Making Artificial Cages with a Ring Protein - American Chemical Society

Chapter 1

TRAPped Structures: Making Artificial Cages with a Ring Protein Downloaded by 80.82.77.83 on November 15, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1252.ch001

Jonathan G. Heddle* Bionanoscience and Biochemistry Laboratory, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland *E-mail [email protected]

Protein cages are hollow containers typically made from multiple copies of a small number of protein subunits. They occur extensively in nature and morphologies can vary but typically they are close to spherical in shape and have diameters in the nanometers to tens of nanometers range. Because central cavities of protein cages are bounded, they offer possibilities for use as nanoreactors or as transport systems in vivo and in fields as diverse as materials science and medicine. It is now possible to produce artificial cage proteins, thus widening their potential further. Here we consider in brief natural and artificial protein cages before focusing on the development of a ring-shaped protein (TRAP) leading up to and including its use as a component of an artificial protein cage.

Naturally occurring protein cages are well known in bionanoscience and perhaps show greatest potential for medical use. This is illustrated by virus capsids, the protein shells of viruses that protect, transport and mediate delivery of viral genetic material to host cells. Natural viruses are of course superbly adapted for this form of delivery, an ability that has been exploited in the development of gene therapy (1) and they can be further modified genetically or chemically to engineer-in desired properties, with recombinant DNA technology allowing the production of non-infectious virus particles free of genetic material (known as virus-like particles, VLP). It is now even possible to use advanced synthetic biology approaches in which completely artificial protein cages are designed, © 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by 80.82.77.83 on November 15, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1252.ch001

typically in silico (albeit with naturally occurring proteins as the basic building blocks). This allows the construction of protein cages that do not exist in nature, thus opening up new potential morphologies and functionalities. The fact that naturally occurring protein cages are easily available and have been widely studied and characterized for decades means that they have benefitted from considerably more research in terms of applications-focused engineering compared to artificial cages. In many cases cages with known high-resolution structures were used, allowing for precise alterations in their structures at well-defined locations. Many excellent reviews in recent years have summarized the advances made in exploiting and developing natural protein cages (2–6). Some highlights include the use of the plant virus cowpea mosaic virus for a range of therapeutic applications (7–9) including, recently as an “in situ vaccine” with promising activity against a number of tumor models (10) wherein simple administration of the unmodified VLP resulted in neutrophil activation. The VLP derived from bacteriophage P22 virus has also shown much promise, particularly as a nano-reactor able to encapsulate functional enzymes (11, 12). Non-virus cages have also been used, most notably ferritin, which has been widely developed. In the cell, ferritin acts as a store of iron, protecting the body from this essential but potentially toxic metal. Ferritin has been shown to be able to mineralize other inorganic materials such as CdSe (13), CoPt (14), TiO2 (15) including some with possible uses in electronics (16). It can also be used to capture potentially useful therapeutic molecules, one example being the encapsulation of doxorubicin and its delivery to tumors (17). In this case the ability of ferritin to be modified with surface peptide sequences specific to cell surface proteins that are up-regulated in some tumors, proved useful (18). Another non-viral cage that has been developed is based on lumazine synthase, an enzyme from Aquifex aeolicus that naturally forms a cage structure. Engineering of this cage has been shown to result in versions able to capture cargo both in vivo (19) and in vitro (20). For example in in vivo work, amino acid residues lining the interior cavity can be mutated to those carrying a negative charge to give a negatively charged interior which is then able to capture a protein (e.g., GFP) that has been modified to contain a short, positively charged amino acid tag (19). Subsequently the protein container’s loading capacity was improved by a directed evolution approach where the cargo (in this case HIV protease) was toxic to the host cell meaning that changes in protein cage sequence resulting in more efficient encapsulation would be favoured. Improved cages were produced after only four rounds of selection (21). More recently lumazine synthase has been shown to be able to act as a nanoreactor, encapsulating an enzyme (ascorbate peroxidase) that is able to polymerise 3,3-diaminobenzidine (22), and it continues to be a promising protein in bionanoscience (23). One step beyond naturally occurring protein cages is the use of designed or artificial protein cages. Here proteins that do not naturally form cages are modified such that cage formation is promoted. As with all examples where designed, artificial versions of natural assemblies are produced, the artificial approach allows the construction of structures and development of capabilities that may not be available in nature and so widens the possibilities beyond those constrained by evolutionary pathways. Such a synthetic biology approach to protein cage 4 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


