Functional and structural principles of the MoxR/VWA chaperone system in target protein recognition and modification.

细胞中的许多基本过程，如DNA的复制和转录、蛋白质的折叠和展开或多蛋白质复合物的组装和拆卸，都依赖于将化学能转化为机械能的能力。AAA+ atp酶，也被称为分子马达，参与了许多这样的功能，并广泛分布于生命的所有三个领域。AAA+马达通常在ATP水解的驱动下重塑其目标大分子的构象。MoxR蛋白是AAA+ atp酶的一个亚家族，在细菌和古细菌中普遍存在。它们被认为是一组分子伴侣，主要参与蛋白质成熟和辅因子插入过程。有趣的是，许多MoxR蛋白在遗传上与含有血管性血友病因子A型（VWA）结构域的蛋白相连，表明它们一起起作用。然而，对MoxR蛋白的一般机制了解甚少，实验数据也有限。最近发现，来自反硝化副球菌的MoxR蛋白NorQ可促进非血红素铁辅助因子插入一氧化氮还原酶（cNOR）中，这是一种参与细菌反硝化链的多辅助因子膜蛋白。尽管具体的金属插入机制仍在很大程度上是推测性的，但已经发现VWA蛋白NorD在这一过程中是必不可少的，并且NorQ和NorD形成了一个复合物。许多研究支持MoxR/VWA蛋白的协同作用，并指出在rubisco、呼吸复合物I、富马酸脱氢酶、CO脱氢酶和甲醇脱氢酶等酶的成熟和调节中具有一般功能。本研究的总体目标是通过表达四个不同的MoxR/VWA对，包括NorQD作为我们的主要模型系统，来表征该伴侣子系统的共同结构和功能特征。我们将确定不同的伴侣复合物单独或与各自的目标相互作用的高分辨率低温电镜结构。这些结构数据将为进一步的诱变分析提供基础，并结合一套动力学技术来阐明这些未被研究的伴侣子的一般机制。这项研究的结果有望为如何在细胞中多种靶酶上有效地进行辅因子组装提供新的见解。更具体地说，我们将扩大我们对非血红素铁辅助因子插入的未知机制的了解。为了设计和生产用于化学工业的新型生物催化剂，有必要加深我们对金属-蛋白质组装的基本机制的理解。此外，许多MoxR/VWA蛋白对与细菌呼吸酶相互作用，这使得该伴侣体系统成为未来抗菌药物的潜在靶点。

Many fundamental processes in the cell such as replication and transcription of DNA, folding and unfolding of proteins or the assembly and disassembly of multi-protein complexes depend on the ability to convert chemical energy into mechanical work. AAA+ ATPases, also referred to as molecular motors, are involved in a great number of such functions and are wide spread within all three domains of life. AAA+ motors commonly remodel the conformation of their target macromolecules driven by ATP hydrolysis. MoxR proteins, a subfamily of AAA+ ATPases, are ubiquitous in bacteria and archaea. They are suggested to represent a group of molecular chaperons, primarily involved in protein maturation and cofactor insertion processes. Interestingly, many MoxR proteins are genetically linked with proteins containing the Von Willebrand Factor Type A (VWA) domain, indicating that they function together. However, the general mechanism of MoxR proteins is only poorly understood and the experimental data is limited. It was recently found that the MoxR protein NorQ from Paracoccus denitrificans facilitates non-heme iron cofactor insertion into nitric oxide reductase (cNOR), a multi-cofactor membrane protein involved in the bacterial denitrification chain. Although the specific metal insertion mechanism remained largely speculative, it was found that the VWA protein NorD is essential for the process and that NorQ and NorD form a complex. A number of studies support a cooperative action of MoxR/VWA proteins and point at a general function in the maturation and regulation of enzymes including rubisco, respiratory complex I, fumarate dehydrogenase, CO dehydrogenase and methanol dehydrogenase.The overall goal of this study is to characterize the shared structural and functional features of this chaperon system by expressing four different MoxR/VWA pairs including NorQD as our main model system. We will determine the high-resolution cryo-EM structures of the different chaperone complexes alone or in interaction with their respective targets. The structural data will be the basis for further mutagenesis analysis combined with a set of kinetic techniques to shed light on the general mechanism of these understudied chaperons.The results of this study are expected to give novel insights into how cofactor assembly can be performed efficiently on a diverse set of target enzymes in the cell. More specifically, we will expand our knowledge of the unknown mechanism of non-heme iron cofactor insertion. In order to design and produce novel biocatalysts for the chemical industry it will be necessary to deepen our understanding of the fundamental mechanisms of metallo-protein assembly. In addition, many MoxR/VWA protein pairs interact with bacterial respiratory enzymes, which makes this chaperon system a potential target for future antimicrobial drugs.