Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures

Abstract

Bacteriophages encode anti-CRISPR suppressors to counteract the CRISPR/Cas immunity of their bacterial hosts, thus facilitating their survival and replication. Previous studies have shown that two phage-encoded anti-CRISPR proteins, AcrF1 and AcrF2, suppress the type I-F CRISPR/Cas system of Pseudomonas aeruginosa by preventing target DNA recognition by the Csy surveillance complex, but the precise underlying mechanism was unknown. Here we present the structure of AcrF1/2 bound to the Csy complex determined by cryo-EM single-particle reconstruction. By structural analysis, we found that AcrF1 inhibits target DNA recognition of the Csy complex by interfering with base pairing between the DNA target strand and crRNA spacer. In addition, multiple copies of AcrF1 bind to the Csy complex with different modes when working individually or cooperating with AcrF2, which might exclude target DNA binding through different mechanisms. Together with previous reports, we provide a comprehensive working scenario for the two anti-CRISPR suppressors, AcrF1 and AcrF2, which silence CRISPR/Cas immunity by targeting the Csy surveillance complex.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

“…As expected, the general arrangement of the 9 polypeptides in the Csy crRNA ·dsDNA is the same as that reported in the cryo-EM structure of Csy crRNA bound simultaneously to inhibitors AcrF1 and AcrF2 by Chowdhury et al (2017) and by Peng et al (2017), with the protein subunits arranged around the crRNA in a helically twisted “G” shape (Figure 1C, 1D). Cas6f is located at the 3′ stem-loop of the crRNA, Cas8f and Cas5f are located at the 5′ handle of crRNA, and six interlocking copies of Cas7f are located along the length of the crRNA spacer.…”

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

“…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 interaction between Csy1 and Csy2 was also detected in Pectobacterium atrosepticum (23).…”

“…Previous cryo-EM structures for the AcrF2-bound P. aeruginosa Csy complex have been determined to moderate resolutions (25,38,47). One study reported that the average resolution was 3.4 Å, and the authors stated that the density of acidic residues on AcrF2 was not sufficient to confidently model side chain positions (25).…”

“…One study reported that the average resolution was 3.4 Å, and the authors stated that the density of acidic residues on AcrF2 was not sufficient to confidently model side chain positions (25). Another study located the Csy1-Csy2-AcrF2 subcomplex but could not model it (38). More recently, Guo et al reported a cryo-EM structure of the complex bound to AcrF2 at a nominal resolution of 3.2 Å (47).…”

“…The authors suggested that AcrF1 prevents target DNA hybridization by interacting with Csy3, and AcrF2 competes with DNA for binding to the Csy1-Csy2 subunit (25). Peng et al also reported the structure of the AcrF1-and AcrF2-bound P. aeruginosa Csy complex, in which the tail (Csy1 and Csy2) of the seahorseshaped complex and the bound AcrF2 were not modeled (38). Guo et al determined several cryo-EM structures for the Csy complex in distinct functional and inhibited states, including its target DNA-bound state and AcrF1-, AcrF2-and AcrF10-bound structures (47).…”