Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE)

Abstract

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

FIGURE 1.

Isolation and characterization of aCASCADE from S. solfataricus. A, colloidal Coomassie-stained SDS-PAGE gel showing the co-purification of Csa2 and Cas5a through streptactin (second lane), Ni-NTA (third lane), and size exclusion chromatography (fourth and fifth lanes) steps. MASCOT identification statistics are listed in supplemental Table S1. B, SYBR Gold-stained UREA-PAGE gel showing RNA co-purification with aCASCADE through all three purification steps. C, alignment of non-redundant cDNA sequences derived from aCASCADE-associated RNA. The co-purifying RNA is CRISPR derived and hails from each of the three S. solfataricus CRISPR types. The labels indicate the CRISPR locus from which the clone is derived. Several clones could not be definitively assigned. The underlined spacer (clone 10) appears to be derived from Sulfolobus icelandicus Rod-shaped virus.

FIGURE 2.

S. solfataricus Cas6 generates crRNA. A, a two-repeat spacer unit CRISPR transcript was cleaved by Cas6 at a single site in each repeat, yielding fragments of 109 and 43 nt for cleavage at repeat 1, 106 and 46 nt for cleavage at repeat 2, and the 63-nt mature crRNA for cleavage at both repeats. B, a synthetic RNA corresponding to a single CRISPR repeat with a 15-unit 5′ extension is cleaved by Cas6 at a single site, generating an 8-nt repeat-derived 5′ extension (“psi-tag”). C, schematic illustrating the 2-repeat transcript and the expected cleavage products. D, schematic illustrating the synthetic substrate.

FIGURE 3.

crRNA and target-DNA binding by a Csa2-Cas5a complex. A, recombinant Csa2 and Cas5a form a complex when co-expressed in E. coli. Coomassie-stained SDS-PAGE gel showing material purified using Ni-NTA affinity chromatography and gel-filtration chromatography (second lane) and further purified using a heparin column (third to 10th lanes). B, EMSA showing the binding of Csa2 alone (second to eighth lanes) and the Csa2-Cas5a complex (ninth to 15th lanes). The first lane shows crRNA alone. Protein concentrations for both Csa2 and Csa2-Cas5a complex were 0.25, 0.5, 1, 2, 3, 5, and 7 μm and the crRNA concentration was 100 nm. C, target-DNA binding by the recombinant complex. Increasing amounts of the Csa2-Cas5a complex were incubated with radiolabeled target single-stranded DNA in the absence of crRNA (lanes 1–5), in the presence of crRNA (lanes 6–10), and in the presence of crRNA and non-target DNA (lanes 11–15). D, TEM images of the helical filaments formed by aCASCADE purified from S. solfataricus. The black bars are 20 nm.

FIGURE 4.

(Embedded 3-D content) The structure of Csa2 reveals a novel domain architecture containing an RNA-recognition motif (chain A shown). A, stereo ribbon diagram of the Csa2 monomer. The RNA-recognition motif is colored violet, the 1–3 domain is red, the 2–4 domain is orange, and the C-terminal subdomain is colored yellow. B, surface representation of Csa2 rotated 90° about the vertical axis relative to A. Sequence motifs that are conserved among Cas7 orthologs are colored orange and residues that are strictly conserved among Csa2 proteins are cyan. The conserved residues cluster on the edge of the RRM and 1–3 domains. The approximate locations of the three disordered loops are indicated by dotted lines and the strictly conserved residues that are located in the disordered loops are indicated by cyan ovals. The two conserved residue clusters are indicated. C, electrostatic surface map of Csa2 calculated using APBS tools and shown in the same orientation as panel B. The color ramp of the surface is from −20 kT/e (red, acidic) to 20 kT/e (blue, basic). D, Csa2 β1 and β7 lack the conserved ssRNA-binding motif found on β1 and β3 of the canonical RRM. These two canonical RNA-binding motifs are aligned with the Csa2 residues in the structurally equivalent positions. The solvent-exposed side chains are indicated with black triangles. The aromatic residues that normally make base-specific contacts are highlighted in blue (60). E, EMSA demonstrating the reduced crRNA-binding activity of the Csa2 H160A variant. Embedded three-dimensional content requires the free Adobe Reader software, version 9 or later, and can be activated by clicking on any part of the figure. The model can be manipulated interactively using the mouse. Options for selecting, rotating, panning, and zooming are available in the toolbar or contextual menu. Parts of the model can be individually accessed and toggled on or off using the model tree. Preset views can be accessed using the dropdown “views” menu. Preset views include a schematic rendering of the Csa2 structure colored as in panel A, two views of a surface rendering of Csa2 with conserved surface features colored as in panel B, and a schematic rendering of Csa2 with conserved residues as shown in Fig. 5. To end three-dimensional viewing, right-click on the model and select “disable content”; for MAC users, Ctrl + click.

FIGURE 5.

The structural similarity between Csa2 and Cas6 is limited to the RNA-recognition motif. A, Csa2 and Cas6 are shown in equivalent orientations based on an SSM Structural alignment. The RRM-like subdomains are colored violet in both structures. The Csa2 1–3 domain is colored red, the 2–4 domain is colored orange, and the C-terminal subdomain is colored yellow. The portions of the Cas6 N-terminal domain that do not exhibit similarity to the Csa2 RRM subdomain are depicted in green and the C-terminal domain in light cyan. The conserved clusters on Csa2 and the putative active site residues on Cas6 are shown as “sticks” colored in dark cyan.

FIGURE 6.

Preliminary structural models for aCASCADE. A, TEM image of aCASCADE helical filaments. B, model of a Csa2 helical assembly with 10 Csa2 protomers per turn of helix. The Csa2 subunits are alternately colored dark and light gray. C, proposed model for aCASCADE. The structural core of the model is formed from 6–8 copies of Csa2(Cas7) in a partial turn of the Csa2 helix (colored as in B). At one end of the Csa2 oligomer is a single copy of Cas5a. The other end may utilize an additional aCASCADE component to cap the growth of the Csa2 oligomer. The crRNA is tentatively placed along the inner surface of the Csa2 helical assembly and is indicated by a dotted line with the repeat portions shown in red and spacer shown in black. D, the model in C is rotated 90° about the vertical axis. E, model for CRISPR-mediated viral defense in S. solfataricus. The CRISPR transcript is processed by Cas6 to generate crRNAs that are loaded into the aCASCADE complex (comprising Csa2, Cas5a, and potentially Csa5 and Csa6) to target viral DNA for degradation. The preferred PAM sequence CCN is shown 5′ of the protospacer. For RNA targeting, crRNAs may be further processed by an unknown 3′ to 5′ exonuclease activity, and loaded into the CMR complex for RNA-directed RNA cleavage.