title: Thermolysin-linearized microcin J25 retains the structured core of the native macrocyclic peptide and displays antimicrobial activity.
abstract: Microcin J25 (MccJ25) is the single macrocyclic antimicrobial peptide belonging to the ribosomally synthesized class of microcins that are secreted by Enterobacteriaceae. It showed potent antibacterial activity against several Salmonella and Escherichia strains and exhibited a compact three-dimensional structure [Blond et al. (2001) Eur. J. Biochem., 268, 2124-2133]. The molecular mechanisms involved in the biosynthesis, folding and mode of action of MccJ25 are still unknown. We have investigated the structure and the antimicrobial activity of thermolysin-linearized MccJ25 (MccJ25-L1-21: VGIGTPISFY10GGGAGHVPEY20F), as well as two synthetic analogs, sMccJ25-L1-21 (sequence of the thermolysin-cleaved MccJ25) and sMccJ25-L12-11 (C-terminal sequence of the MccJ25 precursor: G12GAGHVPEYF21V1GIGTPISFYG11). The three-dimensional solution structure of MccJ25-L1-21, determined by two-dimensional NMR, consists of a boot-shaped hairpin-like well-defined 8-19 region flanked by disordered N and C termini. This structure is remarkably similar to that of cyclic MccJ25, and includes a short double-stranded antiparallel beta-sheet (8-10/17-19) perpendicular to a loop (Gly11-His16). The thermolysin-linearized MccJ25-L1-21 had antibacterial activity against E. coli and S. enteritidis strains, while both synthetic analogues lacked activity and organized structure. We show that the 8-10/17-19 beta-sheet, as well as the Gly11-His16 loop are required for moderate antibacterial activity and that the Phe21-Pro6 loop and the MccJ25 macrocyclic backbone are necessary for complete antibacterial activity. We also reveal a highly stable 8-19 structured core present in both the native MccJ25 and the thermolysin-linearized peptide, which is maintained under thermolysin treatment and resists highly denaturing conditions.
authors.affiliation: Laboratory of Chemistry and Biochemistry of Natural Substances, Department of Regulation, Development and Molecular Diversity, National Museum of Natural History, Paris, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Blond
authors.forename: Michel
authors.initials: M
authors.lastname: Cheminant
authors.forename: Delphine
authors.initials: D
authors.lastname: Destoumieux-Garzón
authors.forename: Isabelle
authors.initials: I
authors.lastname: Ségalas-Milazzo
authors.forename: Jean
authors.initials: J
authors.lastname: Peduzzi
authors.forename: Christophe
authors.initials: C
authors.lastname: Goulard
authors.forename: Sylvie
authors.initials: S
authors.lastname: Rebuffat
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D001430:Bacteriocins / Q000737:chemistry*; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013820:Thermolysin / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; C020411:microcin; D013820:Thermolysin