Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24

Figures

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  Fig. 1. The crystal structure of AcrIF24, a dimer that inhibits the type I-F CRISPR-Cas system. a AcrIF24 but not AcrIF24ΔMD inhibits the in vitro cleavage activity of the type I-F CRISPR-Cas system. Reactions were performed with 0.32 μM Csy complex, 0.16 μM Cas2/3, and 0.04 μM 54-bp dsDNA (5′-FAM in the non-target DNA strand, NTS). AcrIF24 or AcrIF24ΔMD was added with concentrations of 0.16, 0.32, and 0.64 μM following the order indicated by the black triangles. Fraction DNA cleavage was calculated as mentioned in the Methods section. Data are presented as mean values ± SD; n = 3. Two-sided t test was performed. The “*“ indicates that there is a significant difference between the quantitative data (P < 0.05). *P = 0.0199, 0.0018, 0.0008 (left to right). b The crystal structure of AcrIF24 is shown in the cartoon model, together with its schematic illustration. The NTD and CTD are colored in yellow and cyan, respectively. The HTH motif is colored in orange. The unmodelled MD is indicated by the dashed line and colored hot pink in the schematic illustration. c Static light scattering (SLS) studies of AcrIF24 and AcrIF24ΔMD. The calculated molecular weights of the main peaks of the two…

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  Fig. 2. The HTH motif of AcrIF24 binds the Acr promoter with high affinity. a Inverted repeat sequence are conserved in the upstream promoters of AcrIF24 homologs. Genomic context of the acrIF23–acrIF24 locus of Pseudomonas aeruginosa prophage with inverted repeat sequence (shades of orange and red for the two half-sites of the respective repeat). Predicted regulatory sequences are shaded in blue and the predicted transcription start site (+1) is indicated by an arrow. The lower panel shows the alignment of acrIF23–acrIF24 operon promoter from P. aeruginosa prophage with those of other species in which inverted repeats are displayed as above. Invariant residues are indicated by an asterisk. b EMSA shows that AcrIF24 and AcrIF24ΔMD both exhibit high affinity to the IR23-24 DNA. The KD values are 20.18 and 49.06 nM for AcrIF24 and AcrIF24ΔMD, respectively. AcrIF24 displays a weak binding to non-sequence-specific dsDNA (dsDNANS), with a KD value of 3.461 μM. Data are presented as mean values ± SD; n = 3. Raw data for these curves are shown in Supplementary Fig. 3. c EMSA used to test the binding affinities of AcrIF24 for IR23-24 and mutated IR23-24. The sequences of WT and mutated IR23-24…

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  Fig. 3. Cryo-EM structure of the Csy-AcrIF24 complex. a Binding kinetics of AcrIF24 with the Csy complex, measured by surface plasmon resonance (SPR) method. SPR curves (colored curves) were fit kinetically using a 1:1 Langmuir binding model (black lines). The data shown are representative of three independent experiments. b Static light scattering (SLS) studies of the mix of AcrIF24 and the Csy complex under different molar ratios. The predicted composition of each peak based on the calculated molecular weight is shown above the peaks. The calculated molecular weights of the peaks are shown in Supplementary Table 2. c Cryo-EM map of the Csy-AcrIF24 complex with each subunit color-coded. Two views are shown. d Atomic structure of Csy-AcrIF24 in cartoon representation with each subunit colored as in c. Two views are shown. e The AcrIF24 dimer in the Csy-AcrIF24 structure. Cryo-EM density is shown in a slate mesh. The MD of AcrIF24 is marked in a box. f Close-up view of the MD of AcrIF24. Cryo-EM density is shown in slate mesh.

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  Fig. 4. Detailed interactions between AcrIF24 and the Csy complex, which is essential to the inhibition capacity. a Overall structure of the Csy-AcrIF24 complex with one AcrIF24 bound to one Csy complex. The Csy complex is shown in the surface model with each subunit colored as in Fig. 3d. AcrIF24 is shown in cartoon model colored in yellow, hot pink, and cyan for its NTD, MD, and CTD, respectively. b–e Close-up view of the interfaces between AcrIF24 and the Csy complex. The interfaces of AcrIF24NTD-Cas7.6f (b), AcrIF24NTD-Cas7.5f (c), AcrIF24MD-Cas7.4f (d), and AcrIF24MD-Cas7.2f (e) are shown. AcrIF24 is colored as in a, and interacting residues are shown as sticks with cryo-EM density map shown in mesh. f Native gel was used to test the binding between the Csy complex and AcrIF24 or its mutants. Reactions were performed with 0.32 μM Csy complex and AcrIF24 concentrations of 0.16, 0.32, and 0.64 μM following the order indicated by the black triangle. DDRF/AAAA represents the D104A/D105A/R106A/F107A mutant. The gel was stained with Coomassie blue staining. g Mutations of the interface residues of AcrIF24 decreased its inhibition capacity of the in vitro cleavage activity of the type I-F…

