## 5 Non-Conserved _exo-xis_ Region Proteins of Interest
### Bacteriophage \(\varphi 24_{B}\) Gp05 Is a Possible Transcription Factor  
In bacteriophage \(\lambda\), two unique genes, _ea8.5_ and _orf55_, are situated between _ea22_ and _xis_. Both proteins have been presented in this report either as a solved structure (Ea8.5) or a high confidence molecular model (Orf55). Compared to \(\lambda\), the St\({}^{+}\) phages \(\varphi 24_{B}\) and 933W harbor more genes in the _exo-xis_ region along with instances of full or partial gene duplication. Here, a molecular model of the \(\varphi 24_{B}\)_gp05_ (_vb_24B_3c_) gene product is presented as an example of an _exo-xis_ protein with a novel combination of features. It is currently unknown if _gp05_ is expressed and encodes a functional protein. The predicted structures by AlphaFold and RoseTTAFold both describe a protein that is composed of a winged helix-turn-helix (wHTH) DNA binding domain (DBD), followed by a PVQ-repeat leading into a carboxy-terminal coiled-coil domain (Figure 9a). Since the AlphaFold method is capable of modeling oligomers [62], the Gp05 was submitted for prediction as a homodimer. Figure 8: Molecular modeling \(\lambda\) Orf55. High confidence predictions by AlphaFold (pLDDT = 93) and RoseTTAFold (pLDDT = 93) both present Orf55 as a a small \(\alpha/\beta\) protein. From a scan of the PDB for homologs, T7 Gp2 was identified as a candidate. (**a**) Sequence alignment of \(\lambda\) Orf55 and T7 Gp2. The predicted secondary structure is shown underneath the sequence (\(\beta\)-strands, blue arrows; \(\alpha\)-helix, red bar). Sequence identity is denoted by shading. Above the sequence, a # indicates a predicted interaction with the RNAP \(\beta^{\prime}\) subunit and a @ indicates a predicted interaction with the 1.1 domain of the general sigma factor, \(\sigma^{70}\). (**b**) The Rosetta software suite was used to refine a molecular model of Orf55 (green) situated in RNAP. Numerous contacts are made between Orf55 in the model with the \(\beta^{\prime}\) subunit (blue) and \(\sigma^{70}\) (magenta). (**c**) Ribbon and space-filling diagram detail of Orf55 flanked by the RNAP \(\beta^{\prime}\) subunit and \(\sigma^{70}\). From one representative AlphaFold model presented in Figure 9b, an extensive linker region in Gp05 protein would permit its wHTH DBDs to bind sequences spaced tens of bp apart. A search of the PDB for similar wHTH DBDs identified the hyperthermophilic archaeon _Sulfolobus_ AspA chromosomal partitioning protein with 26% identity. The identical amino acids mapped closely to the hydrophobic core supporting the prediction of a wHTH DBD in the \(N\)-terminus of Gp05. The identical amino acids; however, did not include those in third helix that make base-specific contacts to DNA major groove suggesting that Gp05 binds a different sequence. In addition, the wing of Gp05 was shortened to the extent that adjacent minor groove specific contacts would likely not be possible. Thus, if DNA binding by Gp05 did occur, the binding site would be shorter than a typical wHTH domain. A majority of the PVQ-repeat was modeled as a left-handed polyproline helix by AlphaFold, perhaps guided by numerous SH3 domain ligands that populate the PDB. A  
Figure 9: Molecular modeling the multidomain protein Gp05 from the St\(\mathrm{c}^{+}\) phage \(\Phi\)24\({}_{\mathrm{B}}\). **(a)** The protein may be considered in terms of a winged helix-turn-helix (wHTH) DNA binding domain (DBD), a proline-valine-glutamine (PVQ) repeat region and a coiled-coil region. Predicted helices (bars) and strands (arrows) are shown below the sequence. An alignment with a similar wHTH domain from the AspA centromere binding protein (PDB: 5K5R) is shown for comparison. Sequence identity is denoted by shading, a dot indicates amino acids that are predicted to make DNA base contacts.