Berger A L, Welsh M J
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 2000 Sep 22;275(38):29407-12. doi: 10.1074/jbc.M004790200.
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.
囊性纤维化跨膜传导调节因子（CFTR）氯离子通道是ATP结合盒转运蛋白家族的成员之一。该家族最保守的特征是核苷酸结合结构域。与该家族的其他成员一样，这些结构域结合并水解ATP；在CFTR中，这会打开和关闭通道孔。最近相关细菌转运蛋白的晶体结构表明，一个芳香族残基与ATP的腺嘌呤环相互作用以稳定核苷酸结合。CFTR含有六个芳香族残基，它们是协调核苷酸碱基的候选者。我们将每个残基突变为半胱氨酸，并研究其功能后果。没有一个突变破坏通道功能或区分ATP、GTP和CTP的能力。我们还应用[2-（三乙铵）乙基]甲硫基磺酸盐共价修饰引入的半胱氨酸。突变通道CFTR-F429C、F430C、F433C和F1232C与野生型CFTR没有差异，这表明要么这些残基无法被修饰，要么半胱氨酸修饰不影响功能。尽管CFTR-Y1219C的修饰比野生型CFTR更快地使其失活，并且CFTR-F446C的失活是核苷酸依赖性的；但这些突变未能改变门控，这表明Tyr(1219)和Phe(446)对核苷酸结合并不重要。结果表明，ATP结合可能不涉及类似于某些细菌转运蛋白中由芳香族残基对腺嘌呤环的配位。结合早期的研究工作，这项研究指向了一个模型，其中ATP的大部分结合能由磷酸基团贡献。
