# 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
The model is colored based on observed rates of hydrogen-deuterium exchange (HDX) of main-chain amide hydrogens by time resolved HDX mass spectrometry [86]. Rates of exchange are colored as: red = high, yellow = high\(-\)moderate, green = moderate\(-\)low, blue = low. The C-terminal domain (CTD) (left side of the figure) contains a thioredoxin-like motif and exhibits low HDX, indicating a well-structured domain. The N-terminal domain (NTD), predicted to be responsible for interaction with TraH, is much more dynamic, as exhibited by increased HDX. Further analysis is required to pinpoint structural features responsible for F pilus extension and examine the changes in protein structure and dynamics upon TraF’s interaction with TraH. amyloid-like fibrils [91; 93; 94]. The structure of TraC from the F plasmid has not been solved, however Phyre2 analysis shows TraC has high sequence homology to VirB4/TrwB/TraB ATPases which also share structural homology to TraD [20]. Phyre2 also predicts a mixed \(\alpha\)-\(\beta\) secondary structure which aligns confidently with the crystal structure of the CTD of VirB4 from _Thermoanoaterobacter pseudotanolicus_ solved to 2.45 A [95]. This structure features a RecA-like \(\alpha\)/\(\beta\) domain that contains the ATP-binding site and a four-helix bundle domain; a 17 A negative stained EM model of the core T4 SS from pKM101 (of the IncN family) featuring the intact VirB4 homolog TraB displays the NTD binding to the side of the IM core complex, rather than stacking underneath. Cryo-ET models of the core complex from the F-like T4 SS pED208 features TraC; it also assembles to the sides of the core complex as a hexamer of dimers, maintaining the active structure seen in the cytoplasm and when complexed in the IM (Figure 3B) [15]. The structure of TraC\({}_{\text{F}}\) is likely most similar to that seen in the pED208 complex; a high-resolution structure of the protein would be useful in understanding how the Mpf complex of F-like T4 SS forms and would be a new drug target for this system. TraW is a periplasmic protein specific to the F type system that is approximately 23 kDa in size and is thought to play a role both in pilus assembly and conjugative DNA transfer [92]. TraW contains an N-terminal peptidase I signal sequence for periplasmic localization [64]. Mutations in TraW stop the pilus from extending; however, pilus tip formation occurs [14]. A previous study done with a mutated F _traW546_ resulted in abolishment of the F-pilus [96]. This shows that TraW plays an essential role in the pilus extension process; however, its protein interactions are not fully understood. It is hypothesized that TraW interacts with its neighboring protein TrpC [97]. Much like TraW, TrpC is a periplasmic protein, approximately 21.5 or 23.5 kDa in size [98]. Some plasmid systems express _trbC_ as a 212 aa or 23.5 kDa protein while others process the protein to remove the N-terminal signal sequence and produce a 191 aa or 21.5 kDa protein [98]. TrbC from the F-plasmid contains an N-terminal peptidase I signal sequence composed of approximately 21 aa for periplasmic localization [64]. Both TraW and TrbC are very similar in their localization and function, and in some systems, these proteins are expressed as a single polypeptide, indicating that cooperative function may occur between TraW and TrbC. Previous studies performed using conjugative mating assays showed that an interaction between the NTD of TraW and the CTD of TrbC was essential for conjugation to occur [97]. It is possible that this same interaction is required for the assembly of the pilus. When examining sequence alignments of these two proteins and comparing systems that produce two separate proteins and ones that produce one single polypeptide, it is clear that TrbC mimics the NTD of the single polypeptide while TraW mimics the CTD, further indicating an interaction between the CTD of TrbC and the NTD of TraW [97].