Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications
Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications
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DOI：
10.1042/bj20020511
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发表时间：
2002-09-01
影响因子：
4.1
通讯作者：
Riordan, JR
中科院分区：
文献类型：
--
作者：
Gentzsch, M;Aleksandrov, A;Riordan, JR
囊性纤维化跨膜传导调节因子（CFTR）包含两个核苷酸结合结构域（NBD）或ATP结合盒（ABC），其表征膜转运蛋白的大家族。虽然已经确定了来自几种ABC蛋白的这些结构域的三维结构，但CFTR的情况并非如此，因此这些结构域仅基于序列比对来定义。CFTR的NBD 1的功能性C-末端边界通过氯离子通道功能的分析来定位[Chan，Csanady，Seto-Young，Nairn和加兹比（2000）J.Gen.Physiol.116，163 -180]。然而，NBD 2的C-末端和整个蛋白质中更下游的序列之间的边界，这对于其细胞定位和内吞周转是重要的，尚未确定。我们现在已经通过测定渐进C-末端截短对NBD 2通过8-叠氮基-ATP的光标记的影响来做到这一点，这反映了在该结构域的水解以及结合，以及对NBD 2依赖性通道门控本身的影响。以这种方式定义的边界在残基1420和1424之间，其对应于其结构已经确定的对齐的NBD中的最终P链。利用这一信息应有助于产生单分散NBD 2多肽的结构分析，这直到现在还不可能。所建立的边界包括在NBD 2内的四个残基（1413-1416）的疏水补丁，所述四个残基先前显示对于CFTR成熟和稳定性是必需的[Gentzsch和Riordan（2001）J.Biol.Chem.276，1291-1298]。该疏水簇在大多数ABC蛋白中是保守的，并且在与已知结构的那些比对时构成该结构域的倒数第二条β链，其可能参与基本的结构稳定β折叠形成。
The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116,163-180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final P-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413-1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291-1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate beta-strand of the domain which is likely to participate in essential structure-stabilizing beta-sheet formation.