Wei J, Gaut J R, Hendershot L M
Department of Tumor Cell Biollogy, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
J Biol Chem. 1995 Nov 3;270(44):26677-82. doi: 10.1074/jbc.270.44.26677.
In the present study, we produced single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis. These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from BiP. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function. Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to BiP is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary. The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity.
在本研究中，我们在仓鼠BiP的ATP结合位点产生单点突变，分离重组蛋白，并根据它们对ATP和ADP的亲和力、核苷酸结合后发生构象变化的能力以及ATP水解速率对其进行表征。这些分析使我们能够将突变体分为三组：ATP水解（T229G）、ATP结合（G226D、G227D）和ATP诱导构象（T37G）突变体，并测试这些活性在体外ATP介导的蛋白质从BiP释放中的作用。所有这三类突变体仍然能够结合肽，表明核苷酸不参与此功能。向野生型BiP或T229G突变体添加ATP会导致体外结合肽的释放，证实蛋白质释放不需要ATP水解。ATP不会解离G226D、G227D或T37G突变体BiP-肽复合物，这表明ATP与BiP的结合不足以释放结合的肽，但BiP中ATP诱导的构象变化是必要的。鉴定在ATP水解的这些步骤中存在缺陷的BiP突变体将有助于在体内剖析核苷酸在BiP活性中的作用。
