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1.
Proc Natl Acad Sci U S A ; 109(15): 5687-92, 2012 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-22451937

RESUMO

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9-2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.


Assuntos
Doxorrubicina/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Minociclina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Preparações Farmacêuticas/metabolismo , Sítios de Ligação , Biocatálise , Transporte Biológico , Doxorrubicina/química , Minociclina/química , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína
2.
Antimicrob Agents Chemother ; 58(8): 4767-72, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24914123

RESUMO

Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the resistance nodulation and cell division (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å(2) are less well transported than other substrates.


Assuntos
Substituição de Aminoácidos , Antibacterianos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Xenobióticos/química , Antibacterianos/farmacologia , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Doxorrubicina/química , Doxorrubicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Minociclina/química , Minociclina/farmacologia , Simulação de Acoplamento Molecular , Peso Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Xenobióticos/farmacologia
3.
Biochim Biophys Acta ; 1808(9): 2189-96, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21616055

RESUMO

The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.


Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Espectrometria de Massas/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Repetição de Anquirina , Cromatografia em Camada Fina/métodos , Cristalização , Cristalografia por Raios X/métodos , Detergentes/farmacologia , Dimerização , Eletroforese , Eletroforese Capilar , Proteínas de Escherichia coli/química , Ligantes , Micelas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Mutação , Fosfolipídeos/química , Ultracentrifugação/métodos
4.
J Mol Biol ; 429(24): 3863-3874, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-28987732

RESUMO

The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.


Assuntos
Antibacterianos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Conformação Proteica , Sítios de Ligação , Transporte Biológico , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ligação Proteica
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