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Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3

Abstract

Bacteriophages express proteins that inactivate the CRISPR–Cas bacterial immune system. Here we report the crystal structure of the anti-CRISPR protein AcrF3 in complex with Pseudomonas aeruginosa Cas3 (PaCas3). AcrF3 forms a homodimer that locks PaCas3 in an ADP-bound form, blocks the entrance of the DNA-binding tunnel in the helicase domain, and masks the linker region and C-terminal domain of PaCas3, thereby preventing recruitment by Cascade and inhibiting the type I–F CRISPR–Cas system.

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Figure 1: Overall structure of the PaCas3–AcrF3 complex.
Figure 2: Structure of AcrF3 and its interface with PaCas3.

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Acknowledgements

We thank the staff at the BL17U1 and BL18U1 beamlines of Shanghai Synchrotron Radiation Facility (SSRF) for diffraction data collection. This work was supported by grants from the National Natural Science Foundation of China (81322024, 81561130162, 31370722, and 81530068 to Y. Zhu and 81501717 to Y. Zhou), the Natural Science Foundation of Zhejiang Province (LR13C05001 to Y. Zhu), and the Royal Society (NA140239 to Y. Zhu).

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Contributions

X.W. performed the biochemical assays, purification, and crystallization. X.W., D.Y., and Y. Zhu collected the diffraction data and determined the structure. J.-G.X., A.-R.L., J.X., and P.F. prepared reagents. Y. Zhu and Y. Zhou designed the research and wrote the manuscript.

Corresponding authors

Correspondence to Yan Zhou or Yongqun Zhu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Structure of the Cas2 domain of PaCas3.

(a) Cartoon representation of the Cas2 domain of PaCas3.

(b) Structural superposition of the Cas2 domain of PaCas3 with Cas2 from the E. coli K12 strain (PDB ID: 4P6I)18.

(c) Structural superposition of the PaCas3-AcrF3 complex with the Cas1-Cas2 complex (PDB ID: 4P6I)18 (left) and its schematic representation18.

(d) Structural superposition of the PaCas3-AcrF3 complex with the Cas1-Cas2-DNA complex (PDB ID: 5DQZ)6 (left) and its schematic representation.

18. Nuñez, J.K. et al. Nat. Struct. Mol. Biol. 21, 528–534 (2014)

Supplementary Figure 2 Structural comparison of PaCas3 with Cas3 from T. fusca (TfCas3).

All structures are shown as cartoons with cylindrical helices. The HD domain, RecA1, RecA2, the Linker and the CTD are colored in cyan, grey, yellow, blue and red, respectively, for both TfCas3 and PaCas3. The structures of PaCas3 (yellow) and TfCas3 (blue) are superimposed in the right panel.

Supplementary Figure 3 Structure of the HD nuclease domain of PaCas3.

(a) Structure of the HD nuclease domain of PaCas3. Two calcium ions are shown as yellow spheres. The long loops L4, L5 and L6, which are bound to AcrF3, are labeled as indicated.

(b) The catalytic center of HD. Two calcium ions and one water molecule are shown as yellow and red spheres, respectively. The calcium-binding residues are shown as sticks and labeled as indicated.

(c) Structural comparison of the HD of PaCas3 with the HD nuclease domain of Cas3 from Thermobifida fusca (TfCas3). TfCas3 is shown in red. The protruding helix α6 with the long linking loops L5 and L6 are labeled as indicated.

Supplementary Figure 4 ADP bound in the SF2 helicase domain of PaCas3.

(a) ADP is bound in the cleft between the RecA1 and RecA2 domains. ADP and the ADP-binding residues are shown as sticks. The ADP-binding residues from RecA1 are yellow, while the binding residues from RecA2 are grey.

(b) The 2Fo-Fc omit electron density map of ADP contoured at 1.0 σ. ADP is shown as sticks.

Supplementary Figure 5 Gel-filtration chromatography of the AcrF3 dimer, the nuclease activity of PaCas3 and the effects of AcrF3 mutations.

(a) AcrF3 forms a dimer. Size-exclusion chromatography of AcrF3 and Rac1 (residues 1-178) was performed with a HiLoad Superdex 75 16/60 column (GE Healthcare). Rac1 (residues 1-178) has a molecular weight of 19.8 kDa. Full-length AcrF3 (139 amino acid residues) with a 6xHis tag at the N-terminus has a molecular weight of 17.4 kDa. AcrF3 eluted at approximately 64 ml, suggesting that AcrF3 is a dimer weighing approximately 35 kDa.

(b) PaCas3 exhibits a metal ion-dependent nuclease activity. PaCas3 shows nuclease activity towards M13mp18 ssDNA in the presence of 2 mM MnCl2, FeCl3, NiCl2, CaCl2 or MgCl2, and the highest activity was found in the presence of MnCl2 in vitro.

(c) The effects of mutations of the interacting residues in AcrF3 on the inhibition of PaCas3 nuclease activity. Val14 is a residue at the dimerization interface of AcrF3. Trp93, Tyr97 and Asn101 interact with the CTD of PaCas3. Glu62, His65 and Tyr68 interact with RecA2 of PaCas3. Arg58, Arg84 and the six C-terminal residues of AcrF3 are involved in the interactions with the HD and the Linker region. ΔC6 indicates the deletion mutation of the six C-terminal residues of AcrF3.

(d) The effects of mutations of the interacting residues in AcrF3 on the PaCas3-AcrF3 interactions. GST pull-down assays were performed to test the interactions between AcrF3 mutants and PaCas3. The V14D mutation and the triple mutation W93A/Y97A/N101A of AcrF3 impaired the PaCas3-AcrF3 interaction. The E62A/H65A/Y68A and R58A/R84A/ΔC6 mutations slightly affected the PaCas3-AcrF3 interaction.

Supplementary Figure 6 Schematic representation of the detailed interactions of residues of AcrF3 with those of PaCas3.

(a-b) Detailed interactions of AcrF3a (a) and AcrF3b (b) with PaCas3. The interacting residues in AcrF3a and AcrF3b are highlighted in purple and green, respectively. The interacting residues in PaCas3 are highlighted in different colors according to the domains in which the residues are located.

Supplementary Figure 7 Structural comparison of the PaCas3–AcrF3 complex with the TfCas3–DNA complex.

The HD domain, RecA1, RecA2, the Linker region and CTD are colored in cyan, grey, yellow, blue and red, respectively, for both TfCas3 and PaCas3. AcrF3 is represented as a surface and colored as in Figure 1b. The ssDNA in the TfCas3-DNA complex is shown in cartoon. The entrance of the DNA-binding tunnel formed by RecA2 and the CTD in TfCas3 are labeled as indicated (left panel). PaCas3 contains a similar DNA-binding tunnel (middle panel). AcrF3 binding blocks the DNA-binding tunnel in PaCas3 (right panel).

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Note (PDF 2534 kb)

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Wang, X., Yao, D., Xu, JG. et al. Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3. Nat Struct Mol Biol 23, 868–870 (2016). https://doi.org/10.1038/nsmb.3269

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