Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium

Landmesser, Heidi; Stein, Anke; Blüschke, Bettina; Brinkmann, Melanie M.; Hunke, Sabine; Schneider, Erwin

doi:10.1016/s0005-2736(02)00506-0

Cited by 37 publications

(64 citation statements)

References 46 publications

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“…No lag was obvious in the recovery of ATPase activity as a function of MalK concentration in our experiments (Fig. 4A), in agreement with published results for MalK from S. enterica serovar Typhimurium (11). These data suggest that MalK assembly is highly cooperative and that no inactive trimeric complexes accumulate.…”

supporting

confidence: 93%

Functional Reassembly of the Escherichia coli Maltose Transporter following Purification of a MalF-MalG Subassembly

et al. 2005

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supporting

confidence: 93%

Functional Reassembly of the Escherichia coli Maltose Transporter following Purification of a MalF-MalG Subassembly

et al. 2005

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“…Maltose uptake in E. coli requires the malE-encoded periplasmic maltose binding protein and the multisubunit ABC transporter MalFGK 2 (72,226,447). The soluble MalK subunits are tightly associated with the two permease subunits MalF and MalG (354,580).…”

Section: Inhibits Transcription Induction Mechanismmentioning

confidence: 99%

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Deutscher

Francke

Postma

2006

Microbiol Mol Biol Rev

1,187

769

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SUMMARY The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

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“…Gene expression and purification of MalFGK 2 and of MalE were performed according to methods described elsewhere (17,31,32). Spin labeling of the transporter mutants was performed according to ref.…”

Section: Methodsmentioning

confidence: 99%

Conformational plasticity of the type I maltose ABC importer

Böhm

Licht

Wuttge

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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ATP-binding cassette (ABC) transporters couple the translocation of solutes across membranes to ATP hydrolysis. Crystal structures of the Escherichia coli maltose importer (MalFGK 2 ) in complex with its substrate binding protein (MalE) provided unprecedented insights in the mechanism of substrate translocation, leaving the MalE-transporter interactions still poorly understood. Using pulsed EPR and cross-linking methods we investigated the effects of maltose and MalE on complex formation and correlated motions of the MalK 2 nucleotide-binding domains (NBDs A TP-binding cassette (ABC) systems are found in all kingdoms of life, forming one of the largest protein superfamilies (1-4). ABC transporters comprise two transmembrane domains (TMDs) that form the translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Based on biochemical and structural evidence, all ABC transporters are thought to function by an "alternate-access" mode, with the translocation path shuttling between an inward-facing and outward-facing conformation in response to substrate and ATP binding, the latter causing the NBD dimer to close (5).Canonical ABC importers are subdivided into type I and type II based on structural and biochemical evidence (6) and are dependent on extracellular (or periplasmic) substrate binding proteins (SBPs) (4), which play a crucial role in initial steps of the transport cycle (7-10). SBPs generally consist of two symmetrical lobes that rotate toward each other upon substrate binding (11).The type I maltose transporter of Escherichia coli/Salmonella is probably the best understood ABC transporter to date (12). It is composed of the periplasmic maltose binding protein, MalE, the membrane-integral subunits, MalF and MalG, and the nucleotidebinding subunits (NBDs), MalK 2 . The available crystal structures in the pretranslocation, ATP-, and vanadate-trapped states (13-16) have largely contributed to the understanding of the details of the inward-to outward-facing mechanism. The posthydrolytic state has not yet been crystallized, but data exist proposing this state to have a distinct structure from the other three known crystal snapshots (8, 9). MalE interacts with the transporter throughout the nucleotide cycle (15-18), and the X-ray structures revealed the switch of the binding protein from the liganded (closed) to the substrate-free (open) conformation concomitantly with ATP binding to the NBDs. Despite all these structural insights, the response of the transporter to substrate availability is poorly understood. Furthermore, the mechanism behind the stimulation of the ATPase activity of the transporter by unliganded MalE (10, 19) is still elusive (20,21).Our results show that the apo-and ADP-states of the transporter bind both open and closed MalE, but the complex adopts different periplasmic configurations. The ATP-state of the transporter can bind either closed MalE, inducing its opening and release of substrate to MalF or directly unliganded MalE, which generates a futile cycle. I...

show abstract

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium

Cited by 37 publications

References 46 publications

Functional Reassembly of the Escherichia coli Maltose Transporter following Purification of a MalF-MalG Subassembly

Functional Reassembly of the Escherichia coli Maltose Transporter following Purification of a MalF-MalG Subassembly

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Conformational plasticity of the type I maltose ABC importer

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