Crucial Gram-positive type IV secretion system protein TraF is a structural homolog of type VII secretion system...

Type IV secretion systems (T4SS) are found in both monoderm and diderm bacteria. The broad-host-range conjugative plasmid pIP501 from Enterococcus faecalis harbors a T4SS encoding 15 tra genes responsible for the spread of antimicrobial resistance genes among diverse G+ pathogens. Eight Tra proteins (TraB, TraC_B3, TraF, TraH_B8, TraI, TraK, TraL_B6, and TraM_B8) are postulated to form the mating pair formation (MPF) complex representing the central DNA translocation pore. One of these proteins is TraF, a 52.8 kDa transmembrane protein, which lacks any homologs in other well described T4SSs. In this study, TraF was proven to be an essential conjugative transfer protein. The TraF pulldown co-eluted all Tra proteins except TraG_B1 and TraN. Bacterial-two-hybrid assay showed a strong interaction between TraF and TraM_B8. We present a 1.25 Å resolution crystal structure of the N-terminal domain of TraF, which adopts a pseudokinase fold. AlphaFold predictions of full-length TraF with membrane mimetics show a transmembrane protein with two distinct soluble domains. FoldSeek revealed a strong similarity to YukC (EssB), a transmembrane pseudokinase from type VII secretion system (T7SS). YukC was shown to function as an interaction hub by mediating contacts between its pseudokinase domain and other T7SS proteins as part of the central membrane core complex. We postulate that TraF might play an important role in T4SS complex formation.