Differences between cystic fibrosis transmembrane conductance regulator and HisP in the interaction with the adenine ring of ATP.
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is a member of the ATP-binding cassette transporter family. The most conserved features of this family are the nucleotide-binding domains. As in other members of this family, these domains bind and hydrolyze ATP; in CFTR this opens and closes the channel pore. The recent crystal structures of related bacterial transporters show that an aromatic residue interacts with the adenine ring of ATP to stabilize nucleotide binding. CFTR contains six aromatic residues that are candidates to coordinate the nucleotide base. We mutated each to cysteine and examined the functional consequences. None of the mutations disrupted channel function or the ability to discriminate between ATP, GTP, and CTP. We also applied [2-(triethylammonium)ethyl] methanethiosulfonate to covalently modify the introduced cysteines. The mutant channels CFTR-F429C, F430C, F433C, and F1232C showed no difference from wild-type CFTR, indicating that either the residues were not accessible to modification, or cysteine modification did not affect function. Although modification inactivated CFTR-Y1219C more rapidly than wild-type CFTR, and inactivation of CFTR-F446C was nucleotide-dependent; failure of these mutations to alter gating suggested that Tyr(1219) and Phe(446) were not important for nucleotide binding. The results suggest that ATP binding may not involve the coordination of the adenine ring by an aromatic residue analogous to that in some bacterial transporters. Taken together with earlier work, this study points to a model in which most of the binding energy for ATP is contributed by the phosphate groups.
Berger
A L
AL
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Welsh
M J
MJ
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000225:Adenine; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding
D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; C505032:CFTR protein, human; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C043828:histidine permease, Bacteria; D000255:Adenosine Triphosphate; D000225:Adenine