Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium
ABSTRACT The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V(max) of 1.25 micromol P(i)/min/mg and a K(m) of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.
Full-textDOI: · Available from: Melanie Brinkmann, Jun 03, 2015
SourceAvailable from: Sarah A Kuehne[Show abstract] [Hide abstract]
ABSTRACT: CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA_TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA. We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins.Nucleic Acids Research 04/2014; 42(11). DOI:10.1093/nar/gku309 · 8.81 Impact Factor
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ABSTRACT: Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward-facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains (NBDs), a transmembrane domain homodimer, RbsC2, and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed inter-subunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We are able to purify a full complex, RbsABC2, in the presence of stable, transition-state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilizes complex formation between RbsB and RbsC. These observations suggest RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 12/2014; 290(9). DOI:10.1074/jbc.M114.621573 · 4.60 Impact Factor
01/2010; 2010(3). DOI:10.1128/ecosalplus.3.3.3