Team:UCLA/Modeling

From 2013.igem.org





Major Tropism Determinant

Our protein of interest, the Major Tropism Determinant (Mtd) protein, is located on the tail-end fibers of the BPP-1 bacteriophage. This protein is important in that it allows the BPP-1 phage to bind and infect its host species of bacteria- Bordetella, the bacterial species that causes whooping cough. Because Bordetella can vary the expression of its surface proteins, the BPP-1 phage in turn has evolved a way to vary the specificity of the Mtd protein. Through targeted mutagenesis from the phages diversity-generating retroelement (DGR) system, the Mtd protein has shown to be able to support a massive library of protein variants- theoretically up to 9.22*1012 possible variants.

Mtd protein bound to pertactin (yellow). Mtd is composed of 3 monomers- green, blue, and red. Variable amino acids are in purple.


The Mtd gene is a 1146 base pair long sequence that contains two mutagenic islands. There are a total of 22 specific mutagenic nucleotides located in the last ~100 nucleotides on the 3’ end of the sequence, known as the variable repeat (VR). In vivo, there is a nearly identical sequence of nucleotides known as the template repeat (TR) located downstream of the Mtd gene. The DRG system reverse-transcribes a mutant copy of the TR, with mutations confined only to specific adenine residues, which then replaces the VR that encodes the C terminus of the Mtd.


On the quaternary level, the Mtd protein assembles into globular trimeric structure comprised of 380 amino acids in each of its monomers. Out of these 380 amino acids, 12 of them can mutate to a range of different amino acids.


Mtd is a multi-domain protein with a C-lectin type fold at its C terminus. The variable regions of the Mtd form a surface on one face of the pyramid-shaped trimer where binding occurs. The rest of the protein forms a stable scaffold that is constant across different Mtd variants. The Mtd protein binds to the phage tail-fiber at the N-terminus.


Thorough analysis of this protein was conducted through our own 'Protein Calculator' software used concurrently with PyMOL. Similar experiments and analyses, such as the ones conducted with the Mtd protein, can be replicated with any protein containing a C-lectin type fold. Examples include, but are not limited to, aerolysin, invasin, and intimin.


REFERENCES

Overstreet, C M., et al. Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica. Protein Eng. Des. Sel. 145 (2012)