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

Stian Aleksander Helsem¹Kristian Alfsnes²Stephan A. Frye³Alexander Hesselberg Løvestad^1,2Ole Herman Ambur^1*

• ¹Oslo Metropolitan University, Oslo, Norway
• ²Folkehelseinstituttet, Oslo, Norway
• ³Oslo universitetssykehus, Oslo, Norway

The Uptake Signal Sequence (USS) receptor, facilitating acquisition of homologous DNA by natural transformation in Haemophilus influenzae and other 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 (PpdAPast) 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 ten different Pasteurellaceae enriched with divergent USS dialects. PpdAPast was predicted to possess USS-binding specificity by DeepPBS and was found strongly coevolved with USS relative to other orthogroups. PpdAPast was found to share both structural features and functionally involved electropositive residues with other DNA-binding minor pilins and the largely unexplored Escherichia coli/ Enterobacteriaceae PpdA ortholog. One robust DNA binding mode was identified with two alternative and opposite USS orientations. Combining modeled PpdA-USS proximity and coevolved signals showed how the C-terminal region of PpdAPast fitted one of two 180º alternative USS orientations. Root Mean Square Deviations (RMSDs) from molecular dynamics simulations of PpdAPast – USS complexes found confident structures <3 Å in moderately stable trajectories. The ppdA gene, which was previously documented essential for transformation and to constitute part of the competence regulon in Haemophilus influenzae and E. coli, was found in a conserved genomic location with conserved operonic organization across the Pasteurellaceae. Together the in silico results of this study and documented knock-out phenotype makes a strong case for PpdAPast as the USS-receptor and provide future directions for recombinant PpdAPast assays and in vivo experiments of mutants. We propose ComN to be used for these PpdAPast orthologs in compliance with a previously given gene name and the here predicted central role in competence as DNA-receptor with USS-specificity.