Structural and functional characterization of the HrcN-HrcO complex from Xanthomonas citri

The Type III Secretion System (T3SS) is a specialized nanomachine used by Gram-negative plant pathogens to translocate effector proteins into host cells. In Xanthomonas citri, HrcN and HrcO are key components of the export apparatus, with HrcN functioning as an ATPase and HrcO proposed to connect the ATPase to the secretion machinery. Although HrcN is expected to assemble into a hexameric ATPase complex, the molecular organization and stability of the HrcN-HrcO complex remain poorly characterized. Here, we investigate the biochemical and biophysical properties of the HrcN-HrcO complex as a basis for structural characterization by cryo-electron microscopy (cryo-EM). A polycistronic construct encoding HrcN and HrcO was expressed in Escherichia coli, and the proteins were purified by affinity and size-exclusion chromatography. Purification analyses revealed higher-order species and a pronounced tendency to form high-molecular-weight assemblies. Dynamic light scattering showed heterogeneous particle populations, including species compatible with oligomeric assemblies, while SEC-MALS indicated molecular masses substantially higher than the expected 250–300 kDa HrcN-HrcO complex, consistent with heterogeneous oligomerization and aggregation. Importantly, the oligomeric behavior appears to depend on protein concentration, with higher protein concentrations favoring more stable assemblies. Native PAGE/Blue Native-PAGE experiments are being used to resolve and compare these oligomeric states. In parallel, HrcN ATPase activity was detected under optimized assay conditions, supporting the functional integrity of the purified ATPase. Nucleotide-dependent stabilization is also being investigated using the ADP-AlFx transition-state analog, which promotes the trapping of ATPase oligomeric states. Preliminary cryo-EM screening showed dispersed particles with a low incidence of large aggregates, providing a basis for further optimization of sample concentration and biochemical conditions. Together, these results highlight the dynamic oligomeric behavior of HrcN-HrcO and establish a biochemical framework for identifying conditions that stabilize the complex for highresolution structural studies. Understanding how protein concentration and nucleotide state influence assembly may provide important insights into the functional organization of the T3SS in X. citri.