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

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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.