Non-prime- and prime-side profiling of Pro-Pro endopeptidase specificity using synthetic combinatorial peptide libraries and mass spectrometry

Claushuis, B.; Cordfunke, R.A.; Ru, A.H. de; Angeren, J. van; Baumann, U.; Veelen, P.A. van; Wuhrer, M.; Corver, J.; Drijfhout, J.W.; Hensbergen, P.J.

doi:doi:10.1111/febs.17160

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by its substrate speciﬁcity,methods that can extensively characterize substrate speciﬁcity are valuabletools for protease research. Previously, we achieved an in-depth characteri-zation of PPEP prime-side speciﬁcity. However, PPEP speciﬁcity is alsodetermined by the non-prime-side residues in the substrate. To gain a morecomplete insight into the determinants of PPEP speciﬁcity, we character-ized the non-prime- and prime-side speciﬁcity of various PPEPs using acombination of synthetic combinatorial peptide libraries and mass spec-trometry. With this approach, we deepened our understanding of the P3-P30 speciﬁcities of PPEP-1 and PPEP-2, while identifying the endogenoussubstrate of PPEP-2 as the most optimal substrate in our library data. Fur-thermore, by employing the library approach, we...Show moreA group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by its substrate speciﬁcity,methods that can extensively characterize substrate speciﬁcity are valuabletools for protease research. Previously, we achieved an in-depth characteri-zation of PPEP prime-side speciﬁcity. However, PPEP speciﬁcity is alsodetermined by the non-prime-side residues in the substrate. To gain a morecomplete insight into the determinants of PPEP speciﬁcity, we character-ized the non-prime- and prime-side speciﬁcity of various PPEPs using acombination of synthetic combinatorial peptide libraries and mass spec-trometry. With this approach, we deepened our understanding of the P3-P30 speciﬁcities of PPEP-1 and PPEP-2, while identifying the endogenoussubstrate of PPEP-2 as the most optimal substrate in our library data. Fur-thermore, by employing the library approach, we investigated the alteredspeciﬁcity of mutants of PPEP-1 and PPEP-2. Additionally, we character-ized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displaysa PPEP-1-like prime-side speciﬁcity, which was substantiated by the experi-mental data. Intriguingly, another putative PPEP from Clostridioides difﬁ-cile, CD1597, did not display Pro-Pro endoproteolytic activity.Collectively, we characterized PPEP speciﬁcity in detail using our robustpeptide library method and, together with additional structural informa-tion, provide more insight into the intricate mechanisms that govern prote-ase speciﬁcity.
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Non-prime- and prime-side profiling of Pro-Pro endopeptidase specificity using synthetic combinatorial peptide libraries and mass spectrometry