Supplementary Figure 5: Gel-filtration chromatography of the AcrF3 dimer, the nuclease activity of PaCas3 and the effects of AcrF3 mutations.
From: Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3
(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.
