Yang, Lingguang; Zhang, Laixing; Yin, Peipei; Ding, Hao; Xiao, Yu; Zeng, Jianwei; Wang, Wenhe; Zhou, Huan; Wang, Qisheng; Zhang, Yi; Chen, Zeliang; Yang, Maojun; Feng, Yue

NATURE COMMUNICATIONS

2022, 13,

Yang, Lingguang; Zhang, Laixing; Yin, Peipei; Ding, Hao; Xiao, Yu; Zeng, Jianwei; Wang, Wenhe; Zhou, Huan; Wang, Qisheng; Zhang, Yi; Chen, Zeliang; Yang, Maojun; Feng, Yue

Phages use anti-CRISPR proteins (Acrs) to counteract the bacterial CRISPR-Cas systems. Here, the authors characterize AcrIF24, which functions as an Aca (Acr-associated) to repress and regulate its own transcription, dimerizes the Csy complex, blocks the hybridization of target DNA, and tethers non-sequence-specific DNA to the Csy complex. CRISPR-Cas systems are prokaryotic adaptive immune systems and phages use anti-CRISPR proteins (Acrs) to counteract these systems. Here, we report the structures of AcrIF24 and its complex with the crRNA-guided surveillance (Csy) complex. The HTH motif of AcrIF24 can bind the Acr promoter region and repress its transcription, suggesting its role as an Aca gene in self-regulation. AcrIF24 forms a homodimer and further induces dimerization of the Csy complex. Apart from blocking the hybridization of target DNA to the crRNA, AcrIF24 also induces the binding of non-sequence-specific dsDNA to the Csy complex, similar to AcrIF9, although this binding seems to play a minor role in AcrIF24 inhibitory capacity. Further structural and biochemical studies of the Csy-AcrIF24-dsDNA complexes and of AcrIF24 mutants reveal that the HTH motif of AcrIF24 and the PAM recognition loop of the Csy complex are structural elements essential for this non-specific dsDNA binding. Moreover, AcrIF24 and AcrIF9 display distinct characteristics in inducing non-specific DNA binding. Together, our findings highlight a multifunctional Acr and suggest potential wide distribution of Acr-induced non-specific DNA binding.