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

Abstract

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.

Solution structure and dynamics of Xanthomonas albilineans Cas2 provide mechanistic insight on nuclease activity

“…The calculated molecular weight of XaCas2 was 11 492.1 Da, and thus XaCas2 formed a stable dimer in solution. XaCas2 exhibited a nonspecific nuclease activity against dsDNA in a pH-and divalent metal iondependent manner, as previously described [20]. XaCas2 was active between pH 7 and pH 10, but the nuclease activity suddenly dropped at pH 6 ( Fig.…”

“…Fresh purified Xa Cas2 did not show any nuclease activity, indicating that the Xa Cas2 did not bind tightly to endogenous metal ions. We note that Xa Cas2 with the C‐terminal His 6 ‐tag did not show measurable nuclease activity in the presence of the Mn 2+ ion . The intrinsic Mn 2+ binding affinity of the His 6 ‐tag is weak ( K D ~ 800 μ m ), but two His 6 ‐tags attached to the Xa Cas2 dimer possibly have increased affinity and capacity for the Mn 2+ binding, interfering with the nuclease activity .…”

“…It is remarkable that Tyr7 and Asp8 in the metal binding site also exhibited large 15 N R 2 relaxation rates of 47.6 ± 1.3 and 41.9 ± 1.8, respectively, which was comparable to those observed in the linker region. It has been postulated that Cas2 has a rigid‐body hinge motion in the β4–β5 linker region to switch into the active metal‐bound state with nuclease activity . We speculate that the hinge motion might be responsible for the conformational dynamics in the β4–β5 linker region and the metal binding site.…”

“…In the crystal structure of Xa Cas2, Asp8 of the metal binding site is located at the dimer interface (Fig. A) . The Asp8 pair, however, is separated by 11.3 Å between the subunit, and is not able to accommodate a single Mg 2+ ion (Fig.…”

“…The putative hinge motion that brings the Asp8 pair to a close proximity is shown by light blue arrows. [20]. The Asp8 pair, however, is separated by 11.3 A between the subunit, and is not able to accommodate a single Mg 2+ ion (Fig.…”