9zq6

Structure of SpyCas9 in complex with the anti-CRISPR protein AcrIIA26

spinqualitylabelsall models 9zq6, resolution 2.98Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 9zq6 is a 3 chain structure with sequence from Streptococcus and Streptococcus pyogenes M1 GAS. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: Electron Microscopy, Resolution 2.98Å Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 A resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-single-guide RNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central beta-sheet flanked by two alpha-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the protospacer adjacent motif (PAM) duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple anti-CRISPRs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications. Structural basis for inhibition of SpyCas9 by the anti-CRISPR protein AcrIIA26.,Zheng I, Learn B, Bailey S Biochem J. 2026 Mar 4;483(3):289-300. doi: 10.1042/BCJ20250364. PMID:41553775 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

Proteopedia Page Contributors and Editors (what is this?)

OCA