# Protein Dynamics in F-like Bacterial Conjugation
## 5 The Structures of Conjugative DNA Transfer Proteins
### TraD  
The essential coupling protein TraD is a 717 aa IM protein that plays a key role in the transfer of ssDNA during conjugation; it is a component of the transferosome. The most notable feature of the coupling protein is the flexible C-terminal tail that extends into the cytoplasm, considered responsible for relaxosome recognition in the DNA transfer process. The CTD of TraD, more specifically Ala680-Phe717, is highly conserved in other members of the F-like plasmid family [139; 140]. The typical structure of a coupling protein is composed of an N-terminal TM domain and a large cytoplasmic region composed of an \(\alpha\)-helical bundle and a nucleotide binding domain (NBD); these traits have been observed in TraD [55]. Three features common to all F-like TraD proteins include (1) a glycine  
Figure 5: Crystal structures of transferosome proteins from the F (**A–C**) and pED208 (**D**) plasmids. The structures represented with oligomeric states have a monomeric unit expressed as chainbows with the remaining homo multimers represented in brown. (**A**) TraM binds three specific DNA sequences, _sbmA_, _sbmB_ and _sbmC_[137]. The crystal structure of the TraM-_sbmA_ interaction shows cooperative binding is facilitated by DNA distortion, allowing simultaneous binding by both tetramers while still allowing multiple contact points for TraD (PDB ID: 3 ON0). (**B**) The crystal structure of the TraD–TraM interaction (PDB ID: 3D8A) [139]. TraM forms a tetrameric binding protein as represented by the monomer chainbow and the brown tetramer chains, while TraD peptides are represented in red. The C-terminal residue of TraD Phe717 binds to the hydrophobic pocket produced by TraM residues Leu85, Val106, and Ile109. (**C**) The solved crystal structure of the F plasmid TraI relaxase NTD represented in chainbows, with 10 \(\alpha\)-helices and 13 \(\beta\)-strands (PDB ID: 1P4D) [134]. (**D**) The CTD of TraI forms an intertwined tetramer with two domain swapped dimers; one dimer is depicted above (PDB: 3 FLD) [132]. TraI expresses a fold with a compact \(\alpha\)-\(\beta\) domain at the CTD connected to two \(\alpha\)-helices at the NTD by a proline-rich loop.  
residue within the tail which is essential in the formation of a crucial \(\beta\)-turn, (2) an aromatic residue present at the end of the chain, (3) a high percentage of acidic residues [139]. Mutations of the _traD_ gene cause plasmid transfer deficiency [64; 37]. Although the structure of TraD\({}_{\text{F}}\) has not been resolved, the soluble fragment of the TraD homolog TrwB of R388 has been solved by X-ray crystallography; thereby little structural information is present for the TM domain [141]. The hexameric ring structure of TrwB is divided into two domains, one composed of an all \(\alpha\)-helical domain (AAD) and another closer to the membrane composed of an \(\alpha\)/\(\beta\) twisted open-sheet domain [59; 141]. Although purified TraD has been shown to bind DNA in a nonspecific manner, the relaxosome-transferosome interaction observed between TraD and TraM provides a degree of specificity [55].  
The temporary binding between TraD and TraM has been observed by the recruitment of TraM by the flexible C-terminal tail displayed by TraD (Figure 5B) [138; 139]. As previously stated, the C-terminal tetramerization domain of TraM interacts with residues Asp710-Phe717 of the CTD of TraD [37; 137]. This interaction involves hydrophobic and hydrophilic contacts, Phe717 present at the end of the CTD of TraD is observed to interact with the hydrophobic pocket produced by TraM (Leu85, Val106, and Ile109), and the carboxyl group anchors the TraD in place through interaction with Arg110 and Lys76 [139]. This interaction is highly conserved in other F-like plasmids and is essential to successful DNA transfer as it is thought to allow for the coupling of the relaxosome to the transferosome [123; 138; 140].