Pichlo, Christian, Juetten, Linda, Wojtalla, Fabian, Schacherl, Magdalena ORCID: 0000-0002-5478-2509, Diaz, Dolores ORCID: 0000-0002-2406-4952 and Baumann, Ulrich (2019). Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1. J. Biol. Chem., 294 (30). S. 11525 - 11536. ROCKVILLE: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. ISSN 1083-351X

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Abstract

Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP down arrow PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1 ' residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Pichlo, ChristianUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Juetten, LindaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wojtalla, FabianUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Schacherl, MagdalenaUNSPECIFIEDorcid.org/0000-0002-5478-2509UNSPECIFIED
Diaz, DoloresUNSPECIFIEDorcid.org/0000-0002-2406-4952UNSPECIFIED
Baumann, UlrichUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-134592
DOI: 10.1074/jbc.RA119.009029
Journal or Publication Title: J. Biol. Chem.
Volume: 294
Number: 30
Page Range: S. 11525 - 11536
Date: 2019
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Place of Publication: ROCKVILLE
ISSN: 1083-351X
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
STRUCTURAL BASIS; COORDINATION SPHERE; ZINC; CLEAVAGE; BINDING; WATER; ZMP1; SITEMultiple languages
Biochemistry & Molecular BiologyMultiple languages
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/13459

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