[2018] Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor
Journal
Sci Rep 8(1): 3883
Authors
Kim, I., Jeong, M., Ka, D., Han, M., Kim, N.K., Bae, E. and Suh, J.Y.*
*Denotes Corresponding Author
Abstract
Journal
Sci Rep 8(1): 3883
Authors
Kim, I., Jeong, M., Ka, D., Han, M., Kim, N.K., Bae, E. and Suh, J.Y.*
*Denotes Corresponding Author
Abstract
The bacterial CRISPR-Cas system provides adaptive immunity against invading phages. Cas9, an RNA-guided endonuclease, specifically cleaves target DNA substrates and constitutes a well-established platform for genome editing. Recently, anti-CRISPR (Acr) proteins that inhibit Cas9 have been discovered, promising a useful off-switch for Cas9 to avoid undesirable off-target effects. Here, we report the solution structure and dynamics of Listeria monocytogenes AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9). AcrIIA4 forms a compact monomeric αβββαα fold comprising three antiparallel β strands flanked by three α-helices and a short 310-helix. AcrIIA4 exhibits distinct backbone dynamics in fast and slow timescales at loop regions that form interaction surfaces for SpyCas9. In particular, the β1-β2 loop that binds to the RuvC domain of SpyCas9 is highly mobile, and the β1-β2 and α2-α3 loops that bind to the RuvC and C-terminal domains of SpyCas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo-SpyCas9 with KD ~4.8 μM, which compares to KD ~0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9. Since the binary complex between AcrIIA4 and SpyCas9 does not compete with the target DNA binding, it can effectively disable the Cas9 nuclease activity by forming a tight ternary complex in the presence of sgRNA.