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Regulated assembly


Folded protein domains that form oligomers with same or other protein domains could be used to form complex nanostructures. We prepared a new type of polypeptide building blocks composed of dimerization and trimerization domains of proteins, connected by a short flexible peptide linker. This combination should be able to form a polypeptide nanocage or a polypeptide lattice with defined pore size. We designed, prepared and purified fusion protein between the N-terminal gyrase subunit B dimerization domain and trimerization domains composed of CutA1 and foldon. The main advantage of this system is that we can regulate the assembly and disassembly of this material since we can control the dimerization state of the gyrase B domain by adding aminocoumarin antibiotics novobiocin and coumermycin. Both of them bind to the same site on gyrase subunit B. Coumermycin is a pseudodimer and causes gyrase B dimerization while novobiocin binds competitively to the same binding site and disrupts coumermycin-induced dimers. In this way we can assemble or disassemble polypeptide nanostructures, which could be particularly useful to enmesh compounds such as biological drugs or any other application, where controlled release is desired. We demonstrated the ability of the material to assemble and disassemble in the presence of coumermycin and novobiocin respectively and observed the porous structure of the material at the nanoscale by transmission electron microscope.

Figure 1: Basic structural element of the fusion protein gyrB-CutA1, which is trimeric in the solution, due to the trimerization by CutA1 (A). Transmission electron microscopic image of gyrB-CutA1 (B) material crosslinked by coumermycin, which forms hexagonal pores (shown in cyan).

The idea & approach

We can find many different ways of assembling protein structures in nature besides the coiled-coil-driven interactions. Many proteins include dimerization, trimerization and higher order oligomerization domains, which help enzymes and other proteins to interact either to function properly, to stabilize the structure or to bind the subunits in an appropriate stoichiometry.

Some multimerization domains have the ability to regulate the assembly by the presence of small molecules or under the influence of different conditions in the environment. We can utilize this type of property to control the assembly of complex structures.

An example of such an inducible multimerization domain is the N-terminal fragment of bacterial gyrase subunit B. Aminocoumarin antibiotics such as novobiocin and coumermycin bind to the ATP-ase site of gyrase B. Novobiocin tightly binds to the N-terminal part of gyrase subunit B and inhibits its ATPase activity. Coumermycin is structurally a pseudodimer of novobiocin with a short linker between two novobiocin-like segments (Figure 2). Length of the linker between the novobiocin-like segments allows coumermycin to bind to two gyrase B binding sites so that those two polypeptide chains form a tight dimer. Novobiocin, can bind to the same binding site and in competition with coumermycin it can displace it, abolishing coumermycin-induced dimerization (Zhao et al., 2003). With those two compounds we can regulate the assembly and disassembly of fusion polypeptide constructs which include the gyrase B domain.

Figure 2: Aminocoumarin antibiotics: A - coumermycin, B- novobiocin, C – coumermycin binds to two gyrase B binding sites so that those two polypeptide chains form a tight dimmer.

Similar as stated before for the assembly of polypeptide structures the stoichiometry of assembly has to be at least three in order to form two or three-dimensonal polypeptide assemblies, instead of linear chains. On this basis we selected to use trimerization domains as the fusion partner with gyrase B domain. We selected the small bacteriophage T4 fibritin trimerization domain (foldon domain), and CutA1, a trimeric protein that binds heavy metal ions, which can cause additional association (Figure 3).

Figure 3: Molecular representation of a CutA1 (A) and foldon (B) trimer with chains shown in different colours.

Each of those trimerization domains was fused to the 24 kDa fragment of gyrase subunit B (GyrB), connected by a short flexible linker. We could in principle use any other multimerization domain found in nature and put it in a combination with another to create assemblies of different geometry. All of the selected trimerization-dimerization polypeptides additionally include a tag of 6 histidines at N-terminus as added in our new vector (BBa_K245005). Scheme of these constructs (BBa_K245098, BBa_K245050, BBa_K245051) is shown on Figure 4.

Between GyrB and foldon or CutA1 subunits we introduced two or four amino acid linker composed of serine and glycine (SG, SGSG). We decided to vary the length of the linker in order to allow more flexibility of dimerization domains (gyrase B) in comparison to coiled-coils. Length of linker may define if the structure would form a nanocage or a planar lattice.

Figure 4: Scheme of polypeptides containing GyrB and foldon or CutA1 with corresponding BioBrick part numbers.

We constructed a molecular model of GyrB-CutA1 fusion protein, shown on Figure 5. Assembly of a fusion polypeptide by the addition of coumermycin may result in hexagonal assembly forming planar lattice and formation of pores of defined size (Figure 5B).

Figure 5: Hexagonal packing model of GyrB-CutA1 fusion. A: GyrB dimers are shown in blue, green or yellow color, while the trimerization domain of CutA1 is shown in grey. B: Hexagonal packing can cover the planar lattice.

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