Structure and RNA-binding properties of the Type III-A CRISPR-associated protein Csm3

Abstract

The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 Å resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins.

Keywords: :

• RAMP
• RRM domain
• ferredoxin domain
• Cas7
• adaptive immunity

10.4161/rna.26500

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to thank Sutapa Chakrabarti and Marco Hein for critical reading of the manuscript and Jérôme Basquin, Karina Valer-Saldaña, and Sabine Pleyer at the MPI- Martinsried Crystallization Facility. The authors thank the staff of the PXII beamline at the Swiss Light Source for assistance during data collection. This study was supported by the Max Planck Gesellschaft, the DFG Research Group 1680 (FOR1680) to EC and LR, CIPSM to EC and the Schering Foundation fellowship to AH. Author contributions: AH, AS, and LR designed the experiments; AS and JE; crystallized the WT protein; AH and CB solved the structure; AS performed the experiment in Figure 3A; AH performed all other experiments. AH, EC, and LR wrote the manuscript.

Accession Number

The coordinates and the structure factors have been deposited to the Protein Data Bank with the accession code: 4N0L

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/rnabiology/article/26500