Three-dimensional reconstruction of the Saccharomyces cerevisiae multidrug resistance protein Pdr5p.
Pdr5p, the major multidrug exporter in Saccharomyces cerevisiae, is a member of the ATP-binding cassette (ABC) superfamily. Pdr5p shares similar mechanisms of substrate recognition and transport with the human MDR1-Pgp, despite an inverted topology of transmembrane and ATP-binding domains. The hexahistidine-tagged Pdr5p multidrug transporter was highly overexpressed in yeast strains where other ABC genes have been deleted. After solubilization and purification, the 160-kDa recombinant Pdr5p has been reconstituted into a lipid bilayer. Controlled detergent removal from Pdr5p-lipid-detergent micelles allowed the production of peculiar square-shaped particles coexisting with liposomes and proteoliposomes. These particles having 11 nm in side were well suited for single particle analysis by electron microscopy. From such analysis, a computed volume has been determined at 25-A resolution, giving insight into the structural organization of Pdr5p. Comparison with the reported structures of different bacterial ABC transporters was consistent with a dimeric organization of Pdr5p in the square particles. Each monomer was composed of three subregions corresponding to a membrane region of about 50 A in height that joins two well separated protruding stalks of about 40 A in height, ending each one with a cytoplasmic nucleotide-binding domain (NBD) lobe of about 50-60 A in diameter. The three-dimensional reconstruction of Pdr5p revealed a close arrangement and a structural asymmetric organization of the two NBDs that appeared oriented perpendicularly within a monomer. The existence of different angular positions of the NBDs, with respect to the stalks, suggest rotational movements during the catalytic cycle.
Ferreira-Pereira
Antonio
A
Departamento de Analises Clinicas e Toxicologicas, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, CEP 21949-900, Rio de Janeiro, Brazil.
Marco
Sergio
S
Decottignies
Annabelle
A
Nader
Joseph
J
Goffeau
André
A
Rigaud
Jean-Louis
JL
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002462:Cell Membrane / Q000502:physiology; D018360:Crystallography, X-Ray; D018432:Drug Resistance, Multiple; D008854:Microscopy, Electron; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
D018528:ATP-Binding Cassette Transporters; C092438:PDR5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins