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. 2022 Oct 7;298(11):102575. doi: 10.1016/j.jbc.2022.102575

Figure 5.

Figure 5

Interactions between AcrIF4 and the Cas8f HB are essential for the inhibition capacity of AcrIF4.A, detailed interactions between AcrIF4 and the Csy complex. The proteins are colored as in Figure 2A. B, mutations of the interface between the Cas8f HB and AcrIF4 do not affect the binding of AcrIF4 to the Csy complex. Reactions were performed with 6 μM Csy complex or its mutant and 180 μM AcrIF4 or its mutant for 30 min at 37 °C, and then the mixtures were incubated with Ni-NTA beads for 30 min at 4 °C. Samples of input and pull-down were separated using SDS-PAGE after washing three times. Csy M, Cas8f R299G/R302A; AcrIF4 M, AcrIF4 L39/F54G. C, EMSA to test the Cas2/3 recruitment by the Csy or its mutant (Cas8f R299G/R302A) in the presence of AcrIF4 or its mutant (AcrIF4 L39/F54G). Reactions were performed with 1.6 μM Csy complex, 0.1 μM 54-bp dsDNA bubble (5′-FAM in the target DNA strand), 0.8, 3.2, 12.8 μM μM AcrIF4, and concentrations of Cas2/3 were set as 0.8 μM following the order indicated by the black triangle. Reactions were repeated independently three times with similar results. Csy M, Cas8f R299G/R302A; AcrIF4 M, AcrIF4 L39/F54G. D, in vitro DNA cleavage reaction was performed with 0.4 μM Csy mutant, 0.2 μM Cas2/3, 6.4 μM AcrIF4 or its mutant, and 0.04 μM 54-bp dsDNA (5′-FAM in the nontarget DNA strand, NTS). The Csy complex and AcrIF4 were preincubated first, then dsDNA and Cas2/3 were added sequentially. EMSA, electrophoretic mobility shift assay; HB, helical bundle.