Estrutura de modo de ação da microplusina, um peptídeo antimicrobiano quelante de cobre II, do carrapato de boi RHIPICEPHALUS (BOOPHILUS) MICROPLUS

• Processo: 09/16272-4
• Modalidade de apoio: Auxílio à Pesquisa - Publicações científicas - Artigo
• Data de Início da vigência: 01 de dezembro de 2009
• Data de Término da vigência: 31 de maio de 2010
• Área do conhecimento: Ciências Biológicas - Bioquímica - Química de Macromoléculas
• Pesquisador responsável: Sirlei Daffre
• Beneficiário: Sirlei Daffre
• Instituição Sede: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brasil
• Assunto(s): Anti-infecciosos Ressonância magnética nuclear Estrutura de proteína Carrapatos
• Palavra(s)-Chave do Pesquisador: atividade antimicrobiana | carrapato | ligador de cobre | modo de ação | ressonância nuclear magnética | Estrutura de proteína

Resumo

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobialpeptide (AMP) is the first fully characterized member ofa new family of cysteine-rich AMPs with histidine-rich regionsat the N and C termini. In the tick, microplusin belongs to thearsenal of innate defense molecules active against bacteria andfungi. Here we describe the NMR solution structure of microplusinand demonstrate that the protein binds copper II andiron II. Structured as a single-helical globular domain, microplusinconsists of five -helices: 1 (residues Gly-9 to Arg-21),2 (residues Glu-27 to Asn-40),3 (residues Arg-44 to Thr-54),4 (residues Leu-57 to Tyr-64), and5 (residues Asn-67 to Cys-80). TheNandCtermini are disordered. This structure is unlikeany otherAMPstructures described to date.Wealso usedNMRspectroscopy to map the copper binding region on microplusin.Finally, using the Gram-positive bacteria Micrococcus luteus as amodel, we studied of mode of action of microplusin. Microplusinhas a bacteriostatic effect and does not permeabilize thebacterial membrane. Because microplusin binds metals, wetested whether this was related to its antimicrobial activity. Wefound that the bacteriostatic effect of microplusin was fullyreversed by supplementation of culture media with copper II butnot iron II. We also demonstrated that microplusin affects M.luteus respiration, a copper-dependent process. Thus, we concludethat the antibacterial effect of microplusin is due to itsability to bind and sequester copper II. (AU)