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首页> 外文期刊>Journal of Molecular Biology >The flexible attachment of the N-domains to the ClpA ring body allows their use on demand
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The flexible attachment of the N-domains to the ClpA ring body allows their use on demand

机译：N结构域与ClpA环体的灵活连接使它们可以按需使用

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摘要

ClpA is an Hsp100 chaperone that uses the chemical energy of ATP to remodel various protein substrates to prepare them for degradation. It comprises two AAA+ modules and the N-domain, which is attached N-terminally to the first AAA+ module through a linker. On the basis of cryoelectron microscopic and X-ray crystallographic data it has been suggested that the linker confers mobility to the N-domain. In order to define the role of the N-domain in ClpAP-dependent substrate degradation we have generated a AN variant at the protein level by introducing a protease cleavage site. The ClpA molecule generated in this way lacks the N-domain and the associated linker but is impaired only slightly in the processing of substrates that are degraded independently of ClpS. In fact, it shows increased catalytic efficiency in the degradation of ssrA-tagged GFP compared to ClpAwt. The role of the linker attaching the N-domain to the bulk of the molecule was probed by characterizing variants with different lengths of the linker. The degradation efficiency of a ClpS-dependent N-end rule substrate, FRliGFP, is reduced for linkers that are shorter or longer than natural linkers but remains the same for the variant where the linker is replaced by an engineered sequence of equivalent length. These results suggest that the flexible attachment of the N-domains to ClpA allows their recruitment to the pore on demand for certain substrates, while allowing them to move out of the way for substrates binding directly to the pore. (c) 2008 Elsevier Ltd. All rights reserved.
机译：ClpA是一种Hsp100分子伴侣，它利用ATP的化学能来重塑各种蛋白质底物以使其降解。它包含两个AAA +模块和N域，该N域通过链接器N端连接到第一个AAA +模块。根据低温电子显微镜和X射线晶体学数据，已提出连接子赋予N域迁移率。为了确定N-结构域在依赖ClpAP的底物降解中的作用，我们通过引入蛋白酶切割位点在蛋白质水平上产生了AN变体。以这种方式生成的ClpA分子缺少N结构域和相关的连接子，但在处理独立于ClpS降解的底物时仅受到了一点损害。实际上，与ClpAwt相比，它在ssrA标签的GFP降解中显示出更高的催化效率。通过表征具有不同长度的接头的变体来探究接头将N-结构域连接至分子主体的作用。对于比天然接头短或长的接头，依赖ClpS的N端规则底物FRliGFP的降解效率会降低，但对于其中接头被等效长度的工程序列替代的变体，降解效率保持不变。这些结果表明，N结构域与ClpA的柔性连接使它们可以按需将某些底物募集到孔中，同时允许它们移开，使底物直接结合到孔中。 （c）2008 Elsevier Ltd.保留所有权利。

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