title: Structural Insights into Substrate Recognition byClostridium difficileSortase.
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 byClostridium difficilesortase 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.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University Tainan, Taiwan.
authors.forename: Jui-Chieh
authors.initials: JC
authors.lastname: Yin
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University Tainan, Taiwan.
authors.forename: Chun-Hsien
authors.initials: CH
authors.lastname: Fei
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua UniversityHsinchu, Taiwan; Bioinformatics Program, Taiwan International Graduate Program, Academia SinicaTaipei, Taiwan.
authors.forename: Yen-Chen
authors.initials: YC
authors.lastname: Lo
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University Hsinchu, Taiwan.
authors.forename: Yu-Yuan
authors.initials: YY
authors.lastname: Hsiao
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University Tainan, Taiwan.
authors.forename: Jyun-Cyuan
authors.initials: JC
authors.lastname: Chang
authors.affiliation: Molecular Biology Consortium, Advanced Light Source, Lawrence Berkeley National Laboratory Berkeley, CA, USA.
authors.forename: Jay C
authors.initials: JC
authors.lastname: Nix
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University Hsinchu, Taiwan.
authors.forename: Yuan-Yu
authors.initials: YY
authors.lastname: Chang
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua UniversityHsinchu, Taiwan; Physics Division, National Center for Theoretical SciencesHsinchu, Taiwan.
authors.forename: Lee-Wei
authors.initials: LW
authors.lastname: Yang
authors.affiliation: 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.
authors.forename: I-Hsiu
authors.initials: IH
authors.lastname: Huang
authors.affiliation: 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.
authors.forename: Shuying
authors.initials: S
authors.lastname: Wang
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016360:Clostridioides difficile / Q000201:enzymology*; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; C509821:sortase B; D019881:Aminoacyltransferases; D003546:Cysteine Endopeptidases
keywords: Clostridium difficile; crystal structure; fluorescence resonance energy transfer; sortase; substrate specificity