The high temperature requirement A (HtrA) proteases (also termed Deg proteases) play important roles in diverse organisms by regulating protein quality and quantity. One of the 16 Arabidopsis homologs, Deg9, is located in the nucleus where it modulates cytokinin- and light-mediated signalling via degrading the ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4). To uncover the structural features underlying the proteolytic activity of Deg9, we determined its crystal structure. Unlike the well-established trimeric building block of HtrAs, Deg9 displays a novel octameric structure consisting of two tetrameric rings that have distinct conformations. Based on the structural architecture, we generated several mutant variants of Deg9, determined their structure and tested their proteolytic activity towards ARR4. The results of the structural and biochemical analyses allowed us to propose a model for a novel mechanism of substrate recognition and activity regulation of Deg9. In this model, protease activation of one tetramer is mediated by en-bloc reorientation of the protease domains to open an entrance for the substrate in the opposite (inactive) tetramer. This study provides the structural basis for understanding how the levels of nuclear signal components are regulated by a plant protease.

中文翻译：

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Deg9的晶体结构揭示了一种新型的八聚体型HtrA蛋白酶。

高温需求A（HtrA）蛋白酶（也称为Deg蛋白酶）通过调节蛋白质的质量和数量在各种生物中发挥重要作用。16种拟南芥同系物之一Deg9位于细胞核中，可通过降解拟南芥反应调节因子4（ARR4）来调节细胞分裂素和光介导的信号传导。为了揭示Deg9蛋白水解活性的基础结构特征，我们确定了其晶体结构。与成熟的HtrA三聚体构建块不同，Deg9显示了一种新颖的八聚体结构，该结构由两个具有不同构象的四聚体环组成。基于结构架构，我们生成了多个Deg9突变体变体，确定了其结构并测试了其对ARR4的蛋白水解活性。结构和生化分析的结果使我们能够为底物识别和Deg9活性调节的新机制提出一个模型。在该模型中，一个四聚体的蛋白酶激活是通过蛋白酶结构域的整体重新定向来介导的，从而为相反（无活性）的四聚体中的底物打开一个入口。这项研究为理解植物蛋白酶如何调节核信号成分的水平提供了结构基础。