ATP‐dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters

Ames, Giovanna Ferro‐Luzzi; Lecar, Harold

doi:10.1096/fasebj.6.9.1377140

Cited by 112 publications

(60 citation statements)

References 31 publications

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“…Mutation of G319 might slow down conformational changes that are required during the catalytic cycle. This region of the ABC domain may serve an intramolecular signalling function coupling the hydrolysis of ATP with the membrane-spanning components (Ames & Lecar, 1992). Glycines are expected to effect transmission of conformational changes within proteins, since they lack a lateral chain and appear crucial in dynamic functionality.…”

Section: Mutations In the Cydd1 Allele And The Nucleotide-binding Domainmentioning

confidence: 99%

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

Cruz-Ramos

Cook

et al. 2004

Microbiology

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Cytochrome bd is a respiratory quinol oxidase in Escherichia coli. Besides the structural genes (cydA and cydB) encoding the oxidase complex, the cydD and cydC genes, encoding an ABC-type transporter, are required for assembly of this oxidase. Recently, cysteine has been identified as a substrate (allocrite) that is transported from the cytoplasm by CydDC, but the mechanism of cysteine export to the periplasm and its role there remain unknown. To initiate an understanding of structure-function relationships in CydDC, its membrane topography was analysed by generating protein fusions between random and selected residues in the two polypeptides with both alkaline phosphatase and b-galactosidase. CydD and CydC are experimentally shown each to have six transmembrane segments, two major cytoplasmic loops and three minor periplasmic loops; both termini of each protein face the cytoplasm. The cydD1 allele is shown to have two point mutations (G319D, G429E) within the ATP-binding domain of CydD; either mutation alone is sufficient to cause loss or severe reduction of cytochrome bd assembly. A comparative sequence analysis prompted the targeting of residues in CydD for site-directed mutational analysis, which identified (i) the 'start' methionine residue, (ii) essential residues in the ATP-binding site (Walker sequence A) and (iii) a duplicated positively charged heptameric motif, R-G/T-L/M-X-T/V-L-R, in CydD cytoplasmic loop II. The replacement of arginines in these motifs with glycines resulted in Cyd " phenotypes; however, activity could be restored at these positions by replacing the glycine with lysine or histidine and hence returning the positive charge. The conservation of these charges in CydD-like proteins indicates functional importance. Evolutionary aspects of bacterial cyd genes are discussed. INTRODUCTIONEscherichia coli uses two membrane-bound terminal oxidases for aerobic respiration, cytochromes bo9 and bd (Gennis & Stewart, 1996). The genes encoding the cytochrome bd quinol oxidase, cydAB, are expressed both aerobically and anaerobically, but expression is maximal during aerobic stationary phase or in low oxygen environments (Cotter et al., 1990;. Consistent with this expression profile is the extraordinarily high affinity of cytochrome bd for O 2 (K m =3-8 nM; D' Mello et al., 1996). Cytochrome bd comprises two integral membrane polypeptides, subunit I (CydA, 58 kDa) and subunit II (CydB, 43 kDa). Subunit I contains haem b 558 directly involved in ubiquinol oxidation whilst two further haems, b 595 and d, in a catalytic binuclear centre are shared by both subunits (Jünemann, 1997;Borisov et al., 2001). All three haems are probably near the periplasmic side of the membrane (Osborne & Gennis, 1999). Loss of cytochrome bd causes a complex phenotype that extends beyond the lack of a functional oxidase. The Cyd 2 phenotype in E. coli includes (i) an inability to exit aerobically from stationary phase at 37 uC (Siegele et al., 1996); (ii) sensitivity to high temperature (Delaney et al., 1992), (iii) increased...

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Section: Mutations In the Cydd1 Allele And The Nucleotide-binding Domainmentioning

confidence: 99%

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

Cruz-Ramos

Cook

et al. 2004

Microbiology

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“…These latter are flanked by a stretch of sequence, that consists of α-helices , forming the helical domain (Ames and Lecar 1992). Notably, a few residues after the Walker B, many NBDs carry a SALD motif (black) (D-loop), also quite diagnostic but largely ignored so far.…”

Section: Introductionmentioning

confidence: 99%

The motor domains of ABC-transporters

Oswald

Holland

Schmitt

2006

Naunyn Schmied Arch Pharmacol

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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“…The cystic fibrosis transmembrane conductance regulator (CFTR) 1 belongs to the superfamily of ATP-binding cassette transporters, members of which are characterized structurally by two ATP-binding motifs and functionally by their ability to couple ATP hydrolysis to transport of a substrate molecule across a membrane (1,2). Although a role for CFTR in solute transport has been proposed (3)(4)(5)(6), no substrate has yet been identified.…”

mentioning

confidence: 99%

“…ʈ Established Investigator of the American Heart Association. 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NBF, nucleotide-binding fold; PKA, protein kinase A; ␤-ME, ␤-mercaptoethanol; NEM, N-ethylmaleimide; DTT, dithiothreitol; SNAP, S-nitroso-N-acetyl-penicillamine; ATP␥S, adenosine-␥-thiotriphosphate; DTNB, 5,5Ј-dithiobis-2-nitrobenzoic acid; Po, open probability; MOPS, 4-morpholinepropanesulfonic acid; POPE,…”

mentioning

confidence: 99%

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Harrington¹,

Gunderson²,

Kopito³

1999

Journal of Biological Chemistry

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Gating of the cystic fibrosis Cl؊ channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein.With Ca 2؉ as the dominant divalent cation instead of Mg 2؉ , the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca 2؉ . In contrast, "soft" divalent cations such as Cd 2؉ interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.The cystic fibrosis transmembrane conductance regulator (CFTR) 1 belongs to the superfamily of ATP-binding cassette transporters, members of which are characterized structurally by two ATP-binding motifs and functionally by their ability to couple ATP hydrolysis to transport of a substrate molecule across a membrane (1, 2). Although a role for CFTR in solute transport has been proposed (3-6), no substrate has yet been identified. CFTR remains unique among ATP-binding cassette transporters in that it couples ATP hydrolysis to the opening of a Cl Ϫ channel (6, 7), reviewed in (8). The role of ATP hydrolysis in the CFTR gating process was inferred initially from studies of CFTR gating in electrophysiological experiments using poorly hydrolyzable ATP analogs and transition state phosphate analogs (9 -12). Direct measurement of ATPase activity in purified reconstituted CFTR has confirmed that it functions as an ATPase, and the measured turnover rate of the phosphorylated form (0.5-1 s Ϫ1 ) is consistent with a model in which opening and closing of the CFTR channel gate is coupled directly to ATP hydrolysis (13).The role of ATP hydrolysis by the two nucleotide-binding folds (NBF1 and NBF2) in mediating channel gating has been elucidated by studies examining CFTR channels individually mutated in each of the NBFs (14 -16). These papers presented models in which two ATP hydrolytic events, one occurring at NBF1 and one at NBF2, are involved first in opening and then closing the CFTR channel. In one model a single cycle of ATP hydrolysis occurs at NBF1 to directly open the channel (8,14,16,17). Alternatively, a second model proposes that ATP hydrolysis at NBF1 converts the channel into a gatable state from which nucleotide binding at NBF2 can then open the channel (15). In both mod...

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ATP‐dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters

Cited by 112 publications

References 31 publications

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

The motor domains of ABC-transporters

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

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