Determination of the Number of Active GroES Subunits in the Fused Heptamer GroES Required for Interactions with GroEL*
Determination of the Number of Active GroES Subunits in the Fused Heptamer GroES Required for Interactions with GroEL*
复制标题
确定与 GroEL* 相互作用所需的融合七聚体 GroES 中活性 GroES 亚基的数量
DOI：
10.1074/jbc.m709825200
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发表时间：
2008
影响因子：
4.8
通讯作者：
F. Motojima
中科院分区：
文献类型：
--
作者：
T. Nojima;S. Murayama;Masasuke Yoshida;F. Motojima
双七聚体环伴侣蛋白GroEL将变性底物蛋白、ATP和GroES结合到相同的七聚体环上，并将底物封装到GroES下方的中心空腔中，在那里发生有效折叠。GroES是盘状七聚体，并且每个亚基具有GroEL结合环。负责GroES结合的GroEL亚基的残基在很大程度上与参与底物结合的残基重叠，并且当GroES结合到GroEL/底物复合物时GroES可以取代底物的机制仍有待澄清。为了解决这个问题，我们产生了单一的多肽GroES融合七个亚基与各种组合的活性和GroEL结合缺陷的亚基。融合GroES变体的功能测试表明，四个活性GroES亚基是有效形成稳定的GroEL/GroES复合物所必需的，五个亚基是生产性GroEL/底物/GroES复合物所必需的。缺陷GroES亚基数量的增加导致封装和折叠的减慢。这些结果表明存在中间体GroEL/底物/GroES复合物，其中底物和GroES通过共享七个共同的结合位点与GroEL结合。
A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.