Related Experiment Video
Updated: Feb 22, 2026
The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes
Published on: May 25, 2018
The Origins of Specificity in the Microcin-Processing Protease TldD/E
Dmitry Ghilarov1, Marina Serebryakova2, Clare E M Stevenson3
1Centre for Data-Intensive Biomedicine and Biotechnology, Skolkovo Institute of Science and Technology, 143026 Moscow, Russia; Institute of Gene Biology of the Russian Academy of Sciences, 119334 Moscow, Russia; Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
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.
Insights
The TldD/E protease complex from Escherichia coli processes the microcin B17 (MccB17) precursor peptide. This metalloprotease is essential for generating the mature antibiotic by cleaving leader sequences.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Microcin B17 (MccB17) is a peptide toxin produced by Escherichia coli that targets DNA gyrase.
- The biosynthesis of MccB17 involves specific proteins, including TldD and TldE, which are crucial for its activity.
Purpose of the Study:
- To elucidate the structural and functional roles of TldD and TldE proteins in MccB17 biosynthesis.
- To characterize the enzymatic activity and mechanism of the TldD/E complex.
Main Methods:
- Biochemical assays to determine protein interactions and enzymatic activity.
- Crystallographic methods to determine the three-dimensional structure of the TldD/E complex.
- Analysis of substrate cleavage specificity.
Main Results:
- TldD and TldE form a heterodimeric metalloprotease complex.
- The TldD/E complex specifically cleaves the N-terminal leader sequence from modified MccB17 precursor peptides.
- The TldD subunit contains a metal-dependent active site responsible for peptide cleavage.
- Substrate binding is sequence-independent, mediated by beta-sheet interactions with TldD.
Conclusions:
- The TldD/E complex functions as a unique metalloprotease essential for mature MccB17 production.
- The enzyme's mechanism resembles a 'molecular pencil sharpener,' processively truncating unfolded polypeptides.
- Understanding this mechanism provides insights into peptide processing and antibiotic biosynthesis.
Related Concept Videos
01:26Bacterial Translocation and Protein Secretion
01:19Translocation of Proteins into the Mitochondria
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...