Type IV minor pilin ComN predicted the USS-receptor in Pasteurellaceae

The Uptake Signal Sequence (USS) receptor, which facilitates the acquisition of homologous DNA by natural transformation in <i>Haemophilus influenzae</i> and other members of the Pasteurellaceae, remains unknown. Through discriminating functional gene ontology assessment, cellular localization predictions, and deep-learning structural modeling of protein-DNA complexes, prepilin peptidase-dependent protein A (PpdA) was identified as the strongest USS receptor candidate in different Pasteurellaceae family members with divergent USS specificities. Pasteurellaceae PpdA (PpdA_Past) was the only orthogroup modeled by AlphaFold3 (AF3) to form specific complexes with USS significantly better than complexes with sequence-scrambled versions of USS. Further analyses of PpdA-USS complexes using geometric deep learning protein-DNA sequence specificity predictions and coevolution analyses were found to further support PpdA as the USS receptor candidate in 10 different Pasteurellaceae enriched with divergent USS dialects. PpdA_Past was predicted to possess USS-binding specificity by DeepPBS and was found to strongly coevolve with USS relative to other orthogroups. PpdA_Past was found to share both structural features and functionally involved electropositive residues with other DNA-binding minor pilins and the largely unexplored <i>Escherichia coli/Enterobacteriaceae</i> PpdA ortholog. One robust DNA-binding mode was identified with two alternative and opposite USS orientations. Combining modeled PpdA-USS proximity and coevolved signals revealed how the C-terminal region of PpdA_Past fitted one of two 180° alternative USS orientations. Root Mean Square Deviations (RMSDs) from molecular dynamics simulations of PpdA_Past USS complexes found reliable structures under 3 Å in moderately stable trajectories. The <i>ppdA</i> gene, previously documented as essential for transformation and constituting part of the competence regulon in <i>Haemophilus influenzae</i> and <i>E. coli</i>, was found in a conserved genomic location with conserved operonic organization across the Pasteurellaceae. Together, the <i>in silico</i> results of this study and the documented knock out phenotype make a strong case for PpdA_Past as the USS-receptor and provide future directions for recombinant PpdA_Past assays and <i>in vivo</i> experiments with mutants. Here, we propose ComN for use with these PpdA_Past orthologs in compliance with the previously assigned gene name and the predicted central role in competence as a DNA receptor with USS specificity.