Structural and mechanistic basis of ubiquitous bacterial kinase signaling identifies PorX as a noncanonical substrate in Porphyromonas gingivalis

Protein phosphorylation enables bacteria to coordinate regulatory networks underlying virulence and environmental adaptation. The ubiquitous bacterial kinase family comprises atypical kinases with dual serine threonine and tyrosine specificity, yet their structural organization, catalytic mechanisms, and physiological roles remain incompletely defined. In the anaerobic oral pathogen Porphyromonas gingivalis, the sole UbK homolog, UbK1, was previously shown to phosphorylate the orphan response regulator RprY, linking UbK1 to virulence-associated pathways. Here, we present the crystal structure of UbK1, revealing the conserved Walker A, HxDxYR, SPT/S, and EW motifs arranged around the ATP-binding site. Structure-guided mutagenesis establishes essential roles for these motifs in ATP hydrolysis and kinase activity. Phosphosite mapping identifies multiple autophosphorylation sites, with the flexible SPT/S loop showing the strongest occupancy and supporting a model in which loop centered autophosphorylation is a major feature of UbK1 cycling while additional sites arise through intermolecular phosphotransfer in trans. Consistent with this, biochemical assays demonstrate that UbK1 undergoes autophosphorylation both in cis and in trans, arguing against a strictly intramolecular autokinase mechanism. Using conserved gene neighborhood analysis, we identified the orphan response regulator PorX as a previously unrecognized UbK1 substrate, consistent with the relaxed substrate specificity reported for UbK homologs. UbK1 catalyzes PorX transphosphorylation in vitro at a single tyrosine residue within the receiver domain, independent of PorX oligomeric state. In vivo, ubk1 deletion causes a modest reduction in gingipain secretion, whereas mutation of the UbK1 dependent PorX phosphosite does not measurably affect T9SS mediated cargo export, indicating that UbK1-PorX signaling is separable from PorX canonical gingipain secretion output. Together, these findings establish a structural and mechanistic framework for UbK1 function, expand the UbK substrate repertoire, and support a model in which UbK1 contributes to regulatory pathways in P. gingivalis that extend beyond canonical secretion associated outputs.