title: Identification of Api88 Binding Partners in Escherichia coli Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification.
abstract: Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism.
authors.affiliation: †Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and ‡Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Volke
authors.affiliation: †Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and ‡Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.
authors.forename: Andor
authors.initials: A
authors.lastname: Krizsan
authors.affiliation: †Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and ‡Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Berthold
authors.affiliation: †Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and ‡Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Knappe
authors.affiliation: †Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and ‡Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001577:Benzophenones / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000528:radiation effects; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: C488060:4-benzoylphenylalanine; D023181:Antimicrobial Cationic Peptides; D001577:Benzophenones; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D019476:Insect Proteins; C061361:apidaecin; D014443:Tyrosine; D010649:Phenylalanine
keywords: 70 kDa bacterial heat shock protein DnaK; apidaecin; mass spectrometry; proline-rich antimicrobial peptide (PrAMP); protein targets; ribosome