On July 25, 2016, Yongqun Zhu Lab published a research paper online as brief communication in Nature Structural & Molecular Biology entitled “Structural Basis for Cas3 Inhibition by the Bacteriophage Protein AcrF3”.

CRISPR–Cas systems regulate the major adaptive immune defenses of bacteria and archaea against the invasion of foreign nucleic acids. The type I CRISPR–Cas system is the most ubiquitous and is mediatedby the CRISPR RNA–guided surveillance complex Cascade (CRISPR-associatedcomplex for antiviral defense) and the DNA-degradation enzyme Cas3. Cas3 has a histidine-aspartate (HD)-type nuclease domain and a superfamily 2 (SF2) helicase domain. It is recruited by Cascade to R loops, where it unwinds and degrades target DNAs. AcrF3, a protein encoded by gene 35 from phage JBD5, has been reported to specifically inhibit the Cas3 protein ofthe P. aeruginosa PA14 strain (PaCas3) and to counteract the type I–F CRISPR–Cas system. However, the molecular mechanism for the anti-CRISPR activity of AcrF3 is unclear.

To elucidate this mechanism, Yongqun Zhu lab determined a 2.6 Å crystal structure of the PaCas3-AcrF3 complex. In the complex structure, AcrF3 forms a homodimer through hydrophobic interactions. The AcrF3 homodimer binds a concave surface of PaCas3 between the long Linker region and CTD via extensive interactions with the HD domain, the Linker region, RecA2 and CTD domain. The determined structure, together with a series of biochemical assays, uncovered that AcrF3 antagonizes the type I-F CRISPR-Cas system by disrupting all three necessary conditions for PaCas3 recruitment, including prohibiting PaCas3 from accessing the substrate DNA, blocking its recognition by Cse1, and locking PaCas3 in an ADP-bound inactive. The determined PaCas3-AcrF3 complex structure by Yongqun Zhu lab is the first structure of a CRISPR enzyme with an anti-CRISPR protein. It sheds lights on anti-CRISPR mechanisms and the interactions between bacteria and bacteriophages.

This research is accomplished in Life Sciences Institute and Innovation Center of Cell Signaling Network at Zhejiang University. Graduate student Xiaofei Wang carried out all experiments and is the first author of this paper. Dr. Deqiang Yao of National Center for Protein Science Shanghai provided many helps for Xiaofei Wang during data collection and structural model building, and is the co-first author of this paper. Professor Yongqun Zhu and Dr. Yan Zhou are the corresponding author and co-corresponding author of this study, respectively. This work was supported by grants from the National Natural Science Foundation of China(81322024, 81561130162, 31370722, and 81530068 to Y. Zhu and 81501717 toY. Zhou), the Natural Science Foundation of Zhejiang Province (LR13C05001 toY. Zhu), and the Royal Society (NA140239 to Y. Zhu).

Overall structure of the AcrF3-PaCas3 complex

Link:http://www.nature.com/nsmb/journal/vaop/ncurrent/full/nsmb.3269.html