Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases

Abstract

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.

Figure 1.

Structural, biophysical and cleavage activity characterization of the ring nuclease Sis0811. (A) Domain organization of full-length ring nuclease Sis0811. Dash lines represent the sequence fragment removed to obtain the truncated construct Sis0811_Δ268. (B) Cartoon models (each domain is coloured differently) of the dimeric apo Sis0811_Δ268 crystal structure rotated 90 degrees between them along its 2-fold axis. (C) Sis0811 and Sis0811_Δ268 substrate binding assays by ITC. Affinities and thermodynamic values of Sis0811, Sis0811_Δ268 binding events to cA₄ inferred from ITC measurements performed at 25°C. Gibbs free energy (ΔG), enthalpy (ΔH), entropy (–TΔS), equilibrium dissociation constant (K_D) are shown. The protein-cyclic oligonucleotide interaction affinity is defined by the Gibbs energy for binding ΔG = –RT ln K_A = RT ln K_D. The errors are the standard deviation of three independent experiments. (D) Sis0811 and Sis0811_Δ268 substrate cleavage assays analysed by TLC. The panel represents the percentage of the final reaction product (2 ApA>p) generated by SisRN0811 and Sis0811_Δ268. (E) TLC cleavage assay experiment. Arrow reflects the sample migration direction.

Figure 2.

Structural analysis of the ring nuclease SiRe-0811 in complex with its post-catalytic reaction product. (A) Cartoon models of the dimeric Sis0811_Δ268 crystal structure in complex with 2 ApA>p (post-catalytic reaction product from cA₄ substrate).(B) 2F_o – F_c map at the substrate binding pocket superimposed onto its corresponding refined structure. Map displayed at 2.0σ contour value. (C) F_o – F_c omit map at the substrate binding pocket superimposed onto its corresponding 2 ApA>p post-catalytic structure. FoFc omit map is displayed at 4.0σ contour value. (D) LC–MS analysis of SiRe-0811 reaction products. Upper panel: extracted ion chromatograms for m/z 657 (±0.5) (violet trace; ApA>p^–1; retention time 5 min) and m/z 675 (±0.5) (orange trace; ApAp^-1; retention time 6.4 min). Lower panel: extracted ion chromatogram for m/z 1315 (±0.5) (cyan trace; cA₄^-1; retention time 6.8 min). * Symbol means that the hydrolysis of P1 at the cyclic phosphate could generate a P2 product having the phosphate group either at 2′ or 3′.

Figure 3.

Conformational change of the ring nuclease Sis0811 from its apo form to its state in complex with its product reaction. (A) Cartoon/surface models comparing the cA₄ non-bound (upper panels) and its product complex forms (bottom panels). Coloured arrows represent the conformational change path of each corresponding domain from apo structure towards the product reaction bound form. (B) Zoom view at the substrate binding pocket within the apo dimer Sis0811_Δ268 and (C) in complex with its post-catalytic reaction product, depicting their key interacting residues. Each monomer is coloured differently and key side chains residues from each monomer interacting with the 2 ApA>p product (white sticks) are depicted accordingly in sticks.

Figure 4.

Sis0811 reaction product analysis from the designed mutants. (A) 2F_o – F_c map at the substrate binding pocket of the Sis0811_Δ268 (S12A) and Sis0811_Δ268 (S12G/K169G) mutants. (B) LC–MS analysis of the reaction products obtained from Sis0811_Δ268 (S12G), Sis0811_Δ268 (K169G) and Sis0811_Δ268 (S12G/K169G) mutants compared to the wild type. Ion chromatograms extracted for m/z 657 (±0.5) (ApA>p^–1, cA₄^–2, ApApApA>p^–2; retention times 5, 6.8 and 7.1 min), m/z 675 (±0.5) (ApAp ^-1; retention time 6.4 min), m/z 1315 (±0.5) (cA₄^–1, ApApApA>p^–1; retention times 6.8 and 7.1 min) and m/z 1333 (±0.5) (ApApApAp^–1; retention time 7.3 min). Chemical formula of the reaction species is depicted. The asterisk symbol (*) means that the hydrolysis of P1/P3 at the cyclic phosphate could generate a P2/P4 product having the phosphate group either at 2′ or 3′.

Figure 5.

Sis0811:2ApA>p–TonCsm6:2ApA>p complex comparison. (A) Top view of the CARF structural alignment from Sis0811:2ApA>p (orange) and TonCsm6:2ApA>p complexes (grey). (B) Zoom view of the key residues involved in catalysis of both complexes. (C) Electrostatic potential representation of the active site cavity in Sis0811:2ApA>p and (D) TonCsm6:2ApA>p post-catalytic complexes.

Figure 6.

Model of Sis0811 catalytic mechanism. The S12 position the 2′-OH of a ribose to initate the nucleophilic atack on the corresponding phosphate and subsequently K169′ stabilize the pentavalent phosphorous formed in the transition state. Then, the 2′-3′cyclic phosphate is stabilized by the S12 OH group, producing the intermediate reaction product P3 (ApApApA>p). Next, the other cleavable phosphodiester bond is attacked by the 2′-OH of the ribose which is close to S12′ that positions it. Thus, K169 stabilize the pentacovalent phosphorous formed in the transition state and the cyclic 2′,3′-cyclic phosphate is stabilised by S12′ OH group generating two molecules of P1 (ApA>p) as final reaction product.