The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence

Abstract

Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.

Figure 1.

Genome context and domain organisation of Can2. (A) Domain organisation of Can1 and Can2. Each has an N-terminal CARF domain and a C-terminal PD-ExK superfamily nuclease domain. The can1 gene has been predicted to arise from a gene duplication of can2 (19) and includes Domain 2 – a divergent inactive nuclease domain. The can2 gene numbering is from S. thermosulfidooxidans. (B) Gene organisation of selected CRISPR type III systems that include a can2 gene. Can2 is found associated with both type III-A (Csm) and type III-B (Cmr) systems, and frequently with other CARF family effector proteins. A common neighbouring gene encodes the Crn2 ring nuclease for degradation of cA₄ (27). Genes labelled as ‘CARF’ encode uncharacterised CARF family proteins. Genes are coloured consistently across the different genomes. Species represented are Sulfobacillus thermosulfidooxidans (Sth), Thioalkalivibrio sufidiphilus (Tsu), Nitrococcus mobilis, Haemophilus haemolyticus and Methylomagnum ishizawai.

Figure 2.

Can2 is activated by cA₄ to degrade scDNA and RNA. (A) Agarose gel analysis of supercoiled plasmid (1.8 nM) nicking and degrading activity by SthCan2 and TsuCan2 (500 nM dimer). Supercoiled plasmid was degraded rapidly by SthCan2 in the presence of cA₄ (1 μM) and MnCl₂ (5 mM). Plasmid was incubated with wild-type SthCan2 at 50°C for 0.5, 1, 2 and 5 min in reaction buffer supplemented with cA₄ and MnCl₂. The nuclease variant E276A/D278A was incubated under the same conditions for 30 min. Wild-type TsuCan2 and its nuclease variant E302A/K304A were incubated under the same conditions for 30 min at 37 or 50°C. Standards corresponding to supercoiled (SC), linear and nicked plasmid are shown after the marker (M) lane. Control lanes C1, C2 and C3 show the reactions incubated for 30 min without protein, MnCl₂ and cA₄, respectively. (B) Single-turnover kinetic analysis of scDNA cleavage by SthCan2 and Can1 (the plot for Can1 is from our previous study (19)). SthCan2 (500 nM dimer) was incubated with scDNA (1.8 nM) under the same conditions as in part A and the reaction was stopped at 10 s, 20 s, 40 s, 1 min, 2 min and 3 min. The cleaved fraction of scDNA was plotted against time and fitted to a single exponential curve as described in Materials and Methods. The rate constant of scDNA cleavage for SthCan2 and Can1 are 3.39 ± 0.57 and 0.81 ± 0.15 min⁻¹, respectively. Values and error bars represent the mean of triplicate experiments and the standard deviation. (C) Plot of fluorescent signals emitted by RNaseAlert substrates when they were cleaved by wild-type SthCan2, TsuCan2 or VC1899. RNaseAlert substrates (30 nM) were incubated with the enzymes (500 nM dimer) in reaction buffer and supplemented with cA₄ (1 μM) and MnCl₂ (5 mM) at 37°C. The fluorescent signal was plotted against time. Values and error bars represent the mean of triplicate experiments and the standard deviation.

Figure 3.

Can2 protects against MGE in vivo. (A) Plasmid challenge assay of mycobacterial type III-A system in E. coli host. E. coli cells harbouring the mycobacterial type III-A interference complex Csm1-5 and TetR targeting spacer were transformed with plasmid containing TsuCan2 effector and tetracycline-resistance gene (Wild-type). Other strains are indicated as ‘No Can2’ where the TsuCan2 is absent, ‘Cyclase variant’ where the interference complex is unable to produce cOA and ‘No targeting crRNA’ where the TetR targeting spacer is replaced with a spacer targeting pUC19 MCS. A 10-fold dilution series of the transformation mixture was applied onto tetracycline selective plates to determine the number of viable transformants. (B) Growth curves of E. coli cells harbouring the interference complex and phage P1 lpa gene-targeting spacer supplemented with TsuCan2 effector (indicated as Wild-type). Cells were grown in LB broth in a 96-well plate with shaking at 37°C and infected with phage P1 at a MOI of ∼1. OD₅₉₅ of the culture was measured every 15 min to plot against time over the 16 h incubation. Other strains are indicated as ‘Cyclase variant’ (green) where the interference complex is unable to produce cOA molecules; ‘No Lpa target’ (black) where lpa gene-targeting spacer is replaced with a spacer targeting pUC19 (MCS); ‘Can2 E302A/K304A’ (grey) which is a TsuCan2 nuclease variant. ‘Control’ (purple) represents wild-type cells (orange) incubated without phage infection under the same conditions. Data points represent the mean of eight experimental replicates (four biological replicates with two technical replicates each) with the standard deviation shown. (C) The OD₅₉₅ values of all strains after 4 and 10 h growth are shown, coloured as in panel B. Statistical analysis was carried out with RStudio using the unpaired Welch two sample test to calculate P-values. NS (not significant) indicates P-values > 0.05 and *** indicates P-values < 1E–05.

Figure 4.

Structure of Can2 bound to cA₄ activator. (A) Two views of the Can2 dimer in cartoon representation. The CARF domain is shown in cyan and the nuclease domain in blue. A molecule of cA₄ is bound across the CARF dimer, which is shown as spheres (carbon in yellow, oxygen in red, nitrogen in blue, phosphate in magenta). (B) Surface representation of Can2 dimer with the same colouring as (A).

Figure 5.

Structural comparison of Can2 with Can1. (A) Superimposition of CARF domain dimer from Can2 (cyan) bound to cA₄ (yellow) with the CARF domains of Can1 bound to cA₄ (PDB: 6SCE) (pink). The Can2 CARF domain dimer superimposes with the Can1 CARF domains with an RMSD of 2.6 Å over 256 Cα atoms. (B) Superimposition of nuclease domain from Can2 (blue) and Can1 (pink) (RMSD of 3.2 Å over 139 Cα atoms). (C) Superimposition cA₄ molecules bound to Can2 (yellow) and Can1 (pink) shown in stick representation (carbon in yellow/pink, oxygen in red, nitrogen in blue, phosphate in magenta; RMSD 3.1 Å over 88 atoms). Each AMP is numbered (A1-A4).

Figure 6.

Structural comparison of cA₄ bound to Can2. Divergent stereo representation of cA₄ (in stick representation; carbon in yellow, nitrogen in blue, oxygen in red; phosphorus in orange) in complex with the Can2 CARF domain dimer (in stick representation; each monomer in the dimer is coloured green or magenta). Each AMP is numbered (A1–A4). The dotted lines represent hydrogen bond interactions. The residue labels in bold indicate there is a conserved interaction between cA₄ in both Can 1 and Can2.

Figure 7.

Predicted ternary complex of Can2 and cA₄, with dsDNA modelled. (A) Superimposition of the nuclease domain of Can2 (blue) with the nuclease domain of EndoMS in complex with dsDNA (PDB: 5GKE) in cartoon representation (green) (RMSD 4.4 Å over 108 Cα atoms). (B) Close-up view of panel A. The active site residues of Can2 (Glu276 and Asp278) are shown as blue sticks, and the active sites residues (Glu156 and D158A mutant) and metal ion (Mg²⁺) of EndoMS shown as green sticks and sphere respectively.