Sometimes it can be advantageous to combine two proteins into one. One such technique was described by Jennifer Padilla, Christos Colovos, and Todd Yeates back in 2001 (Padilla, et al., 2001). By connecting two proteins, one that dimerized, and another that trimerized, they were able to design synthetic ‘nanohedra’. The way they achieved this was by extending a C-terminal α-helix at the end of one protein by another α-helix ‘linker’, directly into the N-terminal α-helix of another protein:

The resulting molecule spontaneously self-assembled into an artificial molecular assembly that resembles a cage.

Yeates’s group more recently published a variant on this techinique (Liu, et al., 2018) that connected a third protein onto one of the proteins in a synthetic cage:

This third protein was an antibody mimetic known as a DARPin, or Designed Ankyrin Repeat ProteIN. Ankyrins are a class of naturally occurring binding proteins responsible for a variety of functions, such as cell signaling, regulation, and maintaining the structural integrity of the cell. By repeating a simple secondary structural motif, and using techniques such as ribosome display or phage display, it is possible to genetically engineer proteins of varying sizes to bind to a desired ‘antigen’. See for example in the 2° structure of PDB (Berman et al., 2007) entry 3zu7 how the “helix-helix-turn-turn” pattern in the STRIDE description repeats four times:

This DARPin binds to MAP kinase ERK2:

This DARPin was combined with the symmetric cage to create a new symmetric protein, DARP14 (PDB ID 6c9k), which contains the DARPin chain from PDB entry 3zu7, again formed by linking the C-terminal α-helix of on

Why would you want to attach a third protein to a symmetric cage? One of the most exciting recent developments in structural biology is the emergence of single particle cryo-electron microscopy, which thanks to new detector technology is able to resolve more detail than ever. In fact, it is starting to challenge X-ray crystallography in its ability to resolve atomic detail. One of the advantages of cryo-EM over X-ray crystallography is that it is not necessary to crystallize the protein of interest, something that is not always guaranteed to happen. Finding the right crystallization conditions often comes down to luck. By connecting a smaller protein to a larger symmetric cage via a rigid α-helical linker, Yeates and co-workers have shown it is possible to obtain the structure of a protein smaller than the theoretical 50 kDa limit. Furthermore, by fusing an antibody mimic onto the rigid scaffold of a symmetric cage, we now have a general platform to bind a fourth protein of interest to the DARPin, opening up a new avenue for protein structure determination.

Figure Credits

The first two figures are from Padilla et al. (2001) and Liu et al., (2018); the third figure is from the RCSB PDB website; and the last two images were created using PyMOL (Schrödinger et al., 2015).

References

Berman H, Henrick K, Nakamura H, & Markley JL (2007) The worldwide Protein Data Bank (wwPDB): ensuring a single, uniform archive of PDB data. Nucleic acids research 35(Database issue): D301-303.

Padilla JE, Colovos C, Yeates TO (2001) Nanohedra: Using symmetry to design self-assembling protein cages, layers, crystals, and filaments. Proc Natl Acad Sci USA, 98: 2217-2221.

Liu Y, Gonen S, Gonen T, Yeates TO (2018) Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system. Proc Natl Acad Sci USA, 115: 3362-3367.

Schrödinger (2015) The PyMOL Molecular Graphics System), Version 1.8.