Insights into the dual functions of AcrIF14 during the inhibition of type I-F CRISPR–Cas surveillance complex

Figures

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  Figure 1.. The crystal structure of AcrIF14. (A) AcrIF14 inhibits the in vitro DNA cleavage activity of the type I-F CRISPR system. Reactions were performed with 1 μM Csy complex, 0.4 μM Cas2/3 and 50 nM 54-bp dsDNA (5′-FAM in the non-target strand, NTS). AcrIF14 was added with concentrations of 0.5, 1 and 2 μM, respectively. The experiment was repeated three times with similar results. (B) The crystal structure of AcrIF14 shown in cartoon model. The NTD and CTD are shown in cyan and green, respectively. Two perpendicular views are shown with secondary structures labeled. (C) Sequence alignment of AcrIF14 homologs. Residues with 100% identity, over 75% identity, over 50% and over 33% identity are shaded in dark blue, pink, cyan and yellow, respectively. Secondary structural elements of AcrIF14 are shown above the sequences, colored as in Figure 1B. AcrIF14 residues highlighted in Figures 3B–D and 6A are marked with triangles and asterisks, respectively.

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  Figure 2.. The cryo-EM structure of the Csy–AcrIF14 complex. (A) Cryo-EM map of Csy–AcrIF14 with each subunit colored respectively. Two copies of AcrIF14 are colored in cyan and green, for its NTD and CTD, respectively, as in Figure 1B. (B) The structure of Csy–AcrIF14 in cartoon representation with each subunit colored as in (A). (C) Structural superimposition among the Csy-AcrIF1/9/14 structures. Only the Csy within the Csy–AcrIF14 complex is shown in surface model and colored as in (B). AcrIF1 and AcrIF9 are shown in cartoon model and colored wheat and light blue, respectively. AcrIF14 is shown and colored as in Figure 1B. (D) The interactions between AcrIF14.1 and the Csy complex. AcrIF14.1 is colored as in Figure 1B and shown in surface model. (E) The interactions between target DNA and the Csy complex in the Csy-dsDNA structure (PDB code: 6NE0).

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  Figure 3.. Detailed interactions between AcrIF14 and the Csy complex. (A) Overview of the binding between AcrIF14.1 and the Csy complex. The three interfaces are marked in circles. (B–D) Close-up views of the interfaces marked in circles in (A), with B, C, and D representing interface 1, 2 and 3, respectively. Interacting residues are shown in sticks, and polar interactions are shown as red dashed lines. (E) Ni-pull-down assay of the AcrIF14 mutants to disrupt the binding with the Csy complex. 8 μM His-tagged Csy complex was incubated with 40 μM AcrIF14 or its mutants, analyzed through SDS-PAGE stained with CBS. The experiment was repeated three times with similar results. (F) The interaction mutations of AcrIF14 decreased its inhibition capacity in the in vitro DNA cleavage activity. Reactions were performed with 1 μM Csy complex, 0.05 μM 54-bp dsDNA (5′-FAM in the NTS) and 0.4 μM Cas2/3. AcrIF14 or its mutants were added with concentrations of 1, 1.5 and 2 μM, respectively, when needed. The experiment was repeated three times with similar results.

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  Figure 4.. AcrIF14 induces strong non-specific DNA binding activity in the Csy complex. (A) EMSA used to test the effects of AcrIF14 on the binding between Csy and dsDNASP. Reactions were performed with 4 μM Csy complex, 0.25 μM 54-bp dsDNA (5′ -FAM in the target DNA strand, TS), and AcrIF14 concentrations of 4, 8, 16 and 32 μM following the order indicated by the black triangle. (B, C) EMSA used to test the dsDNASP/dsDNANS binding of the Csy and Csy–AcrIF14 complex. Increasing concentrations of Csy or Csy–AcrIF14 complex (0.25/0.5/1/2/4/8 μM) were added to 0.25 μM 54-bp dsDNASP (5′-FAM in the TS) or dsDNANS (5′-FAM in the non-specific DNA strand). (D, E) Competition EMSA to test the binding sites of dsDNASP and dsDNANS. (D) 4 μM Csy or Csy–AcrIF14 complex was incubated with 0.25 μM 54-bp dsDNASP (5′ -FAM in the TS), then increasing concentrations of unlabeled dsDNANS (0.25/0.5/1/2 /4/8 μM) were added. (E) The procedure was the same as (D), except that dsDNANS was 5′-FAM labeled and incubated first, and unlabeled dsDNASP was added. (F) The interaction mutants of AcrIF14 disrupt its ability to induce non-sequence-specific DNA binding. Reactions were performed in the same system as (A),…

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  Figure 5.. The PAM recognition loop and RBC of Cas8f are involved in non-specific DNA binding. (A) Model of the Csy–AcrIF14–dsDNASP complex. Csy–AcrIF14 is shown in cartoon representation and the density of the dsDNASP is shown. (B) Close-up view of the binding site of dsDNASP within the Csy–AcrIF14–dsDNASP complex. Hydrogen bonds are shown as red dashed lines. A schematic drawing of the partial dsDNA with visible density is shown in the left bottom. (C) EMSA used to test the effects of N250D and K247E mutations of the Cas8f subunit on the non-specific DNA binding activity induced by AcrIF14 or AcrIF9. Reactions were performed in the same system as Figure 4B, C. (D, E) EMSA used to test the effects of R207E/R219E/R258E mutations of the Csy complex in the non-specific DNA binding activity induced by AcrIF14 or AcrIF9. Reactions were performed in the same system as Figure 4B, C. The experiments in C–E were repeated three times with similar results.

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  Figure 6.. Non-specific DNA binding induced by AcrIF14 is related to its inhibitory capacity. (A) AcrIF14 is shown in electrostatic surface model. Four patches of residues with positive charges are marked with circles. (B) Mutations of the electropositive patches on AcrIF14NTD disrupt the non-specific DNA binding activity. Reactions were performed in the same system as Figure 4A. The experiment was repeated three times with similar results. (C) and (E) Mutations of the electropositive patches on AcrIF14NTD decreased its inhibition capacity in the in vitro DNA cleavage activity. Reactions were performed with 1 μM Csy complex, 0.05 μM 54-bp dsDNASP (5′ -FAM in the NTS), 0.4 μM Cas2/3 and 1 μM AcrIF14 or its mutants (D). 1 μM 54-bp dsDNANS was additionally included in the system in (E). The experiments were repeated three times with similar results and representative results are shown. (D and F) Quantitative results of the experiment in (C) and (E), respectively. Error bars represent SD; n = 3. Paired t test was performed (*P < 0.05).

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  Figure 7.. AcrIF14 and AcrIF9 do not induce non-specific DNA binding by the same mechanism. (A) EMSA used to test the ssDNASP/ssDNANS binding to the Csy–AcrIF14 and Csy–AcrIF9 complex. Increasing concentrations of Csy–AcrIF14 or Csy–AcrIF9 complex (0.25/0.5/1/2/4/8 μM) were added to 0.25 μM ssDNASP (5′-FAM in the TS) or ssDNANS (5′-FAM in the non-specific DNA strand). (B) Competition EMSA to test the ssDNA binding of Csy–AcrIF9. Reactions were performed in the same system as Figure 4D, except that ssDNASP and ssDNANS but not dsDNASP and dsDNANS were added. (C, D) Competition EMSA to test the binding sites of ssDNASP and ssDNANS by Csy–AcrIF14. Reactions were performed in the same system as Figure 4D and E, except that ssDNASP and ssDNANS but not dsDNASP and dsDNANS were added. All the above experiments were repeated three times with similar results.