Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM
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Structure solved at 3.42 Å resolution showing AcrF8 bound to Csy complex
"we obtained the structures of Csy complex bound with its inhibitors: AcrF9, AcrF8, or AcrF6, with the overall resolution of 2.57 Å, 3.42 Å, or 3.15 Å, respectively"
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AcrF8 binds to the Cas7f spiral backbone of the surveillance complex
"AcrF8 is a 92-residue protein, with a single copy occupying the cavity surrounded by Cas5f, Cas7.4–7.6f, and Cas8f"
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Makes direct contacts with the crRNA scaffold within the complex
"The distance between the residues T29, I31, A32, N33 of AcrF8 and the nucleobases of three nucleotides (U[+21], U[+22], G[+23]) of crRNA is less than 4 Å, which is close enough to form multiple hydrogen bonds and nonbonded interactions"
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Mutations at I31A and A32G abolish inhibitory activity
"we recombinantly expressed the AcrF8 containing a mutation at position 31 (I31A) or position 32 (A32G), which leads to an increase in the cleavage of DNA substrate compared with the WT AcrF8"
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Prevents DNA hybridization through steric occlusion
"the above three nucleotides are located just after the kink caused by the thumb of Cas7.5f, whose interactions with AcrF8 thereby form a continuous 4-nt region that would interfere with the DNA-crRNA hybridization"