Figure 4. Anti-CRISPR protein AcrF1 binds to residues on Cas7f that are essential for crRNA-guided recognition of target DNA.
(A) Cartoon showing the location of three lysine residues (blue circles containing the letter K) on two adjacent Cas7f proteins that form a binding site for AcrF1. (B) Schematic of the Csy complex and the Acr proteins illustrating interactions between Acr and Cas proteins. The diameter of lines connecting subunits scales with buried surface. (C) Structure of two adjacent Cas7f proteins shown as gray (Cas7.6f) and cyan (Cas7.5f) surfaces. The three lysine residues (K85, K254, and K257) highlighted in panel A are shown in blue. AcrF1 is shown in dark red. Above is an expanded view of AcrF1.2 interacting with K85 on the thumb of Cas7.6f. Left is an expanded view of the acidic α2 from AcrF1.2 nestled against the positively charged residues on the extended web domains of Cas7.6f and Cas7.5f, respectively. Side chains involved in specific interactions are labeled. The disordered loop containing K257 is shown as a dotted C-α trace. (D) Surface plasmon resonance performed with wild type and mutant Csy complexes. Mutations in Cas7f (i.e. K85A or K254A/K257A) perturb AcrF1 binding. (E) AcrF1.1 binds to the thumb of Cas7.4f and AcrF1.2 binds to the thumb of Cas7.6f. In these two subunits (gray), but not the other four Cas7f proteins (cyan), the extended web is folded over the crRNA, restricting access to the guide. (F) Electrophoretic mobility shift assays performed with radiolabeled dsDNA substrates show that Cas7f K85A and K254A/K257A mutations perturb crRNA-guided DNA binding. Error bars, s.d.; n = 3.