Zhou Zhen, Wang Xiaohui, Liu Hao-Yang, Zou Xiaoqin, Li Min, Hwang Tzyh-Chang
Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO 65211, USA.
J Gen Physiol. 2006 Oct;128(4):413-22. doi: 10.1085/jgp.200609622. Epub 2006 Sep 11.
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.
囊性纤维化跨膜传导调节因子（CFTR）是ABC（ATP结合盒）转运蛋白家族的成员，是一种氯离子通道，其活性受蛋白激酶依赖性磷酸化的控制。磷酸化的CFTR的开放和关闭（门控）与CFTR的两个核苷酸结合结构域（NBD1和NBD2）处的ATP结合和水解相关联。最近的研究表明，开放通道构象反映了CFTR的两个NBD的头对尾二聚化，正如在其他ABC转运蛋白的NBD中所见（韦尔加尼等人，2005年）。这两个ATP结合位点在NBD二聚化动力学中是否发挥同等作用，进而在CFTR通道门控中发挥同等作用，仍未确定。基于NBD的晶体结构、序列比对和同源建模，我们鉴定出两个关键的芳香族氨基酸（NBD1中的W401和NBD2中的Y1219），它们与结合的ATP的腺嘌呤环配位。将W401残基转化为甘氨酸（W401G）对开放速率对[ATP]的敏感性影响很小，但Y1219残基处的相同突变显著降低了对ATP的表观亲和力，降低了50倍以上，表明这两个ATP结合位点在通道开放中具有不同作用。然而，W401G突变缩短了开放时间常数。对我们数据的能量分析表明，NBD1而非NBD2处ATP结合的自由能对开放状态的能量学有显著贡献。CFTR的两个NBD的这种动力学和能量不对称表明在通道门控期间NBD的不对称运动。通道的开放由NBD2位点处的ATP结合引发，而NBD1位点处NBD二聚体的分离构成通道关闭的限速步骤。