production has met with significant success in recent years and the field holds much promise. However, designing protein structures is not easy; the protein folding “paradox” (24) suggested that it is essentially impossible to accurately predict the amino acid sequence that will fold into a desired structure. It is now known that the problem may not be as insurmountable as initially thought (25) and in fact, great progress has been made in protein structure prediction (26). Nevertheless designing protein structures de novo is still extremely challenging and often not practicable. Therefore current approaches make novel structures by utilizing existing proteins that are stable enough to allow further modification to endow them with the ability to assemble into desired cage structures. Note that this approach, utilizing proteins, is distinct from a self-assembled peptide method whereby cages (or other) structures are built up from smaller, peptide subunits. Such a peptide strategy is useful in its own right and it was recently reported that a mixture of two short coiled-coil bundles could self-assemble into a large (~100 nm) diameter peptide cage (27). In general, cages made from proteins may be expected to have greater sequence/structural redundancy compared to peptide structures, allowing a larger number of modifications per monomer building block. In the design of protein cages there have been to date, two main approaches. In the first, naturally occurring proteins with different rotational symmetries are fused together with linker amino acid sequences to produce tandem proteins. In order for the preferred symmetries of the individual domains of these newly formed proteins to be satisfied, they form cage-like assemblies, typically with standard symmetries (icosahedral, octahedral etc.) This fusion approach was first shown in 2001 by Padilla et al. (28) who fused together bromoperoxidase (a homotrimer) and M1 matrix protein from influenza virus (a homodimer). Twelve such subunits assembled into a tetrahedral arrangement with the trimers forming the three-fold axes of the tetrahedron. This approach relies on existing protein-protein interfaces with the designed structures acting essentially as passive “rods” to link together the different proteins. Indeed, the structure of a modified version of this tetrahedron showed the importance of the linker as they demonstrated considerable bending and distortion (29). A second route to achieve assembly of artificial cages is to use computational approaches coupled with protein structural information where, instead of relying on existing protein-protein interactions, new interacting interfaces are designed into proteins of appropriate symmetry. For example the typical soccer-ball (icosahedral) shape would require twelve pentagons and twenty hexagons. Two proteins, one a pentamer with 5-fold rotational symmetry and one a hexamer with 6-fold rotational symmetry could in principle be designed with protein-protein interfaces between monomer edges to promote self-assembly into the icosahedral arrangement. In protein work this usually involves the design of a favourable interface (typically employing hydrophobic and/or electrostatic interactions) to allow stable packing between protein building blocks. This method has been demonstrated with much success by the group of David Baker (30–32). King et al (30) pioneered the approach, first choosing simple octahedral or tetrahedral point group symmetries as a model. Both include three fold symmetry axes, so by choosing homotrimer proteins, the three-fold protein and model axes could be aligned, allowing prediction 5 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


and subsequent optimization of the resulting protein-protein interfaces between monomers of neighboring timers. The first experiments confirmed production of a 13 nm diameter octahedral cage (made from 24 subunits of modified propanediol utilization polyhedral body protein from Salmonella enterica) and an 11 nm diameter tetrahedral cage (made from 12 copies of putative acetyltransferase SACOL2570 from Staphylococcus aureus) (30). Since then the size and complexity of protein cages has advanced rapidly with highlights including the use of two different proteins (trimers or dimers) that self-assemble to form tetrahedral (31); a 60-subunit icosahedral cage notable for its high thermostability (stable to over 80° C) (32) and the impressive design of a large number of protein cages in silico with the actual production and structural characterization of several of them. These cages range from 24 to 40 nm in diameter (up to 2.8 MDa) with 120 copies of two different protein subunits held together by designed interfaces (33). Some of these cages are notable in that, for the first time, they constitute a tightly packed wall separating the inner cavity of the cage from the outside. This compares to previous cages, which had a significant number of large “holes.” In this respect some of the newest cages more closely resemble virus capsids. In our research with TRAP (trp RNA-binding attenuator protein, Figure 1) we are beginning to develop a third approach in which an inorganic nanoparticle is used to promote interactions between protein rings to form stable protein cages with unusual properties. Although still ongoing, this research suggests a potentially much more limited role for protein-protein interfaces and appears to result in highly stable cage proteins. Our method utilizes metal particles and indeed engineering of proteins so that they interact with metals to assemble into spectacular higher order structures such as crystalline arrays (34, 35) and nanotubes (36) has already been shown as well as a re-engineered ferritin whose ability to form a cage is inducible by copper (37).

