title: Membrane-Active Epithelial Keratin 6A Fragments (KAMPs) Are Unique Human Antimicrobial Peptides with a Non-αβ Structure.
abstract: Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs). Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS) micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation ofStaphylococcus aureusandPseudomonas aeruginosaby scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics.
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Cleveland, OH, USA.
authors.forename: Judy T Y
authors.initials: JT
authors.lastname: Lee
authors.affiliation: Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center Omaha, NE, USA.
authors.forename: Guangshun
authors.initials: G
authors.lastname: Wang
authors.affiliation: Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Yu Tong
authors.initials: YT
authors.lastname: Tam
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute and Lerner Research Institute, Cleveland ClinicCleveland, OH, USA; Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve UniversityCleveland, OH, USA.
authors.forename: Connie
authors.initials: C
authors.lastname: Tam
keywords: antimicrobial peptides; epithelial cells; innate immunity; keratins; peptide structure and function