CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system

Suji Hong,Donghyun Ka,Seo Jeong Yoon,Nayoung Suh,Migyeong Jeong,Jeong-Yong Suh,Euiyoung Bae

doi:10.1074/jbc.ra117.001611

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against bacteriophages. In type I-F CRISPR-Cas systems, multiple Cas proteins (Csy1-4) compose a surveillance complex (Csy complex) with CRISPR RNA (crRNA) for target recognition. Here, we report the biochemical characterization of the Csy1-Csy2 subcomplex from Xanthomonas albilineans, including the analysis of its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage that infects Pseudomonas aeruginosa The X. albilineans Csy1 and Csy2 proteins (XaCsy1 and XaCsy2, respectively) formed a stable heterodimeric complex that specifically bound the 8-nucleotide (nt) 5'-handle of the crRNA. In contrast, the XaCsy1-XaCsy2 heterodimer exhibited reduced affinity for the 28-nt X. albilineans CRISPR repeat RNA containing the 5'-handle sequence. Chromatographic and calorimetric analyses revealed tight binding between the Acr protein from the P. aeruginosa phage and the heterodimeric subunit of the X. albilineans Csy complex, suggesting that AcrF2 recognizes conserved features of Csy1-Csy2 heterodimers. We found that neither XaCsy1 nor XaCsy2 alone forms a stable complex with AcrF2 and the 5'-handle RNA, indicating that XaCsy1-XaCsy2 heterodimerization is required for binding them. We also solved the crystal structure of AcrF2 to a resolution of 1.34 Å, enabling a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. Our results provide information about the order of events during the formation of the multisubunit crRNA-guided surveillance complex and suggest that the Acr protein inactivating type I-F CRISPR-Cas systems has broad specificity.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against bacteriophages
XaCsy1 and XaCsy2 form a stable heterodimeric complex. It has previously been shown in P. aeruginosa that Csy1 and Csy2 form a heterodimeric subunit, which is localized at the periphery of the Csy complex [22, 24, 25, 36, 38, 47]
The results obtained in the present study provide information about the order of events during the formation of the multisubunit Csy complex in type I-F CRISPR-Cas systems

Summary

Results

It has previously been shown in P. aeruginosa that Csy and Csy form a heterodimeric subunit, which is localized at the periphery of the Csy complex [22, 24, 25, 36, 38, 47]. It is likely that the assembly of XaCsy and XaCsy is required for establishment of the binding interface for the 5Ј-handle of the crRNA This interpretation is consistent with the structural data of the P. aeruginosa Csy complex, which shows that the 5Ј-handle part of the crRNA interacts closely with both Csy and Csy proteins [25, 47]. To analyze the interactions of AcrF2 with the individual components of the XaCsy1-Csy heterodimer, we performed analytical SEC with the separately purified XaCsy and His6MBP–tagged XaCsy proteins (Fig. 5). The individual components of the XaCsy1-Csy heterodimer alone did not bind to the 5Ј-handle of crRNA, whereas a mixture of the separately purified proteins resulted in a significant RNA band shift (Fig. 3E).

Number of water molecules

Discussion

Experimental procedures

Analytical SEC