On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA

Biemans-Oldehinkel, Esther

doi:10.1093/emboj/cdg581

Cited by 60 publications

(68 citation statements)

References 86 publications

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“…This finding is reminiscent of type I ABC importers with one or two solute-binding domains fused to one TMD (26). The analysis of such a transporter, the OpuA complex of Lactococcus lactis, revealed that two SBPs cooperatively interact but one is sufficient for transport (27).…”

Section: Discussionmentioning

confidence: 94%

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...

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Section: Discussionmentioning

confidence: 94%

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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“…14 C]glycine betaine to a final concentration of 51 M (more than 10-fold above the K m for transport and K D for binding) (40). At given time intervals, 26-l samples were taken and diluted with 2 ml of ice-cold isotonic assay buffer.…”

Section: Methodsmentioning

confidence: 99%

Cystathionine β-Synthase (CBS) Domains 1 and 2 Fulfill Different Roles in Ionic Strength Sensing of the ATP-binding Cassette (ABC) Transporter OpuA

Karasawa

Erkens

Berntsson

et al. 2011

Journal of Biological Chemistry

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The cystathionine ␤-synthase module of OpuA in conjunction with an anionic membrane surface acts as a sensor of internal ionic strength, which allows the protein to respond to osmotic stress. We now show by chemical modification and cross-linking studies that CBS2-CBS2 interface residues are critical for transport activity and/or ionic regulation of transport, whereas CBS1 serves no functional role. We establish that Cys residues in CBS1, CBS2, and the nucleotide-binding domain are more accessible for cross-linking at high than low ionic strength, indicating that these domains undergo conformational changes when transiting between the active and inactive state. Structural analyses suggest that the cystathionine ␤-synthase module is largely unstructured. Moreover, we could substitute CBS1 by a linker and preserve ionic regulation of transport. These data suggest that CBS1 serves as a linker and the structured CBS2-CBS2 interface forms a hinge point for ionic strength-dependent rearrangements that are transmitted to the nucleotide-binding domain and thereby affect translocation activity.

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“…Membrane vesicles of the L. lactis Opu401/pNZOpuA derivative were isolated as described (13), and fused 1:10 with preformed liposomes, composed of dioleoyl-phosphatidylethanolamine (DOPE), dioleoyl-phosphatidylglycerol (DOPG), and dioleoyl-phosphatidylcholine (DOPC). The hybrid membranes were obtained by two freeze/thaw cycles, followed by extrusion through a polycarbonate filter (200 nm pore size); for details on the preparation of liposomes and the freeze-thaw-extrusion steps, we refer to Geertsma et al (14).…”

Section: Synthesis Of Hybrid Membranesmentioning

confidence: 99%

“…For osmotically activated transport, the hybrid membranes were diluted to a lipid concentration of 4.5-12 mg/ml into assay buffer (90 mM potassium P i , pH 7.0 (unless specified otherwise), supplemented with different concentrations of KCl). Following incubation for 2 min at 30°C, the transport reaction was initiated by the addition of [ 14 C]glycine betaine (Amersham Biosciences) to a final concentration of 31 M (more than 10-fold above the K m for transport and K D for binding) (13). At given time intervals, 40-l samples were taken and diluted with 2 ml of ice-cold isotonic assay buffer.…”

Section: Transport Assaysmentioning

confidence: 99%

Engineering of Ion Sensing by the Cystathionine β-Synthase Module of the ABC Transporter OpuA

Mahmood¹,

Biemans-Oldehinkel²,

Poolman

2009

Journal of Biological Chemistry

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We have previously shown that the C-terminal cystathionine ␤-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I in ). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mM KCl (or NaCl), supplemented to medium of 30 mM potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5-7°C and the temperature coefficient Q 10 was lowered from 8 to ϳ1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state.In their natural habitats microorganisms are often exposed to changes in the concentration of solutes in the environment (1). A sudden increase in the medium osmolality results in loss of water from the cell, loss of turgor, a decrease in cell volume, and an increase in intracellular osmolyte concentration. Osmoregulatory transporters such as OpuA in Lactococcus lactis, ProP in Escherichia coli, and BetP in Corynebacterium glutamicum diminish the consequences of the osmotic stress by mediating the uptake of compatible solutes upon an increase in extracellular osmolality (2-4). For the ATP-binding cassette (ABC) 5 transporter OpuA, it has been shown that the system, reconstituted in proteoliposomes, is activated by increased concentrations of lumenal ions (increased internal ionic strength) (2,5,6). This activation is instantaneous both in vivo and in vitro and only requires threshold levels of ionic osmolytes. Moreover, the ionic threshold for activation is highly dependent of the ionic lipid content (charge density) of the membrane and requires the presence of so-called cystathionine ␤-synthase (CBS) domains, suggesting that the ionic signal is transduced to the transporter via critical interactions of the protein with membrane lipids.The ABC transporter OpuA consists of two identical nucleotide-binding domains (NBD) fused to CBS domains and two identical substrate-binding domains fused to transmembrane domains. The NBD-CBS and substrate-binding domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively. T...

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On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA

Cited by 60 publications

References 86 publications

Conformational plasticity of the type I maltose ABC importer

Conformational plasticity of the type I maltose ABC importer

Cystathionine β-Synthase (CBS) Domains 1 and 2 Fulfill Different Roles in Ionic Strength Sensing of the ATP-binding Cassette (ABC) Transporter OpuA

Engineering of Ion Sensing by the Cystathionine β-Synthase Module of the ABC Transporter OpuA

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