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ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.

SUBMITTER: Randak CO

PROVIDER: S-EPMC3779764 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

Randak Christoph O CO Dong Qian Q Ver Heul Amanda R AR Elcock Adrian H AH Welsh Michael J MJ

The Journal of biological chemistry 20130806 38

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome prote ...[more]

PMID: 23921386

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Project description:BackgroundThe ATP-Binding Cassette (ABC) functional superfamily includes integral transmembrane exporters that have evolved three times independently, forming three families termed ABC1, ABC2 and ABC3, upon which monophyletic ATPases have been superimposed for energy-coupling purposes [e.g., J Membr Biol 231(1):1-10, 2009]. The goal of the work reported in this communication was to understand how the integral membrane constituents of ABC uptake transporters with different numbers of predicted or established transmembrane segments (TMSs) evolved. In a few cases, high resolution 3-dimensional structures were available, and in these cases, their structures plus primary sequence analyses allowed us to predict evolutionary pathways of origin.ResultsAll of the 35 currently recognized families of ABC uptake proteins except for one (family 21) were shown to be homologous using quantitative statistical methods. These methods involved using established programs that compare native protein sequences with each other, after having compared each sequence with thousands of its own shuffled sequences, to gain evidence for homology. Topological analyses suggested that these porters contain numbers of TMSs ranging from four or five to twenty. Intragenic duplication events occurred multiple times during the evolution of these porters. They originated from a simple primordial protein containing 3 TMSs which duplicated to 6 TMSs, and then produced porters of the various topologies via insertions, deletions and further duplications. Except for family 21 which proved to be related to ABC1 exporters, they are all related to members of the previously identified ABC2 exporter family. Duplications that occurred in addition to the primordial 3 → 6 duplication included 5 → 10, 6 → 12 and 10 → 20 TMSs. In one case, protein topologies were uncertain as different programs gave discrepant predictions. It could not be concluded with certainty whether a 4 TMS ancestral protein or a 5 TMS ancestral protein duplicated to give an 8 or a 10 TMS protein. Evidence is presented suggesting but not proving that the 2TMS repeat unit in ABC1 porters derived from the two central TMSs of ABC2 porters. These results provide structural information and plausible evolutionary pathways for the appearance of most integral membrane constituents of ABC uptake transport systems.ConclusionsAlmost all integral membrane uptake porters of the ABC superfamily belong to the ABC2 family, previously established for exporters. Most of these proteins can have 5, 6, 10, 12 or 20 TMSs per polypeptide chain. Evolutionary pathways for their appearance are proposed.

Dataset Information

ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

Publications

ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

Similar Datasets

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