RESUMO
Membrane proteins are challenging targets for crystallization and structure determination by X-ray crystallography. Hurdles can be overcome by antibody-mediated crystallization. More than 25 unique structures of membrane protein:antibody complexes have already been determined. In the majority of cases, hybridoma-derived antibody fragments either in Fab or Fv fragment format were employed for these complexes. We will briefly introduce the background and current status of the strategy and describe in detail the current protocols of well-established methods for the immunization, the selection, and the characterization of antibodies, as well as the cloning, the production, and the purification of recombinant antibodies useful for structural analysis of membrane proteins.
Assuntos
Clonagem Molecular/métodos , Fragmentos de Imunoglobulinas/química , Proteínas de Membrana/química , Proteínas Recombinantes/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Cristalização/métodos , Cristalografia por Raios X/métodos , Escherichia coli/genética , Imunização , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/isolamento & purificação , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos Endogâmicos BALB C , Pichia/genética , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Atovaquone, a substituted hydroxynaphthoquinone, is a potent antimalarial drug that acts by inhibiting the parasite's mitochondrial cytochrome bc1 complex (cyt bc1). Mutations in cyt bc1 confer atovaquone resistance. Here we describe the X-ray structure of mitochondrial cyt bc1 from Saccharomyces cerevisiae with atovaquone bound in the catalytic Qo site, at 3.0-Å resolution. A polarized H-bond to His181 of the Rieske protein in cyt bc1 traps the ionized hydroxyl group of the drug. Side chains of highly conserved cytochrome b residues establish multiple non-polar interactions with the napththoquinone group, whereas less-conserved residues are in contact with atovaquone's cyclohexyl-chlorophenyl tail. Our structural analysis reveals the molecular basis of atovaquone's broad target spectrum, species-specific efficacies and acquired resistances, and may aid drug development to control the spread of resistant parasites.
