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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	Antitoxin autoregulation of M. tuberculosis toxin-antitoxin expression through negative cooperativity arising from multiple inverted repeat sequences Izaak Beck , Ben Usher , Hannah G Hampton , Peter C. Fineran , Tim R. Blower Biochemical Journal DOI: 10.1042/BCJ20200368 Diamond Proposal Number(s): [18598] Open Access Show Abstract ▼ Abstract: Toxin-antitoxin systems play key roles in bacterial adaptation, including protection from antibiotic assault and infection by bacteriophages. The type IV toxin-antitoxin system AbiE encodes a DUF1814 nucleotidyltransferase-like toxin, and a two-domain antitoxin. In Streptococcus agalactiae, the antitoxin AbiEi negatively autoregulates abiE expression through positively co-operative binding to inverted repeats within the promoter. The human pathogen Mycobacterium tuberculosis encodes four DUF1814 putative toxins, two of which have antitoxins homologous to AbiEi. One such M.tuberculosis antitoxin, named Rv2827c, is required for growth and whilst the structure has previously been solved, the mode of regulation is unknown. To complete the gaps in our understanding, we first solved the structure of S. agalactiae AbiEi to 1.83 Å resolution for comparison with M.tuberculosis Rv2827c. AbiEi contains an N-terminal DNA binding domain and C-terminal antitoxicity domain, with bilateral faces of opposing charge. The overall AbiEi fold is similar to Rv2827c, though smaller, and with a 65º difference in C-terminal domain orientation. We further demonstrate that, like AbiEi, Rv2827c can autoregulate toxin-antitoxin operon expression. In contrast to AbiEi, the Prv2827c promoter contains two sets of inverted repeats, which bind Rv2827c with differing affinities depending on the sequence consensus. Surprisingly, Rv2827c bound with negative co-operativity to the full Prv2827c promoter, demonstrating an unexpectedly complex form of transcriptional regulation.	Jun 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems Yiming Cai , Ben Usher , Claude Gutierrez , Anastasia Tolcan , Moise Mansour , Peter C. Fineran , Ciarán Condon , Olivier Neyrolles , Pierre Genevaux , Tim R. Blower Science Advances 6 DOI: 10.1126/sciadv.abb6651 Diamond Proposal Number(s): [18598] Open Access Show Abstract ▼ Abstract: Toxin-antitoxin systems are widespread stress-responsive elements, many of whose functions remain largely unknown. Here, we characterize the four DUF1814-family nucleotidyltransferase-like toxins (MenT1–4) encoded by the human pathogen Mycobacterium tuberculosis. Toxin MenT3 inhibited growth of M. tuberculosis when not antagonized by its cognate antitoxin, MenA3. We solved the structures of toxins MenT3 and MenT4 to 1.6 and 1.2 Å resolution, respectively, and identified the biochemical activity and target of MenT3. MenT3 blocked in vitro protein expression and prevented tRNA charging in vivo. MenT3 added pyrimidines (C or U) to the 3′-CCA acceptor stems of uncharged tRNAs and exhibited strong substrate specificity in vitro, preferentially targeting tRNASer from among the 45 M. tuberculosis tRNAs. Our study identifies a previously unknown mechanism that expands the range of enzymatic activities used by bacterial toxins, uncovering a new way to block protein synthesis and potentially treat tuberculosis and other infections.	Jul 2020
I04-Macromolecular Crystallography	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 Journal Of Structural Biology 213 DOI: 10.1016/j.jsb.2021.107752 Diamond Proposal Number(s): [24948] Open Access Show Abstract ▼ 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.	Sep 2021

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