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Front Cell Infect Microbiol. 2016 Nov 22;6:160. eCollection 2016.

Structural Insights into Substrate Recognition by Clostridium difficile Sortase.

Author information

1
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University Tainan, Taiwan.
2
Institute of Bioinformatics and Structural Biology, National Tsing Hua UniversityHsinchu, Taiwan; Bioinformatics Program, Taiwan International Graduate Program, Academia SinicaTaipei, Taiwan.
3
Department of Biological Science and Technology, National Chiao Tung University Hsinchu, Taiwan.
4
Molecular Biology Consortium, Advanced Light Source, Lawrence Berkeley National Laboratory Berkeley, CA, USA.
5
Institute of Bioinformatics and Structural Biology, National Tsing Hua University Hsinchu, Taiwan.
6
Institute of Bioinformatics and Structural Biology, National Tsing Hua UniversityHsinchu, Taiwan; Physics Division, National Center for Theoretical SciencesHsinchu, Taiwan.
7
Department of Microbiology and Immunology, College of Medicine, National Cheng Kung UniversityTainan, Taiwan; Center of Infectious Disease and Signaling Research, National Cheng Kung UniversityTainan, Taiwan.

Abstract

Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26-PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB.

KEYWORDS:

Clostridium difficile; crystal structure; fluorescence resonance energy transfer; sortase; substrate specificity

PMID:
27921010
PMCID:
PMC5118464
DOI:
10.3389/fcimb.2016.00160
[Indexed for MEDLINE]
Free PMC Article

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