Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.
Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.
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DOI：
10.1085/jgp.201010399
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发表时间：
2010-05
期刊：
影响因子：
--
通讯作者：
Hwang TC
中科院分区：
文献类型：
--
作者：
Tsai MF;Li M;Hwang TC
囊性纤维化跨膜传导调节因子（CFTR）是三磷酸腺苷（ATP）结合盒（ABC）超家族的成员，是ATP门控氯离子通道。与其他ABC蛋白一样，CFTR包含两个核苷酸结合结构域（NBD），NBD 1和NBD 2，每个结构域容纳ATP结合位点。通常认为CFTR的打开-关闭循环（每次在1秒内完成）是由NBD 2中的快速ATP结合和水解事件驱动的。在这里，通过用配体交换方案在真实的时间内记录CFTR电流，我们证明了在许多这些门控循环中，NBD 1恒定地被稳定结合的ATP或8-N3-ATP分子占据数十秒。我们提供的证据表明，这种紧密结合的ATP或8-N3-ATP也与NBD 2的特征序列中的残基相互作用，这是NBD 1-NBD 2界面发生事件的一个警示信号。CFTR的开放状态已被证明代表两个ATP结合的NBD二聚体。我们的研究结果表明，在NBD 2中ATP水解后，通道关闭成“部分NBD二聚体”状态，其中NBD界面保持部分关闭，防止ATP从NBD 1解离，但允许水解产物的释放和下一个ATP的结合发生在NBD 2中。然后，CFTR的打开和关闭可以与NBD二聚体的形成和“部分”分离偶联。NBD 1中紧密结合的ATP分子偶尔会从部分二聚体状态中解离出来，导致NBD的无核苷酸单体状态。我们的数据，连同CFTR的NBD的其他结构/功能研究，表明这个过程是不可逆的，这意味着部分二聚体状态或单体状态的通道通过不同的途径进入开放状态。因此，我们提出了一个门控模型CFTR有两个不同的周期。我们的研究结果的其他ABC蛋白的结构和功能的意义进行了讨论。
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.
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