Crystal structure of the anti-CRISPR repressor Aca2

Ben Usher,Nils Birkholz,Izaak N Beck,Robert D Fagerlund,Simon A Jackson,Peter C Fineran,Tim R Blower

doi:10.1016/j.jsb.2021.107752

Ben Usher, Nils Birkholz + Show 5 more

Open Access

https://doi.org/10.1016/j.jsb.2021.107752

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Abstract

Bacteria use adaptive CRISPR-Cas immune mechanisms to protect from invasion by bacteriophages and other mobile genetic elements. In response, bacteriophages and mobile genetic elements have co-evolved anti-CRISPR proteins to inhibit the bacterial defense. We and others have previously shown that anti-CRISPR associated (Aca) proteins can regulate this anti-CRISPR counter-attack. Here, we report the first structure of an Aca protein, the Aca2 DNA-binding transcriptional autorepressor from Pectobacterium carotovorum bacteriophage ZF40, determined to 1.34 Å. Aca2 presents a conserved N-terminal helix-turn-helix DNA-binding domain and a previously uncharacterized C-terminal dimerization domain. Dimerization positions the Aca2 recognition helices for insertion into the major grooves of target DNA, supporting its role in regulating anti-CRISPRs. Furthermore, database comparisons identified uncharacterized Aca2 structural homologs in pathogenic bacteria, suggesting that Aca2 represents the first characterized member of a more widespread family of transcriptional regulators.

Highlights

Bacteria are under constant threat of invasion by bacteriophages and other mobile genetic elements (MGEs)
Examining a single protomer shows that the proposed N-terminal domain (NTD) HTH and relative C-terminal domain (CTD) are small clusters of secondary structure elements abutting and joined by a longer backbone α-helix, α4, such that the protomer forms a single globular protein (Fig. 1F)
We previously proposed that upon phage infection, anti-CRISPR expression initiates strongly and Aca2 will switch off antiCRISPR production once the host defence has been shut down, poten tially to reduce toxic side-effects of AcrIF8 (Birkholz et al, 2019)

Summary

Introduction

Bacteria are under constant threat of invasion by bacteriophages (phages) and other mobile genetic elements (MGEs). We and others recently showed that Aca and Aca, as well as Aca encoded in an acrIIC3–aca operon, serve as repressors of their respective promoters (Birkholz et al, 2019; Stanley et al, 2019). These findings and the pervasive presence of helix-turn-helix (HTH) domains in all known Aca proteins suggest that Aca proteins generally function to repress, or at least to regulate, anti-CRISPR production. Bacteria may use their own Aca-like regu lators to inhibit anti-CRISPR deployment by phages, thereby maintain ing CRISPR-Cas defense (Osuna et al, 2020; Stanley et al, 2019)

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