Our first steps in modelling the subsequent parts of Finding Diphthy iGEM-UI 2018 in silico is by constructing all 3D models via I-Tasser server.^1,2,3 The extension of the product file is .pdb, that could be read by the server. The chimera molecules which we need to predict their modelling are HB-EGF/TAR (Heparin Binding Epidermal Growth Factor- TAR chemotaxis), CheA signalling protein, Che-Y signalling protein, LuxAB dimerized luciferase subunits, and eYFP (enhanced yellow fluorescent protein), as well as DiphTox (modified diphtheria exotoxin). Since CheA and CheY are required to be linked with LuxB or eYFP, we have cited one of the universal linker, that is ‘GGGSGGGGSGGGGSG’ peptides, according to Sun S et al.

In choosing the best combination, we use FoldX option via YASARA molecules viewer to calculate the ∆G of each molecule, searching for the smallest free energy (regarding its stability in vivo). All those sequences are also submitted to I-Tasser server for projecting their 3D models qualitatively. The following results would conclude that our cytoplasmic signalling combinations are CheY-eYFP and LuxB-CheA.

Characterisation or purification of those proteins would promote the usage of His-tag; therefore, insertion of His-tag inside the sequence is essential. To ensure the slightest change of tertiary structures of each protein, we would need to find out the secondary structure and surface accesibility via NetSurfP ver. 1.1 analyser (http://www.cbs.dtu.dk/services/NetSurfP/). We would insert His-tag sequence in either no available specific protein domain or the coiled secondary structure of protein to minimize any interruptions. Here is our affitoxin data from NetSurfP server.Result from the NetSurf server, we choose C-terminus side, because it most likely turns/coils around (indicated by has high number on the most right column is closest to 1), and it is freely exposed (indicated by most left column has E alphabet)

Performing structural similarity between original molecule and the one inserted with His-tag sequence have been done by MUSTANG server that built in via YASARA molecule viewer.⁵ The output would be distance calculation between interacting atoms called RMSD (Root-mean-square deviation). Following tables are summaries of the molecular similarity analysis. From the data that described above, all the combinations are acceptable, except LuxA, since its possible combination has high RMSD. The threshold is relative, but several literatures define the RMSD value of 2 as threshold for structure similarity.^5,6,7

In HB-EGF, the part that serves as binding domain for diphtheria exotoxin predominantly located in the extracellular environment. Therefore, the domain, expands between 20^th – 160^th amino acid, was selected from natural HB-EGF protein. On the other hand, the Tar domain that are functions to establish intracellular chemotactic signalling includes NdeI cutting-site (around 257^th amino acid) until the utmost C-terminal of the protein (the 553^rd amino acid).8-11 By those factors, our team also selected Tar domains involving the 1st – 33^rd and 191^st – 553^rd amino acid as part of chimeric protein.

HB-EGF/Tar Receptor Modelling

Figure 1. The selected segment of Tar protein. The functional intracellular domain of Tar is shown as yellow box, blue box is transmembrane domain and orange box is periplasmic domain. Selected Tar domain expands from 1st -33^rd amino acids and 191^st -553^rd amino acids. Modification of binding domain is located between 33^rd – 191^st amino acids

Our team have predicted the HB-EGF/Tar protein orientation in the Escherichia coli membrane. For this purpose, server TMHMM and OPM Membrane, are utilized to predict protein orientation.^12,13 Conceptual hypothesis about the chimera protein is that it should begin its orientation of C-terminus in cytoplasm, then continued to fold into transmembrane and extracellular sites, as well as re-folding towards cytoplasm.

Figure 2. The graph above explains the result of HB-EGF/Tar orientation, which began from C-terminus (left) to N-terminus (right).¹² Y-axis pictured the possibility of n^th amino acid on protein located somewhere between transmembrane (red part), intracellular (blue line), and extracellular (pink line). There is also a diagram located above the graph that represent the most possible location of each domain (with elongated box).

From the results, it could be concluded that the protein was oriented as expected in the hypothesis. Therefore, the usage of chimera protein is predicted to be functional anatomically.

Figure 3.Molecular comparation of HB-EGF native protein (left) with the HB-EGF/Tar fusion (right).^13,14 The pink-coloured domain is intracellularly located as the N-terminus, yellow-coloured domain for the transmembrane one. Then, purple-coloured could be a sign as the extracellular domain, finally folding into transmembrane and back to cytoplasm with orange-coloured and cyan-coloured domain respectively.

After deciding sequence combination of amino acids in modelled chimera HB-EGF/Tar protein, analyzing the interaction of both fusion protein and diphtheria exotoxin is extremely important to ensure functional ligand-receptor system. The basic concept of interaction modelling is that the protein will be bound to each other well if it causes the ‘environment’ energy (termed by E parameter; calculated by formula below) being lowered down. In this part, our team sent the respective sequence to ClusPro website for further analyzing.¹⁵

Note: E_rep and E_attr denote as repulsive and attractive contributions to the van der Waals interaction energy. Additionally, E_elec means an electrostatic energy that occur during both protein interaction. E_DARS is a pairwise structure-based potential constructed by the Decoys of the Reference State (DARS) approach, and it primarily represents desolvation contributions, i.e., the free energy change due to the removal of the water molecules from the interface.¹⁵

Table 4. Comparation of E parameter of native and chimera protein of HB-EGF interacted with affitoxin.

HB-EGF Protein	Median Energy (kcal/mol)	Lowest Energy (kcal/mol)
Native	-944.3	-994.3
Chimera	858.2	934.4

Figure 5.HB-EGF natural receptor and Affitoxin 3D interaction modelling result.

The result of interaction modelling is quantified as energy score based on the formula above. Referring to figure 4 and 5, we might expect that the affitoxin (cyan) would bind to both native and chimeric HB-EGF receptor that are both located in the extracellular (green). It is indicated by higher energy score of interaction between chimeric HB-EGF/Tar receptor-Affitoxin than that of to HB-EGF natural receptor-Affitoxin (Table 4). This means that the chimeric receptor could bind towards affitoxin as good (or even better) than the original one.

Beside the cell’s ability to detect toxin, our team also need to ensure the signaling machine works well. Our team also modelled the interaction between LuxA dan LuxB (that we fused with CheA). From figure 6 and 7, we might expect that both proteins are still able to interact normally after combining them with FRET unit (CheA or CheY protein).

Figure 6.LuxA and LuxB-CheA (LuxAB-CheA) 3D interaction modelling result.

Figure 7..LuxA and LuxB 3D interaction modelling result.