The Origins of Specificity in the Microcin-Processing Protease TldD/E

Dmitry Ghilarov, Marina Serebryakova, Clare E.M. Stevenson, Stephen J. Hearnshaw, Dmitry S. Volkov, Anthony Maxwell, David M. Lawson, Konstantin Severinov

    Research output: Contribution to journalArticlepeer-review

    21 Citations (Scopus)

    Abstract

    TldD and TldE proteins are involved in the biosynthesis of microcin B17 (MccB17), an Escherichia coli thiazole/oxazole-modified peptide toxin targeting DNA gyrase. Using a combination of biochemical and crystallographic methods we show that E. coli TldD and TldE interact to form a heterodimeric metalloprotease. TldD/E cleaves the N-terminal leader sequence from the modified MccB17 precursor peptide, to yield mature antibiotic, while it has no effect on the unmodified peptide. Both proteins are essential for the activity; however, only the TldD subunit forms a novel metal-containing active site within the hollow core of the heterodimer. Peptide substrates are bound in a sequence-independent manner through β sheet interactions with TldD and are likely cleaved via a thermolysin-type mechanism. We suggest that TldD/E acts as a “molecular pencil sharpener”: unfolded polypeptides are fed through a narrow channel into the active site and processively truncated through the cleavage of short peptides from the N-terminal end. Ghilarov et al. crystallized the microcin-processing E. coli metalloprotease TldD/E in complex with various substrates, and used this structural information to explain the unusual specificity of the enzyme.

    Original languageEnglish
    Pages (from-to)1549-1561.e5
    JournalStructure
    Volume25
    Issue number10
    DOIs
    Publication statusPublished - 3 Oct 2017

    Keywords

    • CcdAB
    • DNA gyrase
    • metalloprotease
    • microcin B17
    • peptidase
    • ribosomally synthesized modified peptides
    • RiPP
    • toxin-antitoxin
    • X-ray crystallography

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