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by claude@2026-06, 2026-06-24
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The study investigated the structural organization and catalytic mechanism of UbK1, the lone atypical “ubiquitous bacterial kinase” in the anaerobic oral pathogen Porphyromonas gingivalis, and identified its physiological substrates and phosphorylation dynamics. Using a crystal structure plus structure-guided mutagenesis, the authors mapped conserved ATP-binding and catalytic motifs, determined that UbK1 has multiple autophosphorylation sites with the flexible SPT/S loop showing the highest occupancy, and used biochemical assays to show autophosphorylation occurs both in cis and in trans rather than strictly intramolecularly. Through phosphosite mapping and conserved gene neighborhood analysis, they identified PorX as a noncanonical UbK1 substrate and demonstrated UbK1 transphosphorylates a single tyrosine residue in PorX’s receiver domain in vitro independent of PorX oligomeric state. In vivo, deletion of ubk1 modestly reduced gingipain secretion, while mutation of the UbK1-dependent PorX phosphosite did not measurably affect T9SS cargo export, supporting separability from canonical secretion outputs. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
Abstract
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.
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Abstract
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.
Competing Interest Statement
The authors have declared no competing interest.
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