Negative stain transmission electron microscopy (TEM) was employed by
Nathanael Lintner to visualise the structural organisation of the native aCASCADE
sample, purified from S. solfataricus. The complex was arranged in right-handed
helices with 14 nm pitch, forming protein filaments with 6 nm width and variable length (figure 4.23 A). For complete experimental details and processed images refer to
Lintner et al. (2011). These filaments were observed only in the presence of Cas5a and
crRNA, while Csa2 alone was shown to be predominantly monomeric/dimeric in solution. The excess of Csa2 over Cas5a in both the native and recombinant purified
samples is comparable to the over-representation of CasC in the E. coli CASCADE,
where it forms a semicircular “backbone” with 6 subunits. It is possible therefore, that the primary component of the helices is Csa2, with Cas5a and the other accessory co- purifying proteins (Csa5, Cas6, Csa4) serving to stabilise or control the nucleation and growth of the complex. The variable length of the helical assembly could also explain the inconsistent behaviour of the aCASCADE complex on the analytical size exclusion column and our inability to estimate the molecular weight. The open symmetry displayed in these assemblies is in contrast to the closed symmetry observed in the asymmetric unit of the crystal, and because the former were observed in the presence of the natural Csa2 protein partner, they are thought to represent biologically relevant arrangements.
Interestingly, it was observed that the length of the Csa2 monomer (65 Å) is
comparable to the width of the helix, allowing multiple copies of the Csa2 structure to be modelled onto the helix. Two structural models were proposed by N. Lintner and M. Lawrence to account for the potential functional role of the helical assemblies (figure 4.23 B). In the first model, the extended filaments bind multiple crRNA units and are used to screen target DNA simultaneously, perhaps by wrapping around it and
inducing the formation of R-loops as observed for the E. coli CASCADE. In the second
model, a shorter arch-shaped assembly composed of limited Csa2 subunits is binding a single crRNA, and sub-stoichiometric amounts of Cas5a and perhaps Csa5/Cas6 form the nucleation/termination ends of this partial helix. This second model is
reminiscent of the arrangement of the CasC backbone in the E. coli CASCADE, and
would constitute a more flexible effector complex to patrol the cell for invading DNA, with the added advantage of adjustable length according to spacer length. In any case, it was observed that the groove of the helical assembly is large enough to
accommodate either dsDNA or an RNA/DNA hybrid (see Lintner et al. 2010).
Our biochemical data suggest that Csa2 is the primary RNA-binding
component of the Csa2-Cas5a complex, exhibiting a higher affinity for crRNA over control sequences. However, Csa2 can bind the control RNA substrate suggesting a general sequence-independent binding ability, perhaps as a result of its basic surface
patches. In an effort to analyse the crRNA elements responsible for this specific interaction, it was observed that both the conserved 5’ psitag and the 3’ handle contributed to the binding specificity, since the protein exhibited comparable affinity for substrates missing one or the other element. Since there is an obvious need for the bases of the spacer sequence to be solvent-exposed and accessible for basepairing with target DNA, sequence-specific interactions must be restricted to the conserved repeat-derived sequences. The 5’ psitag sequence seems to be a general feature of crRNAs in all systems studied so far, highlighting its importance as a universal recognition signal of cRNA by the effector Cas proteins. The Csa2-Cas5a complex must also be able to screen invader dsDNA for appropriate targets and stabilise a crRNA/DNA heteroduplex. A crystal structure of crRNA-bound Csa2 (±Cas5a) would help elucidate these mechanistic problems.
Figure 4.23: Quaternary structural models of native aCASCADE
(A) The right-handed helical structures of aCASCADE visualised by TEM. (B) Model of the potential arrangement of Csa2 monomers in the observed helical assembly, and roles of the accessory subunits. The Csa2 subunits composing the crRNA-supporting core of a partial helix are coloured in alternating dark and light grey. Copies of Cas5a and/or Csa5/Cas6 could be involved in inducing / terminating the polymerisation of Csa2. Adapted from Lintner et al. (2011).
In contrast to the observation made by Gudbergsdottir et al. (2011) that the
protospacer adjacent motif (PAM) is required for in vivo targeting in S. solfataricus, we
do not observe such a requirement in the minimal molecular system identified here.
The same conclusion was reached by Manica et al. (2011) in their genetic studies of in
Cas5a ? crRNA ? Cas6 or Csa5? Csa2n Cas5a ? Cas6 or Csa5? 90º 16 n m 9 nm crRNA A B
vivo interference in S. solfataricus, where the protospacer of choice did not contain a PAM sequence in its original context. Since the recombinant Csa2-Cas5a complex is
lacking some of its in vivo partners (Csa5, Csa4, Cas3, HD nuclease) we are not in a
position to make definite assumptions about the importance of the PAM motif,
especially since it is identified in sequence analyses of the S. solfataricus protospacers
(Lillestol et al. 2009). It cannot be ruled out that one of these accessory proteins (Csa5,
Csa4) are responsible for this specific interaction, and perhaps it is required in order for the final step to occur, namely the recruitment of Cas3 and HD nuclease and the final degradation of the target DNA. It could also be the case that the PAM is recognised in a dsDNA substrate, but the molecular basis of such a possibility would require interactions between Csa2 and both the DNA strands simultaneously. It is also possible that this motif plays a key role during the adaptation stage and is essential for the recognition and selection of new spacers, in which case it may be recognised and bound by other Cas components.