Composite

Part:BBa_K2607001:Design

Designed by: Andrea Laurentius   Group: iGEM18_UI_Indonesia   (2018-09-25)


HB-EGF/Tar Receptor (HT) Device


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1294
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1434


Design Notes

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-33rd amino acids and 191st-553rd amino acids. Modification of binding domain is located between 33rd–191st amino acids.

In HB-EGF, the part that serves as binding domain for diphtheria exotoxin predominantly located in the extracellular environment. Therefore, the domain, expands between 20th–160th 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 257th amino acid) until the utmost C-terminal of the protein (the 553rd amino acid). By those factors, our team also selected Tar domains involving the 1st–33rd and 191st–553rd amino acid as part of chimeric protein (Figure 1).

Figure 2. The graph above explains the result of HB-EGF/Tar orientation, which began from C-terminus (left) to N-terminus (right). Y-axis pictured the possibility of nth 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).
Figure 3. Molecular comparation of HB-EGF native protein (left) with the HB-EGF/Tar fusion (right). 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.

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 (Figure 2 and 3). 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. 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 4. Formula for environment energy (E). Erep and Eattr denote as repulsive and attractive contributions to the van der Waals interaction energy. Additionally, Eelec means an electrostatic energy that occur during both protein interaction. EDARS 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).

After deciding sequence combination of amino acids in model 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 in Figure 4) being lowered down. In this part, our team sent the respective sequence to ClusPro website for further analyzing.

Figure 5. HB-EGF/Tar receptor-DiphTox 3D interaction modelling result.
Figure 6. HB-EGF natural receptor and DiphTox 3D interaction modelling result.

The result of interaction modelling is quantified as energy score based on the formula above. Referring to Figure 5 and 6, we might expect that the DiphTox (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-DiphTox than that of to HB-EGF natural receptor-DiphTox (Table 1). This means that the chimeric receptor could bind towards DiphTox as good (or even better) than the original one.

Table 1. Comparation of E parameter of native and chimera protein of HB-EGF interacted with DiphTox.

HB-EGF Protein

Median Energy (kcal/mol)

Lowest Energy (kcal/mol)

Native

-944.3

-944.3

Chimera

-858.2

-934.4


Source

The binding domain of HB-EGF is a native in Homo sapiens, while the transmembrane/intracellular domain of Tar chemotaxis receptor is a part of Escherichia coli.

References

  1. Kanchan, K., Linder, J., Winkler, K., Hantke, K., Schultz, A. and Schultz, J. (2009). Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases. Journal of Biological Chemistry, 285(3), pp.2090-2099.
  2. Ward, S., Delgado, A., Gunsalus, R. and Manson, M. (2002). A NarX-Tar chimera mediates repellent chemotaxis to nitrate and nitrite. Molecular Microbiology, 44(3), pp.709-719.
  3. Melchers, L. S., Regensburg-Tuïnk, T. J., Bourret, R. B., Sedee, N. J., Schilperoort, R. A. and Hooykaas, P. J. (1989). Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens. The EMBO Journal, 8(7), pp.1919-1925.
  4. Weerasuriya, S., Schneider, B. M. and Manson, M. D. (1998). Chimeric Chemoreceptors in Escherichia coli: Signaling Properties of Tar-Tap and Tap-Tar Hybrids. Journal of Bacteriology, 180(4), pp.914-920
  5. Cbs.dtu.dk. (2018). TMHMM Server, v. 2.0. [online] Available at: http://www.cbs.dtu.dk/services/TMHMM/ [Accessed 22 Jul. 2018].
  6. Lomize M.A., Pogozheva I,D, Joo H., Mosberg H.I., Lomize A.L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res., 2012, 40(Database issue):D370-6
  7. Kelley LA et al. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10, pp.845-858.
  8. Kozakov D, Hall DR, Xia B, Porter KA, Padhorny D, Yueh C, Beglov D, Vajda S. The ClusPro web server for protein-protein docking. Nature Protocols.2017 Feb;12(2):255-278 ; pdf