# Protein Dynamics in F-like Bacterial Conjugation
## 3 Structures Involved in Pilin Processing, Pilus Extension, and Retraction
### Pilus Assembly and Extension Proteins
#### 3.2.3 Pilus Extension--TraF, TrbB, TraP, TraC, TraW, and TrbC  
TraF is a 26 kDa periplasmic protein necessary for F pilus extension, and deletions at either termini abolish F pili formation eliminating conjugative gene transfer [86]. The NTD of TraF exhibits increased structural dynamics and is responsible for interactions with TraH; the CTD (Glu127-Leu247) contains a thioredoxin-like fold (Figure 4). Through Phyre2 modeling the structure of TraF is predicted to have at least three \(\alpha\)-helices bordered by a four-strand \(\beta\)-sheet [20]. It is hypothesized that both TraF and TrbB proteins have chaperone capabilities based on the conserved thioredoxin-like region [86]. TraF has been shown to directly bind to TraH; if this interaction is inhibited, pilus extension is abolished. Furthermore, TraF is considered to have further vital functions due to the abolishment of conjugative function after mutations in the C-terminal domain; this may be due to the predicted complex formation between periplasmic proteins TraF, -H, -W, TrbB, and TrbC, and potentially, TraU. Of these proteins, TraF, -H, -U, and -W appear to associate with the OM when in the context of the complete transfer apparatus [87]. An interaction between TraF and TraV is hypothesized to localize the periplasmic complex with the core complex; if proven, the point of contact would provide a critical site of T4 SS association and therefore be a useful drug target [87]. TraF is homologous to another periplasmic protein from the tra operon of the F plasmid TrbB, however TrbB contains the C-X-X-C thioredoxin motif while TraF displays a thioredoxin-like fold but does not have the C-X-X-C motif [88]. TrbB is a 161 aa periplasmic protein that has been shown to act as a disulfide isomerase, and is assumed to help mediate pilus extension as a protein chaperone [89; 90]. The structure of TrbB is predicted to be unique amongst disulfide bond isomerases; the N-terminal domain of TrbB is predicted to be largely unstructured (similar to TraF) and is required for proper function, however it does not act as a dimerization domain [89]. TrbB also requires the host protein DsbD to maintain its active, reduced state. The enzymatic activity of TrbB activity is lower for common redox substrates when compared to that of the host disulfide isomerase DsbC; this lowered activity is considered to enable substrate specificity for interacting partners of TrbB. Neither TraF nor TrbB from F-like T4 SS have solved structures, which would aid in understanding their unique functions. TraP is a 21.5 kDa protein predicted to have both termini in the cytoplasm with a periplasmic loop flanked by TM regions from Leu25\(-\)Trp49 and Leu118\(-\)Pro141 in the IM [76]. TraP is thought to be an accessory protein, which enhances stability of the TM complex involved in pilus polymerization, as mutations in _traP_ seem to have minimal negative effect on pilus extension; the protein is not required for F-conjugation in _E. coli._ The disorder content of the protein's secondary structure is predicted to be 24% by Phyre2 [20]. ATPases are essential components in T4 SS as they supply energy for various processes in conjugation, namely pilus extension and transportation of the transferosome. Due to its role as one of two ATPases of the F-like T4 SS (the other being TraD), TraC is considered to be the characteristic protein for mating pair formation (Mpf); the _traC_ gene has been used for the classification of F-like plasmids [13]. TraC is an 875 aa protein that is expressed cytoplasmically; however, it is chaperoned to the IM through interactions with other IM T4 SS components, specifically TraB, -E, and -L [14; 91; 92]. If over-expressed TraC forms inclusion bodies with reversible solubilization, which can be indicative of the presence of either intrinsic disordered regions (IDRs) or \(\beta\)-strands that allow for the assembly of  
Figure 4: A predicted model of TraF, known to be required for F pilus extension beyond the OM.