Stratford Fiona L L, Ramjeesingh Mohabir, Cheung Joanne C, Huan Ling-Jun, Bear Christine E
Programme in Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8.
Biochem J. 2007 Jan 15;401(2):581-6. doi: 10.1042/BJ20060968.
CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory 'R' domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1-NBD2 heterodimer.
囊性纤维化跨膜传导调节因子（CFTR）是ABC（ATP结合盒）膜蛋白超家族的成员，除了两个跨膜结构域（MSD）和调节性“R”结构域外，还拥有两个核苷酸结合结构域（NBD）。CFTR的两个NBD被模拟为异二聚体，通过在NBD界面的两个位点结合ATP而稳定。有人提出，ATP水解仅发生在其中一个位点，因为假定的催化碱基仅在CFTR的NBD2（Glu1371）中保守，而在NBD1中相应的残基是丝氨酸，即Ser573。此前，我们表明，与NBD1和NBD2相对应的CFTR片段可以纯化并共同重组形成具有ATP酶活性的异二聚体。在本研究中，我们表明这两个NBD片段在体内形成复合物，支持该模型系统用于评估Glu1371在ATP结合和水解中的作用。目前的研究表明，突变的NBD2（E1371Q）保留了NBD2的野生型核苷酸结合亲和力。另一方面，这种取代消除了共纯化复合物形成的ATP酶活性。有趣的是，在NBD1中用谷氨酸残基取代非保守的Ser573并没有赋予异二聚体额外的ATP酶活性，这表明多个残基在催化位点形成中起着至关重要的作用。这些发现提供了首个生化证据，表明沃克B残基Glu1371在NBD1-NBD2异二聚体赋予的ATP酶活性中起主要作用。
