Nucleotide Dependent Monomer/Dimer Equilibrium of OpuAA, the Nucleotide-binding Protein of the Osmotically Regulated ABC Transporter OpuA from Bacillus subtilis

Horn, Christian; Bremer, Erhard; Schmitt, Lutz

doi:10.1016/j.jmb.2003.09.079

Cited by 45 publications

(86 citation statements)

References 60 publications

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“…In our experiment, we also observed that the nucleotide binding affinity strongly decreases with temperature. At temperatures close to the growth temperature of S. solfataricus, the nucleotide binding affinity of GlcV is comparable to what has been observed for the NBDs of other ABC transporters, i.e., values in the micromolar range (15, 27,28,33). However, at low temperatures, the nucleotides bind with nanomolar affinity.…”

Section: Discussionsupporting

confidence: 61%

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

et al. 2006

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ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

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Section: Discussionsupporting

confidence: 61%

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

et al. 2006

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“…For the competitive binding assay, after the proteins were incubated with radioactive nucleotides, different concentrations of non-radioactive ATP or GTP were added to compete on ice for 10 minutes, and then UV-irradiation was performed as described (44). The competitive binding data were fitted to the linear regression by the Prism (GraphPad Software Inc., Ober-Olm, Germany) to obtain the best fit and the half of effective concentration, which were displayed as R 2 and EC 50 , respectively.…”

Section: Atp/gtp Uv Cross-linking and Competitive Bindingmentioning

confidence: 99%

Molecular Basis for Differential Nucleotide Binding of the Nucleotide-Binding Domain of ABC-Transporter CvaB

et al. 2006

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The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP, and indeed highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide binding domain predicted that the aromatic stacking region of CvaB (Y 501 DSQ-loop) had a role in the differential binding of nucleotides, and the Ser503 and Gln504 provided potential hydrogen-bonds to GTP but not to ATP. Site-directed mutagenesis of the Y 501 DSQ-loop: mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen-bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature-effect assays on nucleotide binding and hydrolysis further indicated that the double-mutant protein had largely eliminated the difference to substrate ATP and GTP and behaved more closely to the NBD of typical ABC transporter HlyB. Therefore, we conclude that the Ser 503 and Gln 504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference. *To whom correspondence should be addressed: Phone, 404-651-3109; fax, 404-651-2509; e-mail, biopct@langate.gsu.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptEscherichia coli, ColV is secreted by a dedicated type-I exporter system across both the cytoplamic and outer membranes directly into the environment outside the cells. At least three protein components participate in the ColV export system: two plasmid-born proteins CvaA and CvaB in the cytoplasmic membrane and a host chromosomal gene product TolC in the outer membrane (2,3).The cytoplasmic membrane protein CvaB plays pivotal roles in the ColV export system. CvaB belongs to the family of ATP-binding cassette transporters (4,5), like the α-hemolysin exporter HlyB (6), the Cystic Fibrosis trans-membrane conductance regulator (7), and the multidrug resistance P-glycoprotein (8). The N-terminal proteolytic domain of CvaB processes the leader region of ColV precursor at the double-glycine site (9-11), the trans-membrane domains in the middle of CvaB form the cytoplasmic channel (3,9,12), and the C-terminal nucleotide-binding domain supplies energy for ColV secretion. Although the sequence of the proteolytic Nterminal domain of CvaB is unique among ABC transporters (11), the C-terminal nucleotide binding domain (NBD) retains a high level of homology with other family members (4,5,13). Several highly conserved ABC-NBD motifs are found in the CvaB sequence, su...

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“…In contrast to the half size transporters, full size transporters have all 4 subunits encoded by one gene (TMD-NBD-TMD*-NBD*). Other variations are fused NBDs such as RbsA, (Zaitseva et al 1996), fused TMDs [FhuC,B, (Groeger and Koster 1998)] or NBDs with an additional domain (such as OpuAA) (Horn et al 2003).…”

Section: Introductionmentioning

confidence: 99%

The motor domains of ABC-transporters

Oswald

Holland

Schmitt

2006

Naunyn Schmied Arch Pharmacol

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133

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The transport of substrates across a cellular membrane is a vitally important biological function essential for cell survival. ATP-binding cassette (ABC) transporters constitute one of the largest subfamilies of membrane proteins, accomplishing this task. Mutations in genes encoding for ABC transporters cause different diseases, for example, Adrenoleukodystrophy, Stargardt disease or Cystic Fibrosis. Furthermore, some ABC transporters are responsible for multidrug resistance, presenting a major obstacle in modern cancer chemotherapy. In order to translocate the enormous variety of substrates, ranging from ions, nutrients, small peptides to large toxins, different ABC-transporters utilize the energy gained from ATP binding and hydrolysis. The ATP binding cassette, also called the motor domain of ABC transporters, is highly conserved among all ABC transporters. The ability to purify this domain rather easily presents a perfect possibility to investigate the mechanism of ATP hydrolysis, thus providing us with a detailed picture of this process. Recently, many crystal structures of the ATP-binding domain and the full-length structures of two ABC transporters have been solved. Combining these structural data, we have now the opportunity to analyze the hydrolysis event on a molecular level. This review provides an overview of the structural investigations of the ATPbinding domains, highlighting molecular changes upon ATP binding and hydrolysis.

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Nucleotide Dependent Monomer/Dimer Equilibrium of OpuAA, the Nucleotide-binding Protein of the Osmotically Regulated ABC Transporter OpuA from Bacillus subtilis

Cited by 45 publications

References 60 publications

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

Molecular Basis for Differential Nucleotide Binding of the Nucleotide-Binding Domain of ABC-Transporter CvaB

The motor domains of ABC-transporters

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