AI-2–mediated quorum sensing of E. coli is directly connected to sugar metabolism via the phosphocarrier HPr protein of PTS.

Significance: Auxin is the central hormone that governs diverse developmental processes in plants. Auxin response is regulated by auxin response transcription factor (ARF) and Aux/IAA transcriptional repressor. ARF and Aux/IAA form homo-oligomers and also hetero-oligomers for transcriptional regulation of auxin-response genes. Mechanistic understanding of how ARF and Aux/IAA change their association states is not well established. This work reports, to our knowledge, the first structure of the oligomerization domain of IAA17, and describes the key determinant that dictates the switch between homo- and hetero-oligomers. While Aux/IAA and ARF use a common scaffold and interface for homotypic and heterotypic associations, the charge composition at the interface determines the affinity and the oligomerization states. Based on the results, we propose a refined model of auxin-induced transcriptional regulation.

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 K_D ~4.8 μM, which compares to K_D ~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.

Clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas systems constitute the adaptive immunity of bacteria and archaea, degrading nucleic acids of invading phages and plasmids. In response, phages employ anti‐CRISPR (Acr) proteins as a counterdefense mechanism to neutralize the host immunity. AcrIIC3 directly inhibits target DNA cleavage of type II‐C Cas9 of Neisseria meningitidis. Here, we show that AcrIIC3 interacts with the HNH nuclease domain of N. meningitidis Cas9 to inhibit its nuclease activity in an allosteric manner. The crystal structure of the AcrIIC3–HNH complex reveals that AcrIIC3 binds opposite the active site on the HNH nuclease domain. AcrIIC3 employs a unique interface for HNH, allowing it to discriminate between Cas9 orthologs, which contrasts with the broad spectrum of Cas9 inhibition by AcrIIC1. Interface residues of HNH provide key electrostatic and hydrophobic interactions that determine the host specificity of AcrIIC3. Mutations that replace HNH interfaces of N. meningitidis Cas9 with those of Geobacillus stearothermophilus Cas9 or Campylobacter jejuni Cas9 significantly attenuate AcrIIC3 binding, illustrating that the divergent interaction surface confers the host specificity of AcrIIC3. Our study demonstrates that the variable sequences of binding interface can define the target specificity of Acr proteins, suggesting potential applications in Cas9 control for gene editing.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide bacteria with RNA-based adaptive immunity against phage infection. To counteract this defense mechanism, phages evolved anti-CRISPR (Acr) proteins that inactivate the CRISPR-Cas systems. AcrIIA1, encoded by Listeria monocytogenes prophages, is the most prevalent among the Acr proteins targeting type II-A CRISPR-Cas systems and has been used as a marker to identify other Acr proteins. Here, we report the crystal structure of AcrIIA1 and its RNA-binding affinity. AcrIIA1 forms a dimer with a novel two helical-domain architecture. The N-terminal domain of AcrIIA1 exhibits a helix-turn-helix motif similar to transcriptional factors. When overexpressed in Escherichia coli, AcrIIA1 associates with RNAs, suggesting that AcrIIA1 functions via nucleic acid recognition. Taken together, the unique structural and functional features of AcrIIA1 suggest its distinct mode of Acr activity, expanding the diversity of the inhibitory mechanisms employed by Acr proteins.