SEE ALSO CRISPR-Cas

Cas2

Structural and dynamic insights into the role of conformational switching in the nuclease activity of the Xanthomonas albilineans Cas2 in CRISPR-mediated adaptive immunity^[1]

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute a microbial, adaptive immune system countering invading nucleic acids. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition into CRISPR loci. In subtype I-C CRISPR-Cas systems, Cas2 proteins are metal-dependent double-stranded DNA (dsDNA) nucleases, and a pH-dependent conformational transition has been proposed as a prerequisite for catalytic action. Here, we report the crystal structure of Xanthomonas albilineans Cas2 (XaCas2) and provide experimental evidence of a pH-dependent conformational change during functional activation. XaCas2 crystallized at an acidic pH represented a catalytically inactive conformational state in which two Asp8 residues were too far apart to coordinate a single catalytic metal ion. Consistently, XaCas2 exhibited dsDNA nuclease activity only under neutral and basic conditions. Despite the overall structural similarity of the two protomers, significant conformational heterogeneity was evident in the putative hinge regions, suggesting that XaCas2 engages in hinge-bending conformational switching. The presence of a Trp residue in the hinge region enabled the investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analyses further suggested that conformational activation proceeded via a rigid-body hinge-bending motion as both D8E and hinge mutations significantly reduced nuclease activity. Together, our results reveal strong correlations between the conformational states, catalytic activity, and hinge dynamics of XaCas2, and provide structural and dynamic insights into the conformational activation of the nuclease function of Cas2.

Overall, the structural features of XaCas2 (5h1o) were similar to those of other subtype I-C Cas2 structures, including the protomer fold, the dimer interface, and the conformational state. The contained an N-terminal ferredoxin fold consisting of a four-stranded antiparallel β-sheet (β1–4) and two α-helices (α1, α2), and a C-terminal segment including a 3₁₀-helix (η1) and a β-strand (β5). The to extend the four-stranded β-sheet. The two XaCas2 protomers form a . Hinge regions (residues 72–78) are indicated. XaCas2 protomers A and B are colored in yellow and cyan, respectively. Dimerization of XaCas2 buried 1346 A˚² of the surface area and involved polar interactions between the two protomers including . These interactions are conserved in the other subtype I-C Cas2 structures.

In present crystal structure, XaCas2 seemed to adopt a catalytically inactive conformational state, as was also true of several other subtype I-C Cas2 structures evaluated previously. In the XaCas2 dimer, the two were too far apart to allow them to coordinate a single catalytic metal ion together. The distance between the side chains of the two Asp8 residues was 11.3 A˚, and no metal ion was found near the residues. It is thus very likely that XaCas2 must undergo conformational switching to bring the two Asp8 residues sufficiently close to allow them to coordinate the single metal ion required for catalysis. Previous studies on subtype I-C Cas2 proteins revealed that their catalytic functions were strongly pH-dependent as nuclease activities decreased significantly at acidic pHs.

Strikingly, despite the high similarity in overall structure, the two XaCas2 protomers exhibited significant conformational heterogeneity within the putative hinge regions. When the , a substantial structural deviation was noted in the hinge region (residues 72–78). Without these residues, the RMSD of the Ca atomic positions between the two XaCas2 protomers was only 0.3 A˚, whereas the RMSD value for residues 72–78 was 3.5 A˚. This indicated that structural variation was significant within the putative hinge region only. .

• .

In particular, .

• .

In . In contrast, . The accessible surface area of Trp75 was 191 A˚² in protomer A, but only 41 A˚² in protomer B. The conformational heterogeneity persisted to Val79, after which the β5 strand commenced and the two protomers again exhibited high-level structural similarity.

The structural heterogeneity observed in the putative hinge regions is probably caused by differences in crystal contacts within the lattice. However, it is plausible to assume that the two distinct conformations are physiologically relevant, and may represent two different hinge structures corresponding to active and inactive conformational states of XaCas2 at different pHs. It was necessary to explore whether the hinge regions adopted different conformations depending on the pH. Trp75 was located in the middle of the hinge region. Trp is a dominant source of protein fluorescence, and its fluorescence is strongly affected by the microenvironment. Thus, Trp fluorescence can be used to monitor conformational transitions in proteins if Trp residue(s) experience local environmental changes in different conformational states. In the crystal structure of XaCas2, the microenvironment of Trp75 differs dramatically between the two protomers. residue of the hinge regions of the two XaCas2 protomers. Side chains of the Trp75 residues in protomers A and B are shown in blue and red, respectively. , but in .

Cas5c

• Cas5c (Cas5d): 4f3m (Bacillus halodurans), 3kg4 (Mannheimia succiniciproducens), 3vzh (Streptococcus pyogenes), 3vzi (Xanthomonas oryzae)^[2].