Cleaving like a pro: specificity, structure, and function of Pro-Pro endopeptidases

Abstract

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), have the unique ability to cleave their substrates between two proline residues. However, their specificity extends to surrounding residues as well. We have developed a method to determine the specificity of PPEPs in great detail. For this, we treated a specially designed synthetic combinatorial peptide library with PPEPs and analyzed the resulting products using mass spectrometry. This information was linked to the structures of PPEPs, allowing for the detailed description of the mechanisms that govern PPEP specificity.

Furthermore, we examined the protein CD1597 from C. difficile. This protein initially appears to resemble a PPEP, but in our experiments, it did not show proteolytic activity. Several experiments were conducted to investigate changes in a cd1597 mutant strain. In many cases, the bacterium did not seem to be affected by the absence of CD1597, but using a mass spectrometry-based proteomics approach, we identified a link to sporulation.

Finally, a new model has been proposed for the biosynthetic pathway responsible for producing the Type A glycan found on the flagella of C. difficile. This model can be tested in the future and may aid in the development of vaccines against pathogenic bacteria.