Sequential Action of ATP-dependent Subunit Conformational Change and Interaction between Helical Protrusions in the Closure of the Built-in Lid of Group II Chaperonins
Sequential Action of ATP-dependent Subunit Conformational Change and Interaction between Helical Protrusions in the Closure of the Built-in Lid of Group II Chaperonins
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DOI：
10.1074/jbc.m805303200
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发表时间：
2008-12-12
影响因子：
4.8
通讯作者：
Yohda, Masafumi
中科院分区：
文献类型：
--
作者：
Kanzaki, Taro;Iizuka, Ryo;Yohda, Masafumi
ATP驱动II组伴侣蛋白的构象变化，从开盖底物结合构象到闭盖构象，以将未折叠的蛋白质包封在中心腔中。这种构象变化的详细机制仍然未知。为了阐明亚基的构象变化的环内合作行动，我们构建了热球菌伴侣蛋白复合物含有突变亚基在有序的方式，并检查其折叠和构象变化的能力。伴侣蛋白复合物包含野生型亚基和突变亚基，这些亚基具有受损的ATP依赖性构象变化能力或ATP水解活性，一个接一个地，表现出高的蛋白质重折叠能力。突变体亚基的效应与环中的数量和顺序相关。相反，使用缺乏螺旋突起的突变体严重影响了功能。有趣的是，这些突变体伴侣蛋白复合物也表现出ATP依赖性的构象变化所证明的小角X-射线散射，蛋白酶消化，和荧光团的荧光变化连接到螺旋突起的尖端。然而，它们的构象变化可能是短暂的。他们捕获变性蛋白质，即使在ATP的存在下，而ATP的加入削弱了野生型伴侣蛋白的能力，以保护柠檬酸合酶从热聚集。这些结果表明，ATP结合/水解导致的亚基的独立的构象变化，并进一步的构象变化的完全关闭的盖子是诱导和稳定的螺旋突起之间的相互作用。
ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.