8E2W

Structure of CRISPR-Associated DinG

Summary for 8E2W

• Entry DOI: 10.2210/pdb8e2w/pdb
• Descriptor: CasDinG (1 entity in total)
• Functional Keywords: crispr, ding, helicase, atpase, dna binding protein
• Biological source: Pseudomonas aeruginosa
• Total number of polymer chains: 1
• Total formula weight: 81111.81

Authors

Domgaard, H.,Jackson, R.N. (deposition date: 2022-08-16, release date: 2023-06-21, Last modification date: 2024-04-03)

Primary citation

Domgaard, H.,Cahoon, C.,Armbrust, M.J.,Redman, O.,Jolley, A.,Thomas, A.,Jackson, R.N.
CasDinG is a 5'-3' dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity.
Nucleic Acids Res., 51:8115-8132, 2023

PubMed Abstract: 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.

PubMed: 37395408
DOI: 10.1093/nar/gkad546

Experimental method

X-RAY DIFFRACTION (2.95 Å)