acrIF23–24 locus was found. In comparison, the aca9 gene was found downstream of acrIF23 in a D. carbinolicus prophage. Adapted from6. (b) AcrIF23 and AcrIF24 inhibits substrate DNA cleavage by the type I-F CRISPR-Cas system in the in vitro DNA cleavage assay. (c) Quantitative result of the experiment in (b). Error bars represent SD; n = 3. Two-sided t test was performed (*P < 0.05). (d) EMSA results reveal that AcrIF24 inhibits substrate DNA binding to Csy whereas AcrIF23 does not. Note: Csy–AcrIF24 is capable of binding DNA independent of PAM and spacer sequences, resulting in a high molecular weight shift. * indicates a faint band where two copies of Csy bound to DNA, similar to previous report14 (doi.org/10.1093/nar/gkab092). (e) Quantitative result of the experiment in (d). Error bars represent SD; n = 3. Two-sided t test was performed (*P < 0.05). (f) EMSA results reveal that Cas2/3 recruitment by Csy is abolished by AcrIF24 but not by AcrIF23.
Extended Data Fig. 2 Biochemical and biophysical analysis of AcrIF23 and AcrIF24.
(a) Size exclusion chromatography (SEC) profiles of Csy, Csy–AcrIF24, Csy(Cas8f K247E/N250D)–AcrIF24 and Csy–AcrIF24–DNA complexes. Fractions of Csy–AcrIF24–DNA as indicated were analyzed by SDS-PAGE (to visualize proteins) and 10% Native PAGE gel (to visualize DNA), results of which are shown below the SEC profiles. SDS-PAGE of fractions of Csy(Cas8f K247E/N250D)–AcrIF24 is also shown below the SEC profiles. (b) SEC profiles (top) and SDS-PAGE (bottom) of AcrIF24 and its domain deletion mutants. (c) SEC profiles (left) and SDS-PAGE (right) of AcrIF24 mutants.
Extended Data Fig. 3 Cryo-EM data processing workflow for Csy–AcrIF24.
(a) A representative raw cryo-EM micrograph of Csy–AcrIF24. (b) Representative 2D class averages. (c–e) Three major 3D classes were identified from 2D classification and heterogenous refinement: Csy–AcrIF24 (c), Csy–AcrIF24 dimer (d), and AcrIF24 only (e). (f) Major 3D classes from the 3D classification of Csy–AcrIF24. Class 2 shows missing density for Cas5f, and Cas8f subunits. (g) 3D refinement for particles from the 3D classification of Csy–AcrIF24 as indicated. (h) The plot of the global half map FSC of Csy–AcrIF24 indicates an average resolution of 3.2 Å. (i) Two focused refinements with a soft mask either in the head or tail of the Csy complex to improve local resolutions. (j) The final Csy–AcrIF24 map generated by combining maps from focused refinement. (k) 3D refinement for particles from the 3D classification of Csy–AcrIF24 dimer as indicated. (l) The plot of the global half map FSC of Csy–AcrIF24 dimer indicates an average resolution of ~ 3.5 Å. (m) Two focused refinements with a soft mask to each Csy complex bound to AcrIF24 to improve local resolutions. (n) The final Csy–AcrIF24 dimer map generated by combining maps from focused refinement.
Extended Data Fig. 4 Structure of Csy–AcrIF24.
(a) Structure of AcrIF24 protomer color-coded as in Fig. 2a. EM density map is shown in orange mesh, and the atomic model of AcrIF24 is shown in sticks. (b) The protein sequence and secondary structure of AcrIF24. Residues involved in the dimeric interaction and tested by mutagenesis are marked with triangles. (c) Structural superimposition between AcrIF24 NTD domain and MMOD (PDB: 6D7K). (d) Interaction between HTH and NTD, with hydrophobic residues in the interface highlighted. (e)Structural superimposition between AcrIF24 HTH domain and Aca1 (PDB: 7CJK). (f) MID-MID dimerization interface of AcrIF24 as in Fig. 2e, with corresponding EM density shown in mesh. (g) HTH-HTH dimerization interface of AcrIF24 as in Fig. 2h, with corresponding EM density shown in mesh. (h) Interaction between AcrIF24-NTD and Csy as in Fig. 3c, with corresponding EM density shown in mesh. (i) Interaction between AcrIF24-MID and Csy as in Fig. 3e, with corresponding EM density shown in mesh.
Extended Data Fig. 5 Effect of AcrIF24 mutants on DNA cleavage and binding by Csy.
(a,b) In vitro DNA cleavage assays (a) and EMSAs (b) with Csy and target DNA in the presence of wild-type or domain deletion mutants of AcrIF24. (c,d) In vitro DNA cleavage assays (c) and EMSAs (d) with Csy and target DNA in the presence of wild-type AcrIF24 or AcrIF24 with mutations of residues in the dimerization interfaces. (e,f) In vitro DNA cleavage assays (e) and EMSAs (f) in the presence of wild-type AcrIF24 or AcrIF24 with mutations of residues involved in Csy interaction.
Extended Data Fig. 6 SAXS analysis of AcrIF23 and AcrIF24.
