The N-terminal domain of Type IV-A1 CRISPR-associated DinG is vulnerable to proteolysis.

CasDinG is an ATP-dependent 5'-3' DNA helicase essential for bacterial Type IV-A1 CRISPR associated immunity. CasDinG contains an essential N-terminal domain predicted to bind DNA. To better understand the role of the N-terminal domain, we attempted to co-crystallize CasDinG with DNA substrates. We successfully crystallized CasDinG in a tightly packed, crystal conformation with previously unobserved unit cell dimensions. However, the structure lacked electron density for a bound DNA substrate and the CasDinG N-terminal domain. Additionally, the tight crystal packing disallowed space for the N-terminal domain, indicating that the N-terminal domain was proteolyzed before crystallization. Follow up experiments revealed that the N-terminal domain of CasDinG is proteolyzed after a few days at room temperature, but is protected from proteolysis at 4°C. These data provide a distinct x-ray crystal structure of CasDinG and indicate the essential N-terminal domain of CasDinG is prone to proteolysis.

CasDinG is a 5'-3' dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity.

CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Here, we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP-dependent 5'-3' DNA translocase that unwinds double-stranded (ds)DNA and RNA/DNA hybrids. The crystal structure of CasDinG reveals a superfamily 2 helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these domains, we identified the preferred PAM sequence for the type IV-A system (5'-GNAWN-3' on the 5'-side of the target) with a plasmid library and performed plasmid clearance assays with domain deletion mutants. Plasmid clearance assays demonstrated that all three domains are essential for type IV-A immunity. Protein expression and biochemical assays suggested the vFeS domain is needed for protein stability and the arch for helicase activity. However, deletion of the N-terminal domain did not impair ATPase, ssDNA binding, or helicase activities, indicating a role distinct from canonical helicase activities that structure prediction tools suggest involves interaction with dsDNA. This work demonstrates CasDinG helicase activity is essential for type IV-A CRISPR immunity as well as the yet undetermined activity of the CasDinG N-terminal domain.