Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallographically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment.

中文翻译：

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通过对其C末端片段构象进行突变修饰，将功能不确定的同五聚体蛋白PbaA转化为蛋白酶体激活剂。

最近的生物信息学分析鉴定了古细菌中的蛋白酶体组装伴侣样蛋白PbaA和PbaB。PbaB形成同型四聚体并起蛋白酶体激活剂的作用，尽管存在明显的C端蛋白酶体激活基序，PbaA也不与蛋白酶体相互作用。我们发现，与具有突出的C末端片段的PbaB同型四聚体相反，PbaA的闭合构象主要形成封闭的构象，其C末端片段紧靠核心结构域。这促使我们基于功能强大的结构框架创建了一种新型的蛋白酶体激活剂。我们构建了一组嵌合蛋白，包括PbaA的同五聚体支架和PbaB的C末端片段，并对其进行蛋白酶体激活测定以及小角度X射线散射和高速原子力显微镜检查。结果表明，通过用相应的PbaB无序片段代替晶体学上无序的PbaA C端片段，可以增强开放构象和随后的蛋白酶体活化活性。而且，这些作用仅通过将两个谷氨酸残基掺入到PbaA的无序的C-末端区段中就可能产生，这可能是由于带负电荷的区段之间的静电排斥。因此，我们通过修饰其C末端区段成功赋予了功能不确定的蛋白质以蛋白酶体激活活性。