RESUMO
The bacterial heterodimeric ATP-binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug-resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide-binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug-mimetic azido-ethidium. Surprisingly, our analyses of azido-ethidium-labelled PatAB peptides point to ethidium binding in the PatA nucleotide-binding domain, with the azido moiety crosslinked to residue Q521 in the H-like loop of the degenerate nucleotide-binding site. Investigation into this compound and residue's role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium-dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium-like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non-competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide-binding domains.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Streptococcus pneumoniae , Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/metabolismo , Etídio/metabolismo , Hidrólise , Nucleotídeos/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMO
The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lipídeos de Membrana/metabolismo , Thermus thermophilus/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sítios de Ligação , Cardiolipinas/análise , Cardiolipinas/metabolismo , Enterococcus/enzimologia , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Lipídeos de Membrana/análise , Modelos Moleculares , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/metabolismo , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.