Analysis of nucleotide binding to p97 reveals the properties of a tandem AAA hexameric ATPase
Analysis of nucleotide binding to p97 reveals the properties of a tandem AAA hexameric ATPase
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DOI：
10.1074/jbc.m709632200
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发表时间：
2008-05-16
影响因子：
4.8
通讯作者：
Freemont, Paul S.
中科院分区：
文献类型：
--
作者：
Briggs, Louise C.;Baldwin, Geoff S.;Freemont, Paul S.
p97是内质网相关降解和细胞器生物发生中的必需伴侣，包含两个AAA结构域（D1和D2），并组装成稳定的六聚体。我们提出了一个定量分析的核苷酸结合的D1和D2结构域的p97，第一个详细的研究核苷酸结合到两个AAA域的这种类型的AAA+ ATP酶。我们报道了腺苷5 '-O-（硫代三磷酸）（ATP γ S）与D1和D2的结合亲和力相似，但ADP与D1的结合亲和力高于D2，这为D2中较高的ATP酶活性提供了解释。化学计量学测量表明，虽然ADP和ATP γ S都可以饱和D1中的所有6个核苷酸结合位点，但6个D2位点中只有3-4个可以同时结合ATP γ S。ATP γ S结合触发至少三个单体的下游协同构象变化，这涉及保守的精氨酸指，并且是ATP水解所必需的。
p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5'-O-(thiotriphosphate) (ATP gamma S) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATP gamma S can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATP gamma S simultaneously. ATP gamma S binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.