X-Ray Crystal Structure of the CRISPR-Associated Protein Csx3

Sharidan Brown, Colin C. Gauvin, Alexander A. Charbonneau, Nathaniel Burman, C. Martin Lawrence. Department of Chemistry and Biochemistry, Montana State University, 103 Chemistry and Biochemistry Building, Bozeman, MT 59717.

CRISPR-Cas is an adaptive prokaryotic immune system found in bacteria and archaea. These systems contain a variety of CRISPR-associated (Cas) proteins that play essential roles in spacer acquisition, transcription and processing of crRNA, and degradation of the invading nucleic acid. The Cas protein Csx3, is part of a Type III CRISPR-Cas system found in Archaeoglobus fulgidus. Type III CRISPR systems utilize the signature multidomain protein Cas10, which upon target recognition catalyzes the synthesis of cyclic tetra-adenylate (cA4), an intracellular signaling molecule produced in response to viral infection. Csx3 is reported to show low level RNase activity, and it was speculated that Csx3 works in conjunction with additional Cas proteins to degrade RNA products. The Lawrence Lab recognized that like other Cas proteins such as Csa3, Csm6, and Csx1, Csx3 adopts a CARF domain fold, suggesting its activity is regulated by cyclic oligonucleotides. Here we show that cA4, as well as other RNA ligands, bind to Csx3 with high affinity. Further we have found that Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly attenuates this cA4 signal. Lastly, we have determined the 3D structure of Csx3 by x-ray crystallography at 1.8 Å resolution (PDB ID: 6VJG). This high resolution Csx3 structure reveals a CARF domain fold with extended C-terminal arms that might orchestrate formation of a larger macromolecular complex. Currently, the mechanistic basis for ring nuclease activity and cA4 binding are being investigated using site-directed mutagenesis. Overall, our work suggests Csx3 functions within CRISPR-Cas as a counter balance to Cas10 to regulate the duration and amplitude of the cA4 signal, providing an off-ramp from the programmed cell death pathway in cells that successfully cure viral infection.

Additional Abstract Information

Presenter: Sharidan Brown

Institution: Montana State University

Type: Poster

Subject: Biochemistry

Status: Approved

Time and Location

Session: Poster 1
Date/Time: Mon 1:30pm-2:30pm
Session Number: 2131