Peroxidasin Structure and Interaction Partners

Human peroxidasin 1 (PXDN) is a multidomain protein that plays a crucial role in stabilizing the highly specialized basement membranes. It catalyzes the oxidation of bromide to hypobromous acid, which in turn promotes the formation of a unique covalent sulfilimine bond within the collagen IV network. This rare crosslink not only enhances the mechanical strength of the basement membrane but also supports its function as a scaffold for cell adhesion and signaling. Despite its importance, the structural organization and specific functions of several peroxidasin-typical domains—such as the leucine-rich repeat domain, immunoglobulin domains, and the C-terminal von Willebrand factor type C module-remain poorly understood. These domains are believed to mediate protein–protein interactions and contribute to cell adhesion, but their precise roles are yet to be elucidated. Objective 1 of this project is to investigate whether peroxidasin, or its individual domain constructs, directly or indirectly interacts with other basement membrane components. However, the limited structural data available on PXDN presents a significant barrier to understanding the spatial arrangement of its domains. Binding studies will be performed using surface plasmon resonance and isothermal titration calorimetry to characterize interactions with extracellular matrix components. To bridge this knowledge gap, Objective 2 is to determine the three-dimensional structure of full-length peroxidasin in presence of synthetic and/or physiologic binders . Structural studies will be conducted using X-ray crystallography and cryo-electron microscopy. A key innovation of this project lies in the use of truncated PXDN constructs for domain-specific binding assays. This approach will help pinpoint which domains are responsible for interacting with particular basement membrane molecules. In parallel, Br-tyrosine peptide mapping via mass spectrometry will provide complementary information about the binding interface. Ultimately, resolving the full-length structure of peroxidasin by cryo-EM will offer novel insights into domain dynamics and intermolecular interactions. This work will be carried out in collaboration with Prof. Kristina Djinović-Carugo at the European Molecular Biology Laboratory (EMBL), Grenoble, as an international partner