The amino-terminal domain of ClpB supports binding to strongly aggregated proteins
The amino-terminal domain of ClpB supports binding to strongly aggregated proteins
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DOI：
10.1074/jbc.m505653200
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发表时间：
2005-10-14
影响因子：
4.8
通讯作者：
Zolkiewski, M
中科院分区：
文献类型：
--
作者：
Barnett, ME;Nagy, M;Zolkiewski, M
细菌热休克蛋白、ClpB和DNAK形成一个双环蛋白系统，有效地重新激活聚集的蛋白。ClpB发生核苷酸依赖的自结合，形成环状低聚物。ClpB辅助的蛋白质聚集体的解离与底物通过寡聚体ClpB的中央通道的移位有关。在转位步骤之前的事件，如ClpB对聚集体的识别，还没有被探索，聚集结合位点在ClpB中的位置一直在讨论中。我们研究了在DNAK、DNAJ和GRPE存在的情况下，ClpB及其N端截短变异体ClpB Delta N对聚集的葡萄糖-6-磷酸脱氢酶(G6PDH)的重新激活作用。我们发现，随着G6PDH聚集体尺寸的增大，ClpB Delta N的伴侣活性显著低于全长ClpB。使用一个在两个ATP结合模块(E279Q/E678Q)中都有Walker B基序突变的ClpB的“底物陷阱”变体，我们证明了ClpB Delta N与G6PDH聚集体的亲和力显著低于全长ClpB。此外，我们在ClpB的N-末端结构域的表面发现了两个保守的酸性残基，它们支持与G6PDH聚集体的结合。这些N末端残基(Asp-103，Glu-109)对ClpB底物结合能力的贡献与位于ClpB通道入口处的保守Tyr一样多。综上所述，我们为ClpB的N-末端结构域在识别和结合强聚集蛋白中的重要作用提供了证据。
Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpB Delta N in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpB Delta N becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpB Delta N binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.