Structure, Function, And Dynamics Of Xanthomonas Albilineans Cas2 In Type I-C Crispr-Cas System

CRISPRs and CRISPR-associated (Cas) proteins provide a microbial adaptive immunity against phage infection. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in the new spacer acquisition into CRISPR loci. In type 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 prerequisite for the nuclease function. 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, and exhibited dsDNA nuclease activity only under neutral and basic conditions. In the crystal structure, 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 investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analysis further confirmed that the conformational activation of the nuclease function was in play. Together, our results reveal strong correlations among the conformational states, catalytic activity, and hinge dynamics of XaCas2, and suggest that Cas2 may play a dual role as a dsDNA nuclease or as a central adaptor protein within the CRISPR-adaptation complex.