Article

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium

Humboldt-Universität zu Berlin, Berlín, Berlin, Germany
Biochimica et Biophysica Acta (Impact Factor: 4.66). 10/2002; 1565(1):64-72. DOI: 10.1016/S0005-2736(02)00506-0
Source: PubMed

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.

Download full-text

Full-text

Available from: Melanie Brinkmann, Jun 20, 2015
2 Followers
 · 
107 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The ATP binding cassette (ABC-) transporter mediating the uptake of maltose/maltodextrins in Escherichia coli/Salmonella enterica serovar Typhimurium is one of the best characterized systems and serves as a model for studying the molecular mechanism by which ABC importers exert their functions. The transporter is composed of a periplasmic maltose binding protein (MalE), and a membrane-bound complex (MalFGK(2)), comprising the pore-forming hydrophobic subunits, MalF and MalG, and two copies of the ABC subunit, MalK. We report on the isolation of suppressor mutations within malFG that partially restore transport of a maltose-negative mutant carrying the malK809 allele (MalKQ140K). The mutation affects the conserved LSGGQ motif that is involved in ATP binding. Three out of four suppressor mutations map in periplasmic loops P2 and P1 respectively of MalFG. Cross-linking data revealed proximity of these regions to MalE. In particular, as demonstrated in vitro and in vivo, Gly-13 of substrate-free and substrate-loaded MalE is in close contact to Pro-78 of MalG. These data suggest that MalE is permanently in close contact to MalG-P1 via its N-terminal domain. Together, our results are interpreted in favour of the notion that substrate availability is communicated from MalE to the MalK dimer via extracytoplasmic loops of MalFG, and are discussed with respect to a current transport model.
    Molecular Microbiology 01/2008; 66(5):1107-22. DOI:10.1111/j.1365-2958.2007.05982.x · 5.03 Impact Factor
  • [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The maltose transporter MalFGK(2), together with the substrate-binding protein MalE, is one of the best-characterized ABC transporters. In the conventional model, MalE captures maltose in the periplasm and delivers the sugar to the transporter. Here, using nanodiscs and proteoliposomes, we instead find that MalE is bound with high-affinity to MalFGK2 to facilitate the acquisition of the sugar. When the maltose concentration exceeds the transport capacity, MalE captures maltose and dissociates from the transporter. This mechanism explains why the transport rate is high when MalE has low affinity for maltose, and low when MalE has high affinity for maltose. Transporter-bound MalE facilitates the acquisition of the sugar at low concentrations, but also captures and dissociates from the transporter past a threshold maltose concentration. In vivo, this maltose-forced dissociation limits the rate of transport. Given the conservation of the substrate-binding proteins, this mode of allosteric regulation may be universal to ABC importers.
    PLoS ONE 04/2012; 7(4):e34836. DOI:10.1371/journal.pone.0034836 · 3.53 Impact Factor