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Page 8 of 14 Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29

contain two 5E7T_B domains compared to the single domain encoded by the insertions in the Dits
belonging to phages that infect hosts harboring the typical 6073-like eps gene cluster. Representative evolved
[4]
Dit from D2929 was modeled and compared with the classical Dit from M7361 as well as with D6887
[Figure 3]. A clear conservation of the core region from the classical Dit compared to the evolved
counterparts can be observed, with the inserts existing as a distinct region connected to the core region via a
series of flexible loops. This highlights a modular evolution within the protein, further exemplified when
comparing D6887 with D2929, the latter containing two distinctly visible domains within the insert region.
Both domains within the D2929 insert region were predicted to contain motifs involved in carbohydrate
binding, supporting the HHpred results. The proximal domain was predicted to contain two contiguous
regions located at residues 188-259 and 280-321 involved in carbohydrate binding, whereas the distal
domain was predicted to contain one such motif across the amino acid range 391-432. With respect to
D6887, the absence of the D2929 distal domain is immediately apparent in the structure. Alignments of the
proximal domain revealed a 92% structural similarity; however, it was observed that in the case of D6887,
only one carbohydrate-binding motif was predicted, corresponding to the one located in the range 188-259
in D2929. The presence of BppA-like domains within the Dits of some phages also represented a point of
interest, as the accessory base plate protein (BppA) has been shown to contain CBMs involved in phage
binding to the host [8,40,42] . Therefore, a model was constructed of the Dit from phage D4006, predicted to
contain this domain, and was compared to the solved structure for TUC2009 (5E7T_B) [Figure 3D]. Indeed,
it was observed that there exists a structural similarity between the proximal portion of the insert in the
D4006 Dit and 5E7T_B.

Figure 3. Molecular modeling and comparison of select classical and evolved Dit proteins. Structural alignment of the AlphaFold
predicted Dit models for phages D2929 (green) and M7361 (blue), respectively (image A). The region corresponding to the insert
present in D2929 is shown on the left-hand side of the figure, with the locations of the three predicted carbohydrate-binding regions
(two of them corresponding to the proximal domain and one corresponding to the distal domain) highlighted in different colors.
Structural alignment of the D2929 (green) and D6887 (blue) Dit models is provided in panel B on the right-hand side of the figure with
an alignment corresponding only to the insert regions provided in panel C at the bottom. The predicted carbohydrate-binding motif in
D6887 is highlighted in yellow. Panel D at the bottom right shows a structural alignment of the D4006 Dit protein predicted to possess
BppA domains aligned with the minor structural protein 5 (BppA) from the TUC2009 solved baseplate structure (PDB: 5E7T). It can be
clearly seen in the proximal portion of the insert in D4006 that there is a structural similarity with BppA. BppA: accessory base plate
protein; Dit: distal tail protein; PDB: protein data bank.