# Crucial Gram-positive type IV secretion system protein TraF is a structural homolog of type VII secretion system protein EssB/YukC

## TL;DR

This study identifies TraF as a key protein in a bacterial DNA transfer system and finds it structurally similar to a protein from a different secretion system.

## Contribution

TraF is shown to be essential for conjugation and structurally homologous to YukC/EssB from a different secretion system.

## Key findings

- TraF is essential for conjugative transfer and interacts strongly with TraMB8.
- The crystal structure of TraF's N-terminal domain reveals a pseudokinase fold.
- TraF shows structural similarity to YukC/EssB from the type VII secretion system.

## Abstract

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, TraCB3, TraF, TraHB8, TraI, TraK, TraLB6, and TraMB8) 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 TraGB1 and TraN. Bacterial-two-hybrid assay showed a strong interaction between TraF and TraMB8. 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.

Crystal structure of the soluble domain of TraF, a transmembrane T4SS protein, reveals structural homology to the T7SS protein YukC/EssB.