# Protein Dynamics in F-like Bacterial Conjugation
## 6 Exclusion Proteins and their Low Structural Dynamicity
### TraT  
The only protein known to participate in Sfx, TraT is an OM bound protein that is well structured and displays high homology in F-like plasmids [11; 105]. TraT is processed prior to forming its active conformation in the OM; proTraT contains a 21 aa hydrophobic signal sequence that is modified through esterification by a diacylglyceroll glycerol in Cys22, then the signal sequence is cleaved off the N-terminus to form a 233 aa protein [146; 147; 148]. The addition of a third acyl group at the amide forms the mature TraT lipoprotein at 23,709 Da. TraT is considered to be a major OM protein as it is highly expressed in F+ cells with more than 20,000 copies per cell; the protein forms multimeric aggregates to perform its function [105; 147]. ProTraT has been shown to translocate into the OM; however, it does not oligomerize resulting in a lack of function [148]. As TraT is a lipoprotein its strong anchoring into the membrane is formed by intermolecular lipid-lipid interactions and lipid-protein interactions [142; 146]. This provides high structural stability to the protein as TraT can withstand denaturation by high temperatures, solubilization by detergents and proteolytic cleavage by proteases [148].  
In comparing TraT\({}_{\text{F}}\) to TraT, from all known IncF plasmids, the lowest homology between the aa sequence of the mature protein is 80% (in comparing to TraT\({}_{\text{PED208}}\)), and the highest homology is held with TraT\({}_{\text{R100}}\) which only differs by 1 aa [142; 149]. Although residue 120 is a glycine in TraT\({}_{\text{F}}\) and alanine in TraT\({}_{\text{R100}}\), Sfx limits conjugation between cultures containing the two different plasmids 3-fold less than if mated with bacteria containing the same plasmid. Structural predictions indicate that the single residue change causes an extra \(\beta\)-turn that radically alters the local structure of the domain. The 5 aa region from residues Arg116-Gly120 has been experimentally shown to be important for Sfx specificity and is known to be a hydrophilic exposed region in the ECM [150; 151]. Therefore, these residues are considered responsible for the interaction with TraT and its unknown binding partner. TraT was initially proposed to define the specificity of the system through the formation of a 5 membered ring, with the extracellular motif acting as a recognition sequence for an incoming pilus tip and blocks its interaction with OmpA [152]. The sequence variability of pilins cannot provide the required specificity based on experimental results, thus a new mechanism of Sfx was proposed where TraT disrupts the Mps interactions of TraN with OmpA [102; 106; 153]. While evidence of interaction between TraT and OmpA exists, this mechanism has not been confirmed experimentally [106; 142; 152]. An interaction with TraN is plausible as TraT is involved in disaggregating mating pairs after DNA transfer in a mechanism independent from its function in the disruption of donor-donor conjugation [11; 154].  
No high-resolution structural information is available for TraT from F-like plasmids, however TraT\({}_{\text{F}}\) was predicted to have 55% \(\alpha\)-helical content [147]. It is also believed to differ radically in molecular organization in the OM compared to other OM proteins based on its unusually long stretches of uncharged residues (Val99-Ser110 and Tyr118-Asn135) that likely contribute to TM helices. Analysis of TraT\({}_{\text{F}}\) sequence using Phyre2 indicated no homologs have been deposited in the PDB, implying the novelty of the TraT sequence [20]. In analyzing the available structural information, an interesting pattern is seen in which the high structural integrity of the protein results in high sensitivity to slight mutations, which are likely to drastically alter the protein's structure. TraT may be a useful drug candidate as slight structural changes that could be induced by a small molecule would likely result in loss of Sfx function.