A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding

Abstract

Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α-RNP) was purified from the native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α-RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme-substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.

Figure 1.

Cmr-α-mediated ATP conversion and its regulation. (A) Cmr-α converts ATP to a dominant product upon binding to target RNA. The Cmr-α complex (40 nM) was incubated with 1 nM α³²P-ATP and 100 μM ATP in the presence of different RNA oligos (80 nM) and then the samples were analyzed with denaturing gel electrophoresis. The interactions of the Cmr-α complex with different RNA oligos are depicted above the gel. The 5′-handle of crRNA, the spacer sequence of crRNA and the supplemented RNA oligos are shown in red, black and blue, respectively. (B) Pre-incubation of Cmr-α and target RNA abolishes the ATP reaction. The Cmr-α complex (40 nM) and SS1-46 RNA (40 nM) were pre-incubated until 1 nM α³²P-ATP and 100 μM ATP were supplemented into the reaction mixture at indicated delay time points. Then, the samples were further incubated for 20 min, followed by analysis of denaturing gel electrophoresis.

Figure 2.

Identification of the Cmr-α-mediated ATP reaction products. (A) Liquid chromatography (upper panel) and mass spectrometry (two lower panels) analysis of the ATP reaction products by Cmr-α; *: the peak showing a MW of 657.10 in the MS analysis was also present in the reference sample; **: peak 3 with a MW of 1315.22 could be the tail of the main peak. (B) The ATP reaction product (∼40 nM) was treated with 1 U/μl PNK, 0.05 U/μl poly(A) polymerase (PAP), 0.1 U/μl alkaline phosphatase (FastAP) and 0.2 U/μl Nuclease S1, respectively, followed by analysis of denaturing gel electrophoresis. The reactions with PNK, PAP and FastAP were incubated at 37°C for 60 min and the S1 nuclease incubation time was indicated above the gel. If applicable, 1 mM ATP was supplemented into the reaction mixture. (C) The fractions of the peaks from Figure 2A (1: peak 1; 2: peak 2) were labeled with γ³²P-ATP by PNK. The ATP reaction product generated with α³²P-ATP was also loaded as size marker (the first lane).

Figure 3.

c-A4 activates the RNA cleavage activity of SisCsx1 by binding to its CARF domain. (A) Activation of SisCsx1 RNA cleavage by c-A4 and a linear poly(A) oligo (CAAAA). The non-cleaved RNA substrate was quantified after incubation with SiCsx1 in the presence of increasing concentrations of c-A4 or CAAAA. Error bar represents S.D. of three independent experiments. The EC₅₀ value determined for c-A4 and CAAAA is 7.4 and 27.3 nM, respectively. (B) Comparison of the binding affinity of SisCsx1 to c-A4 and CAAAA. The two ligands were incubation with 0.16, 0.5, 1.5 and 4.5 μM SiCsx1 and analyzed by non-denaturing PAGE. The percentage of bound ligand was calculated. Error bar represents S.D. of three independent experiments. (C) Schematic of SisCsx1 domain structure. Conserved amino acid residues subjected to mutagenesis are indicated, including one HEPN domain mutant (M1: R399A-H404A) and three CARF domain mutants (M2: G95L-A97L-A99L, M3: D50L-S51L and M6: H154L). (D) RNA substrate was incubated with the wild-type SisCsx1 or each mutant in the presence of 100 nM c-A4, followed by analysis of denaturing PAGE. (E) Labeled c-A4 was incubated with the wild-type and mutated SisCsx1 proteins, followed by analysis of non-denaturing PAGE.

Figure 4.

Michaelis–Menten modeling of the ATP reaction by Cmr-α–RNP. (A) Four nanomolar Cmr–α was incubated with increasing concentrations of ATP and the ATP incorporation rate (V) was plotted to the substrate concentration [S] (blue line). Error bar represents S.D. of three independent experiments. The V versus [S] relationship was further fitted into the Michaelis–Menten model (red line). F-test shows the P value <0.001 for the simulation. (B) The curves from (A) are shown in a logarithmic scale.

Figure 5.

Cmr-α-mediated ATP incorporation exhibits cooperative substrate binding. Cmr-α of 80 nM (A) or 20 nM (B) was incubated with different concentration ranges of ATP for which ATP incorporation rates (V) were determined and plotted against substrate concentrations [S] (blue line). Error bar represents S.D. of three independent experiments. (C) The logV versus log[S] relationship for the 80 nM Cmr-α experiements (blue dots) and 20 nM Cmr-α experiments (red dots) was fitted into a linear model: 80 nM Cmr-α (blue dashed line) and 20 nM Cmr-α (red dashed line). F-test indicates the P value is <0.001 for both simulations.

Figure 6.

Cmr-α exhibits a weak and divalent metal ion-dependent ATP binding activity. (A) About 1 nM γ³²P-ATP was mixed with 200 nM Cmr-α in the presence of 1 mM EDTA or 5 mM MgCl₂ and incubated at 70°C for 10 min. One set of these samples was supplemented with 300 nM SS1-46 and further incubated for 3 min at 70°C. All samples were analyzed by non-denaturing PAGE. (B) About 1 nM γ³²P-ATP was incubated with increasing concentrations of Cmr-α (50, 100, 200 and 250 nM) in the presence of 5 mM MgCl₂ and analyzed by non-denaturing PAGE.

Figure 7.

Both Palm1 and Palm2 domains of Cmr2α function in c-A4 generation. (A) Schematic of Cmr–2α domain structure. The conserved amino acids in HD, Palm1 and Palm2 domains (subjected to mutagenesis) are indicated. (B) Relative cOA synthesis activity of the wild-type Cmr-α–RNP (WT) and its mutant derivatives. (C) ATP binding affinity of the wild-type Cmr-α–RNP (WT) and its mutant derivatives. Cmr-α^HD, Cmr-α^P1 and Cmr-α^P2: effector complexes carrying substitution mutations in the HD, Palm 1 or Palm 2 domain of Cmr2α. The amount of synthesized cOA/bound ATP by the wild-type Cmr-α complex was arbitrarily set to 1.

Figure 8.

A model for Cmr-α-mediated c-A4 synthesis. Cmr-α is represented with Palm1 (P1), Palm2 (P2) and D2 (D2) domains of the Cmr–2α, three of the five conserved domains identified in the P. furiosus Cmr2 (57). Binding of the cognate target RNA to Cmr-α yields a ternary Cmr-α complex (A). Upon the binding of the first ATP molecule to the ternary Cmr-α, the substrate-enzyme intermediate adopts a conformational change and becomes more accessible to a second ATP molecule (B and C). Nucleophilic attack from the 3′-hydroxyl group (3′-OH) of the first ATP molecule to the 5′-triphosphate group (5′-P) of the second ATP molecule yields a phosphodiester bond between the two nucleotides (D). Cmr-α–RNP translocates on the 2-nt intermediate, freeing one of the ATP-binding sites (E). The process is repeated, leading to the formation of the third and the fourth phosphodiester bond (E and F). Finally, the substrate-free active site in Cmr-α recaptures the first nucleotide of the poly-A4 RNA (F and G) and circularizes the tetraadenylate in a condensation reaction.