title: Structure and mode of action of microplusin, a copper II-chelating antimicrobial peptide from the cattle tick Rhipicephalus (Boophilus) microplus.
abstract: Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.
authors.affiliation: Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, Brazil.
authors.forename: Fernanda D
authors.initials: FD
authors.lastname: Silva
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Rezende
authors.forename: Diego C P
authors.initials: DC
authors.lastname: Rossi
authors.forename: Eliane
authors.initials: E
authors.lastname: Esteves
authors.forename: Fábio H
authors.initials: FH
authors.lastname: Dyszy
authors.forename: Shirley
authors.initials: S
authors.lastname: Schreier
authors.forename: Frederico
authors.initials: F
authors.lastname: Gueiros-Filho
authors.forename: Cláudia B
authors.initials: CB
authors.lastname: Campos
authors.forename: José R
authors.initials: JR
authors.lastname: Pires
authors.forename: Sirlei
authors.initials: S
authors.lastname: Daffre
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010101:Oxygen Consumption; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D048494:Rhipicephalus / Q000737:chemistry*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins; C542675:microplusin protein, Boophilus microplus; D003300:Copper