Biochemical and structural characterisation of the E coli CASCADE

As described in chapter 1, a large multimeric complex composed of Cas type

I-E proteins Cse1-5e (also known as CasA-CasE) was isolated from E. coli and shown

to be implicated in target recognition and interference in this organism (Brouns et al.

2008). The complex was named CRISPR-Associated Complex for Antiviral Defence, or CASCADE. Further biochemical and structural characterisation of CASCADE by Jore et al. (2011) enabled the understanding of the molecular mechanism which mediates specific interference in the context of the CRISPR system. Native mass-spectrometry in combination with propanol-mediated complex dissociation revealed that the

stoichiometry of the intact CASCADE complex is CasA1B2C6D1E1-crRNA1 and has an

experimental mass of 405 kDa, but also revealed the presence of the stable sub-

complexes CasB2C6D1E1-crRNA1 and CasC6D1E1-crRNA1, indicating that CasA is

loosely associated with the other subunits in the periphery of the complex. Transmission electron microscopy and small- angle X-ray scattering revealed the unusual quaternary structure of the full complex, which appears to have an asymmetric “seahorse” shape 10 x 20 nm in size, and comparison with the stable sub- complexes enabled also the elucidation of the complex topology. As can be seen in figure 4.1, CasC is arranged in a semi-circular manner comprising the backbone of the complex, with CasD, CasE and CasA being attached to the “tail” end and the two CasB subunits located at the “nose” end. From the dissociation data it is demonstrated that the crRNA is strictly associated with the CasCDE core complex, and the authors suggest that it is bound either at the “tail” end of the structure, interacting with CasE, CasD and the end of the CasC backbone, in close proximity to the DNA - binding subunit CasA, or along the CasC backbone, thereby defining the

length of the assembly. Conformational change of the complex was observed upon target DNA binding.

The complex-bound crRNA was confirmed as the product of a single cleavage

event by the processing endonuclease CasE, producing 5’ hydroxyl and a 2’, 3’-cyclic phosphate termini. This mature crRNA unit comprises of the 8 nt repeat-derived 5’ psitag, a complete spacer sequence and the remaining 21 nt of the repeat forming a hairpin on the 3’ end. The 5’ handle appears to be a generally conserved feature of the

mature crRNAs in multiple CRISPR systems (Brouns et al. 2008; Hale et al. 2009),

providing a protein binding platform for the effector complexes and perhaps indicating its important role in mediating self-nonself discrimination.

In electrophoretic mobility shift assays CASCADE exhibited a high affinity for ssDNA and dsDNA containing sequences complementary to the complex-bound crRNA (reported Kd values were 8 and 790 nM respectively), and minimal affinity to non-target DNA resulting from the non-specific DNA-binding ability of CasA. It could also bind target ssRNA with a lower affinity. Enzymatic and chemical footprint analysis of the CASCADE binding to ss and dsDNA demonstrated that the molecular basis of the specific target recognition is the formation of an R-loop, whereby the basepairing between the spacer in the crRNA and the protospacer in the target DNA strand leads to displacement of the non-target strand (figure 4.1, C). This process was shown to be ATP-independent, enabling the system to be constantly active in an economic and efficient way without wasting the cell resources. CASCADE alone did not catalyse degradation of the target DNA, in accordance with the initial study where the presence

of Cas3 was also required to inhibit phage proliferation in vivo (Brouns et al. 2008). In

the proposed model, CasA is responsible for the non-specific interaction of the CASCADE with DNA, which enables the sequence-specific scanning of ss and dsDNA species for protospacer matches. Target recognition by the CASCADE-bound crRNA induces the formation of an R-loop (in the case of a dsDNA invader), where Cas3 is recruited by an unknown mechanism and hypothetically catalyses the cleavage of the invader DNA by the HD-nuclease domain. The helicase domain fused to the HD-

nuclease domain in E. coli Cas3 is potentially implicated in unwinding the dsDNA to

facilitate the R-loop formation, or in unwinding the RNA-DNA heteroduplex to enable degradation of the target DNA and perhaps rescue the crRNA. The ability of CASCADE to recognise dsDNA as its primary target is of great physiologic significance, since most invader DNA is in a double stranded form, and therefore provides a fast and effective way to silence potential threats at their source.

CASCADE

components nomenclaturealternative stoichiometry superfamily TIGRfam Function (if known)

CasA Cse1 1 YgcL TIGR02547 unspecific

DNA binding

CasB Cse2 2 YgcK TIGR02548

CasC Cas7, Cse4 6 COG1857 TIGR01869

CasD Cas5e 1 COG1688 TIGR02593

CasE Cas6e, Cse3 1 RAMP TIGR01907 CRISPR

RNA ribonuclease

Table 4.1: Composition of the E. coli CASCADE

Protein names in the first column are by Brouns et al. (2008). Protein names by Haft et al. (2005) and Makarova et al. (2011) are presented in the second column. Superfamilies and TIGRfam models presented according to Makarova et al. (2011).

Figure 4.1: Structure of the E. coli CASCADE and the Csy complex from P. aeruginosa

(A) EM structure of CASCADE revealing the seahorse-shaped complex. (B) Structural model of CASCADE, in the same orientation as the EM image, showing the location and arrangement of the Cas subunits and the bound crRNA. Dimensions refer to the EM image. (C) The R-loop formed by CASCADE, showing the non-complementary strand of the invader DNA displaced by the crRNA, which forms a heteroduplex with the target strand. Position of the protospacer adjacent motif is shown with a yellow box, and the basepaired spacer sequence is highlighted in orange. The stem-loop secondary structure formed by the CRISPR RNA repeat of E. coli is shown in the 3’ end of the processed crRNA. (D) EM projection and (E) SAXS reconstruction of the Csy complex, revealing the crescent-shape particle. (A)-(C) Adapted from Jore et al. (2011), (D)-(E) adapted from Wiedenheft et al. (2011).

A B C PAM crRNA 5ʼ 5ʼ invader dsDNA D E

A homologous complex was recently isolated from Pseudomonas aeruginosa

(Wiedenheft et al. 2011), which harbours a type I-F system consisting of genes cas1,

cas3, csy1, csy2, csy3 and csy4 (cas6f). Mass spectrometry and structural analysis by TEM and SAXS of this 350 kDa ribonucleoprotein complex revealed a subunit

stoichiometry of Csy11∶Csy21∶Csy36∶Csy41∶ crRNA1, and a crescent-shaped structure

120 x 150 Å (figure 4.1 D, E). The backbone of the particle is formed by the six subunits of Csy3, and it is proposed that the crRNA molecule is bound along the arch

of the complex. The main structural difference with the E. coli CASCADE is the lack of

the “tail” observed in CASCADE where CasA is located. The result of the lack of a CasA homologue is that the Csy complex exhibits strict sequence-dependent recognition of the target and it does not have a general sequence-unspecific affinity

for DNA (Wiedenheft et al. 2011). Similar to CASCADE, biochemical characterisation of

the Csy complex also revealed crRNA-mediated target recognition within a ssDNA or a dsDNA molecule and formation of an R-loop. Moreover, the authors demonstrated that the mechanism of target recongition is based in the initial binding of a shorter

seed sequence at the 3’ end of the protospacer (Wiedenheft et al. 2011).