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  Fig. 5. AcrIF24 induces strong non-specific DNA binding activity in the Csy complex. a EMSA was used to test the induction of non-specific DNA binding by AcrIF24 using dsDNASP and dsDNANS. Reactions were performed with 1 μM dsDNASP or dsDNANS, 8 μM Csy, and concentrations of AcrIF24 were set as 1, 2, 4, and 8 μM following the order indicated by the black triangle. The “+” represents 8 μM of AcrIF24. b EMSA was used to test the induction of non-specific DNA binding by AcrIF24 mutants using dsDNASP and dsDNANS. Reactions were performed as in a. DDRF/AAAA represents the D104A/D105A/R106A/F107A mutant. Please see the left and right panels of a for WT controls of the experiments using dsDNASP and dsDNANS, respectively. c–d EMSA was used to test the DNA binding of Csy complex and Csy-AcrIF24 using dsDNASP (c) and dsDNANS (d). dsDNASP and dsDNANS were added as 1 μM, and the concentrations of Csy complex and Csy-AcrIF24 were set as 0.5, 1, 2, 4, 8, and 16 μM following the order indicated by the black triangle. e EMSA was used to test the binding competition between dsDNASP and dsDNANS on Csy or Csy-AcrIF24 complex. Reactions were started with incubation of 1 μM dsDNASP and 10 μM Csy or…

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  Fig. 6. Cryo-EM structure of Csy-AcrIF24-dsDNA complexes. a Atomic structure of Csy-AcrIF24-dsDNASP in cartoon representation with each subunit colored as in Fig. 3d. The bound dsDNASP molecules are shown in surface model colored in yellow. b Structural superimposition between Csy-AcrIF24-dsDNASP and Csy-AcrIF24. The structures of Csy-AcrIF24-dsDNASP and Csy-AcrIF24 are colored in orange and gray, respectively. The dsDNASP is shown in the surface model colored in yellow. c The densities corresponding to dsDNA molecules in the Csy-AcrIF24-dsDNASP structure are shown in yellow mesh. The Csy and AcrIF24 are colored as in Fig. 3d. d Close-up view of the binding site of dsDNASP near the PAM recognition loop in Cas8f in the Csy-AcrIF24-dsDNASP complex. The cryo-EM density maps for dsDNASP, K247 of Cas8f and U1 of the crRNA are shown in mesh. e Close-up view of the interfaces between AcrIF24 and the dsDNASP density within the Csy-AcrIF24-dsDNASP complex. The density corresponding to dsDNA is shown as yellow mesh and N194/R196/K197 is shown in sticks. f The densities corresponding to dsDNA molecules in the Csy-AcrIF24-dsDNANS structure are shown in the slate mesh. The Csy and AcrIF24 are colored…

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  Fig. 7. Structural elements involved in dsDNA binding by the Csy-AcrIF24 complex. a EMSA used to test the non-specific DNA binding ability of Csy-AcrIF24 with mutations in the PAM recognition loop of Cas8f using dsDNASP and dsDNANS. Reactions were performed as in Fig. 5c. Please see the right panels of Figs. 5c, d for WT controls of the experiments using dsDNASP and dsDNANS, respectively. b EMSA used to test the induction of non-specific DNA binding by AcrIF24 or its mutants using dsDNASP and dsDNANS. Reactions were performed as in Fig. 5a. c EMSA used to test the non-specific DNA binding ability of Csy-AcrIF24 with mutations both in AcrIF24 and Csy using dsDNASP and dsDNANS. Reactions were performed as in Fig. 5c. Please see the right panels of Figs. 5c, d for WT controls of the experiments using dsDNASP and dsDNANS, respectively. d Mutations impairing the induction of non-specific DNA binding of AcrIF24 decreased its capacity of inhibition in the in vitro cleavage activity assay. Reactions were performed as in Fig. 1a except that the concentrations of AcrIF24 or its mutants were set as 0.16 μM. Data are presented as mean values ± SD; n = 3. Two-sided t test was performed. The “*“…

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  Fig. 8. The Csy-AcrIF24 complex interacts with dsDNASP and dsDNANS through similar mechanisms. a Schematic illustration of the dsDNASP with different lengths is used in the present study. Red and green lines represent PAM and protospacer sequences, respectively. b–c EMSA was used to test the non-specific DNA binding of Csy-AcrIF24 or Csy-AcrIF9 using dsDNANS with 5′ FAM labels (b) and dsDNASP with 3′ FAM labels (c) of different lengths. Reactions were performed as in Fig. 5c. d–e EMSA was used to test the non-specific DNA binding of Csy-AcrIF24 mutants using dsDNANS with 5′ FAM labels (d) and dsDNASP with 3′ FAM labels (e) of different lengths. Reactions were performed as in Fig. 5c. Please see the right two panels of b for WT controls of the experiments using dsDNANS 35 bp and 45 bp in d, respectively.

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  Fig. 9. AcrIF24 and AcrIF9 show different characteristics in inducing non-specific DNA binding. a–b EMSA used to test the non-specific DNA binding affinity and mode of Csy-AcrIF24 and Csy-AcrIF9 using dsDNASP/dsDNANS (a), or ssDNASP/ssDNANS (b). Reactions were performed as in Fig. 5c. c EMSA used to test the binding ability of AcrIF24 and AcrIF9 using dsDNASP or dsDNANS. Reactions were performed as in Fig. 5c. d Structural superimposition of the Csy-AcrIF24-dsDNASP and Csy-AcrIF9-dsDNANS complexes. Only the Cas8f, Acr, and dsDNA regions are shown. K247 of Cas8f and the residues involved in non-specific DNA binding of AcrIF9 and AcrIF24 are shown in the sphere model. e EMSA was used to test the non-specific DNA binding of Csy-AcrIF9 with mutations in AcrIF9 or Csy using 3′ FAM-labeled dsDNASP or 5′ FAM-labeled dsDNANS. Reactions were performed as in Fig. 5c. Please see the left two panels of Fig. 8c for the WT control of the experiments using 3′ FAM dsDNASP 15 bp and 25 bp, respectively. Please see the left two panels of Fig. 8b for the WT control of the experiments using 5′ FAM dsDNASP 15 bp and 25 bp, respectively.