# Protein Dynamics in F-like Bacterial Conjugation
## 4 Mating Pair Stabilization Proteins and the Dynamics of their Structural Ensemble
### TraN  
The integral OM protein TraN is arguably the primary actor for Mps as it forms essential contacts for recipient cell lipopolysaccharide-LPS recognition prior to formation of the conjugative pore [102; 106]. TraN from the F plasmid has been shown to require outer membrane protein A (OmpA) expressed in the recipient cell for Mps to occur, while TraN\({}_{\text{R100}}\) can perform its action through recognition of LPS moieties alone. Binding to OmpA appears to increase mating efficiency relative to F-like plasmids that feature a TraN homolog that does not bind OmpA. TraN is hypothesized to function as a multimer, but the extent of oligomerization in the T4 SS is not known as the protein is difficult to purify from the biological complex due to the low number of F pili expressed in cells and the many associations TraN forms in large complexes, as attributed to predicted intermolecular disulfide bonds [102]. Cryo-ET structures of the F-like pili pED208 did not include TraN as part of the core complex visualized (Figure 3A-C) [15]. There are 20 conserved cysteines in TraN from F-like plasmids; results suggest that these residues are involved in the behavior of this protein to act similar to an adhesin, despite its low sequence homology to adhesins with known structures [102; 106; 107]. Altering five of six highly conserved cysteines in the C-terminus of the protein eliminates the Mps function of TraN [102]. The number of cysteine residues in the OM segments is uncommon, which causes difficulty in the predictive modeling of TraN, as few OM proteins with solved structures contain many cysteine residues, and the presence of disulfide bonds occurring in TM segments has not been documented in biological environments [102; 108].  
The structure of TraN is largely hydrophobic as it is a polytopic TM protein predicted to have a \(\beta\)-barrel structure, although this is still highly contested based on low homology to TM \(\beta\)-barrel proteins, with loops extending into the ECM and in the periplasm [102; 107]. Models have indicated the presence of 20 TM \(\beta\) sheets in the 584 aa protein (602 aa before C-terminal signal sequence processing during transport to the OM) with 10 extracellular loops and 9 loops, as well as the N-and C-termini, in the periplasm [102; 109]. These loop regions have been shown to perform key interactions on both sides of the membrane. Experiments involving TraN fusions of the loop regions with c-Myc showed 3 main extracellular loops in the N-terminal half of the protein prevented interaction with OmpA, therefore these loops are responsible for the TraN-OmpA interaction [102]. Attempts to demonstrate this interaction by two-hybrid techniques were unsuccessful, as was co-immunoprecipitation from mating cells, probably due to the small number of interacting partners in a mating pair [11]. This suggests that TraN changes conformation in the context of a mating pair and interacts with OmpA, acting as a clamp to stabilize the conjugative junction. The periplasmic loops containing cysteines are predicted to perform disulfide bonding as mediated by periplasmic proteins TraF and/or TrbB, as these parts of the F pilus complex have thioredoxin-like folds [88; 102; 110]. They may mediate important contacts for Mps predicted to occur between the C-terminal region of TraG and TraN [102].