Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR-Cas that degrades the second messenger cA4

Abstract

Cas10 is the signature gene for type III CRISPR-Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn²⁺-dependent deadenylation exoribonuclease. However, its specific role in CRISPR-Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA₄) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA₄ signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR-Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA₄ signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.

Keywords: CARF; CRISPR/Cas; RNase; X-ray crystallography; cyclic nucleotide; cyclic tetra-adenylate; fluorescence; fluorescence lifetime; phosphodiesterase; phosphodiesterases.

Figure 1.

AfCsx3 binds cA₄ with high affinity. A, the fluorescence lifetime of AfCsx3 Trp⁵⁰ was used to follow cA₄ binding. The mean waveform at each cA₄ concentration was modeled as a linear combination of the cA₄-bound and free waveforms, yielding the fraction bound. Each point is the mean of three independent experiments, each with three technical replicates. Error bars represent the standard deviation of the mean. The data were then fit to the one-site single binding function in GraphPad Prism giving K_d = 55 ± 4.5 nm. Importantly, the indicated cA₄ concentrations along the abscissa represent total ligand concentration. Free ligand concentration is significantly less at the lower concentrations of cA₄ used in this assay. Thus, the apparent K_d represents only an upper limit and may well be an underestimate of the affinity. B, note that the ligand concentrations are μm rather than nm as in A. The fluorescence lifetime binding assay shows significantly lower affinity for the small linear RNA with a poly(A) tail, giving a K_d of 1.9 ± 0.2 μm.

Figure 2.

The AfCsx3 fold. Previous structures of Csx3 reported a six-stranded mixed β-sheet with two α-helices. The high-resolution crystal structure described here revealed an ordered C terminus with a third α-helix (α3, dark blue) followed by an additional β-strand (β7, violet). This C-terminal extension crosses the outside, solvent-exposed surface of the β-sheet, allowing β7 to add antiparallel to β1. The fold is thus a mixed seven-stranded β-sheet with β7↑-β1↓-β2↑-α1-β3↑-α2-β4↑-β5↓-β6↑-α3 topology with the α-helices providing right-handed crossovers between the indicated β-strands. In a dimer, α1 and α2 are buried at the subunit interface, whereas α3 is exposed on the surface. Conserved motifs GRXPXW, LXHXXH, and DPR are found in the β3-α2, α2-β4, and β4-β5 loops. Note that the LXHXXH RNase active-site motif lies at the bottom of the protomer, whereas the GRXPXW and DPR motifs involved in cA₄ recognition are at the top.

Figure 3.

The dynamic C-terminal tail. A, the Csx3 dimer packs in sheets or planes within the crystal. B, the interdimer crystal contacts within a plane are anchored primarily by a parallel β-strand hydrogen bond interaction between β₇ (violet) of one subunit and β₆ (light blue) of the neighboring dimer. The ordered β7-strand of the C-terminal tail thus serves to crosslink dimers within the crystal as it “inserts” between β1 (green) of one subunit and β6 of the neighbor. This interaction leads to the formation of continuous extended β-sheets that run the length of the crystal. Visual observations and PISA interface analysis software indicate that these interactions are not substantial enough to support the formation of a stable protein interface in solution. This open symmetry is thus a likely artifact of crystallization conditions and is not indicative of interactions in vivo. That said, the dynamic C-terminal tail clearly has the ability to form interprotein interactions that might mediate protein–protein interactions within the cell.

Figure 4.

AfCsx3 ring nuclease activity. A, TLC analysis showing a representative time course for the degradation of unlabeled cA₄ (150 μm) incubated with Af Csx3 (10 μm dimer) at 50 °C in the presence of 200 μm Mn²⁺. Lanes 1–9 are time points at 0, 0.5, 1, 3, 5 10, 20, 30, and 60 min. Lane 10 is a 60-min incubation in the presence of Mn²⁺ in the absence of Csx3. Lane 11 was loaded with 1.5 nmol of cA₄ standard. B, data from five independent cA₄ ring nuclease activity replicates were averaged, and the data fit to a first order exponential decay model yielding an apparent first order rate constant, k_obs = 4.95/min. The standard deviations at each time point are represented by error bars.

Figure 5.

Structural definition of the CARF domain core. Superposition of the β3-α2-β4-β5 sequence of Csx3 (A) onto the β4-α4-β5-β6 sequence of S. solfataricus Csa3 CARF domain (B) (root-mean-square deviation, 2.9 Å) reveals core secondary structures shared by the two proteins (green). Csa3 and Csx3 each contain this contiguous βαββ super secondary structures in which the first two β-strands are connected by a right-handed helical crossover followed by a reverse turn into the third β-strand. The core helix in this motif is an essential part of the dimer interface; it packs against the two β-strands connected by the reverse turn: β₄β₅ in Csx3 (left) and β₅β₆ in Csa3 (right). In the classic dinucleotide-binding domain, these β-strands are instead connected by a right-handed helical crossover, in which the connecting helix occupies the same space as the helix from the adjacent Csa3 or Csx3 subunit. The reverse turn connecting these β-strands is thus a critical structural feature of the CARF domain dimer interface. In addition, Csx3 places β2 and α1 in positions equivalent to the discontinuous β1 and α3 secondary structural elements of Csa3 (gray).

Figure 6.

RNA density in the 3WZI Csx3 structure accommodates cA₄. A, stereo image. An omit map was generated using the 3WZI model with RNA atoms deleted and contoured at 1.5 σ (green mesh). The omit map was then used to inform modeling the cA₄–Csx3 interaction. Csx3, oriented as in Fig. 5A, is represented as a surface model with nitrogen atoms in blue, oxygen atoms in red, and carbon atoms colored gray, and cA₄ is represented in sticks with carbon atoms colored yellow. B, Csx3 secondary structure elements shown in cartoon representation with select side chains shown as sticks. cA₄ is also shown in stick representation with carbons in beige.