RESUMO
The ATP binding cassette (ABC) transporter LmrA from the bacterium Lactococcus lactis is a homolog of the human multidrug resistance P-glycoprotein (ABCB1), the activity of which impairs the efficacy of chemotherapy. In a previous study, LmrA was shown to mediate ethidium efflux by an ATP-dependent proton-ethidium symport reaction in which the carboxylate E314 is critical. The functional importance of this key residue for ABC proteins was suggested by its conservation in a wider family of related transporters; however, the structural basis of its role was not apparent. Here, we have used homology modeling to define the structural environment of E314. The residue is nested in a hydrophobic environment that probably elevates its pKa, accounting for the pH dependency of drug efflux that we report in this work. Functional analyses of wild-type and mutant proteins in cells and proteoliposomes support our proposal for the mechanistic role of E314 in proton-coupled ethidium transport. As the carboxylate is known to participate in proton translocation by secondary-active transporters, our observations suggest that this substituent can play a similar role in the activity of ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Ácidos Carboxílicos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Transporte Biológico , Citoplasma/metabolismo , Bases de Dados de Proteínas , Resistência Microbiana a Medicamentos , Etídio/química , Concentração de Íons de Hidrogênio , Cinética , Lactococcus lactis/metabolismo , Potenciais da Membrana , Proteínas de Membrana Transportadoras/química , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Plasmídeos/metabolismo , Prótons , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Membrana/química , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cristalografia por Raios X , Dimerização , Resistência Microbiana a Medicamentos , Bombas de Íon/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Prótons , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
In Gram-negative bacteria, drug resistance is due in part to the activity of transmembrane efflux-pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer-membrane protein TolC, which is an allosteric channel, the trimeric inner-membrane proton-antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.