(a) SAXS assay of purified wild type AcrIF24 in solution. The experimental SAXS scattering curve for AcrIF24 is shown as gray points, while calculated curves for the monomeric (red) and dimer (blue) forms of AcrIF24 are shown as solid lines. The fit of each calculated curve to the experimental data is indicated by χ2 values of 194 and 1.4, respectively. (b) A superposition of the AcrIF24 dimer with an ab initio bead model from the SAXS analysis (DAMMIF/N). (c) The ‘estimated molecular weight’ of AcrIF24 as the average of Vc, Vp, Bayes, and Shape/Size. (d) The experimental SAXS scattering curve for AcrIF23 is shown as gray points, while calculated curve for the AcrIF23 monomer model (AlphaFold) (green) is shown as a solid line. The fit of the calculated curve to the experimental data is indicated by the χ2 value of 1.2. (e) A superposition of the AcrIF23 monomeric model (AlphaFold) with the ab initio bead model from the SAXS analysis (DAMMIF/N). (f) The ‘estimated molecular weight’ of AcrIF23 as the average of Vc, Vp, Bayes, and Shape/Size. AcrIF23 has a theoretical molecular weight of 17.5 kDa. (g) SEC-SAXS scattering curve (gray; Dmax of 75.0 Å; Rg of 25.3 Å; MWavg of 36 kDa) for the AcrIF24(H154A/H216A/Y217A/R221A) mutant aligned with the calculated scattering curve for the AcrIF24 monomer (red, χ2 of 1.3). The residual plot from the fit of the experimental data (gray) and fit of the model (red) is shown in the bottom panel. (h) SEC-MALS analysis of the AcrIF24(H154A/H216A/Y217A/R221A) mutant showing a calculated molecular weight consistent with a monomer.
Extended Data Fig. 7 Cryo-EM data processing workflow for Csy–AcrIF24–DNA.
(a) A representative raw cryo-EM micrograph of the Csy–AcrIF24–dsDNA complex. (b) Representative 2D class averages. (c,d) Two 3D classes separated by heterogenous refinement: Csy–AcrIF24 in monomer (c) and dimer (d) states. (e) Three major classes from the heterogenous refinement of Csy–AcrIF24 monomer. (f) Refined map of Csy–AcrIF24 in monomer state. (g) Homogenous refinement for particles from the dimer state. (h) The plot of the global half map FSC of Csy–AcrIF24–dsDNA indicates an average resolution of 3.62 Å. (i) Refined map of Csy–AcrIF24–dsDNA after applying Gaussian filter to show the weak density of DNA.
Extended Data Fig. 8 Csy–AcrIF24 dimer recruits DNA independent of PAM and spacer sequences.
(a) Cryo-EM structure of Csy–AcrIF24 dimer bound to dsDNA. Csy–AcrIF24 subunits are color-coded as Fig. 1a. Two strands of DNA are color-coded in blue and magenta, respectively. (b) Electrostatic potential maps of Csy–AcrIF24 dimer (left) and AcrIF24 dimer (right), showing that DNA is bound to the positively changed surface of the assembly. (c) EMSA assay showing that Csy–AcrIF24 binds to both specific DNA (dsDNAsp) and non-specific DNA (dsDNAns) without a PAM or a spacer sequence. Sequences of dsDNAsp and dsDNAns are shown at the top, with the spacer highlighted and the GG PAM sequence colored in red. (d) EMSA assay showing that Csy (Cas8f K247E/N250D)–AcrIF24 is not capable of binding to both dsDNAsp or dsDNAns. (e,f) Competition EMSA assay to test whether the binding sites of dsDNAsp and dsDNAns overlap. In e, Csy or Csy–AcrIF24 was incubated with dsDNAsp (5′-FAM in the TS), followed by adding increasing concentrations of unlabeled dsDNAns. In f, Csy or Csy–AcrIF24 was incubated with dsDNAns (5′-FAM in the TS), followed by adding increasing concentrations of unlabeled dsDNAsp.
Extended Data Fig. 9 Cryo-EM data processing workflow for Csy–AcrIF24–promoter DNA.
(a) A representative raw cryo-EM micrograph of the Csy–AcrIF24–promoter DNA complex. (b) Representative 2D class averages. (c,d) Two 3D classes separated by heterogenous refinement: Csy–AcrIF24-promoter DNA in monomer (c) and dimer (d) states. (e) Two major classes from the heterogenous refinement of Csy–AcrIF24-promoter DNA monomer. (f) Refined map of Csy–AcrIF24-promoter DNA in monomer state. (g) The plot of the global half map FSC of Csy–AcrIF24–promoter DNA in monomer state indicates an average resolution of ~ 2.7 Å. (h) Two major classes from the heterogenous refinement of Csy–AcrIF24-promoter DNA dimer. (i) Refined map of Csy–AcrIF24-promoter DNA in dimer state. (j) The plot of the global half map FSC of Csy–AcrIF24–promoter DNA in dimer state indicates an average resolution of ~ 2.7 Å.
Extended Data Fig. 10 Structure of AcrIF24–promoter DNA.
(a) Modeling of the 19-bp promoter DNA bound to AcrIF24. Shown are individual base pairs of the 19-bp promoter DNA, with corresponding EM densities in mesh. (b) Structural comparison between AcrIF24–promoter DNA and Aca1–promoter DNA.
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Mukherjee, I.A., Gabel, C., Noinaj, N. et al. Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor. Nat Chem Biol 18, 1417–1424 (2022). https://doi.org/10.1038/s41589-022-01137-w
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DOI: https://doi.org/10.1038/s41589-022-01137-w
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