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Remodeling of a tripartite substrate-binding motif in the HD domain provides the mechanism for activation of CRISPR-Cas10 DNases

Yu, Z.; Wang, F.; She, Q.

2022-11-08 microbiology
10.1101/2022.11.07.515528 bioRxiv
Show abstract

Type III CRISPR systems are endowed with multiple immune activities, including target RNA cleavage, RNA-activated DNA cleavage and the cOA synthesis among which molecular mechanism remains elusive for the DNase. Here, DNase of LdCsm is investigated. Structural modeling revealed two HD loop segments. Cas10 mutants carrying either loop truncation or amino acid substitution in the HD domain are generated and characterized. We found each HD loop contains a substrate-binding site essential for its immunity. In fact, the substrate binding requires a tripartite motif composed of the two loop binding sites and the HD catalytic site. We demonstrate cognate target RNA (CTR) remodels the tripartite motif to activate the LdCsm DNase in two consecutive events: (a) it reduces the flexibility of LD-L1 and facilitates the simultaneous substrate binding at both loops, (b) the bound substrate is then propelled into the catalytic site via LD-L2 oscillation, driving the substrate into the catalytic site for DNA cleavage.

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