Figure 1. Crystal structure of TRAP from G. stearothermophilus (pdb 1qaw (40)). Structure is shown in cartoon format with each monomer numbered individually. Residues discussed in the text are shown as spheres and consist of those involved in nanotube formation, i.e., residues E50 and V69, colored black and dark grey respectively and residue K35 shown in white. Curled arrows indicate a rotation of 90 degrees.

6 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


TRAP (38, 39) is a toroidal protein found in species of Bacillus and related species. The structure and biochemistry of the protein has been well studied and include a number of crystal structures of the tryptophan-liganded protein alone (40, 41) and in complex with RNA (42–44). It is a homo-11mer with an overall diameter of approximately 8 nm and a central hole of approximately 2 nm in diameter (40, 41) (Figure 1). The outer rim of the ring contains numerous charged residues for binding of a specific sequence of mRNA, including a prominent lysine. Each TRAP monomer can also bind tryptophan in a specific binding pocket (40, 45). In vivo the role of the protein is in the control of tryptophan synthesis: A number of enzymes are involved in the synthetic pathway, primarily located in the trp operon (38) and TRAP regulates their transcription by, when liganded with tryptophan, binding to the trp leader sequence (specifically NAG repeats). This leads to formation of a terminator hairpin and transcription termination (38) (though recent work suggests the terminator hairpin may not be necessary) (46). Binding of tryptophan-liganded TRAP also regulates the translation of trpE by promoting formation of a RNA secondary structure that sequesters the ShineDalgarno sequence, thus disfavoring ribosome binding (38). In the absence of tryptophan, TRAP does not bind to the RNA sequences and tryptophan synthesis is allowed to proceed. Our own work with TRAP has included a number of biochemical and structural studies which can provide useful information and inspiration for subsequent engineering of the protein to make novel, artificial structures: We showed that TRAP from different species demonstrated remarkable differences in cooperativity of ligand (tryptophan) binding (47) and we also succeeded in solving the crystal structure of apo-TRAP (48), i.e., a TRAP ring free of bound tryptophan, something which had proved difficult, presumably due to increased flexibility (49) in the tryptophan free form. The resulting structure was interesting as it showed little difference from the liganded protein, suggesting a control of RNA-binding via dynamic rather than major structural changes and we also showed that the effect of ligand binding could be mimicked by low temperature. In other structural work we solved the structure of TRAP in complex with its inhibitor protein Anti-TRAP. Anti-TRAP is a small homotrimer (50) and in vivo it offers an additional layer of control over tryptophan production: it binds to TRAP and inhibits binding to RNA, with Anti-TRAP production itself controlled by levels of charged tRNAtrp. The structure of Anti-TRAP in complex with TRAP (51) showed that it binds around the outer rim of the ring, blocking the key residues involved in RNA binding. One of the monomers of the trimer is not involved in interacting with TRAP, the other two monomers are, with each one of them binding to one TRAP monomer. As a TRAP ring consists of 11 monomers, this would result in at best, five equally spaced Anti-TRAPs binding to ten of the monomers, with one gap. Indeed this stoichiometry has been confirmed from analytical ultracentrifugation and mass spectrometry (51). How could such a non-symmetrical shape be easily crystallised? The answer appears to be that it did not: a minority of TRAP exists in solution in a 12-membered form (in fact a naturally-occurring 12-membered form is known in B. Haoldurans (52)) and it was such a 12-membered form, with exactly six anti-TRAPs bound that crystallised. 7 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


It is in the area of bionanoscience that TRAP has proved particularly interesting. One of the goals of bionanoscience is to design and build novel, artificial structures. As with other areas of synthetic biology, building artificial systems allows fine control, bespoke properties and capabilities beyond those found in nature. In early work we showed that TRAP could be modified with addition of a peptide sequence able to bind titanium (and silicon) surfaces (53) and with cysteine in the central hole for binding to gold. This resulted in a TRAP protein able to capture gold particles and localize them on a silicon surface for subsequent integration into a prototype MOS capacitor (54). We have also been able to engineer TRAP so that it forms rings with altered symmetry (55) (“symmetry engineering”) and protein nanotubes (56) as well as hollow cages (57, 58). In the early symmetry engineering work we converted wild type TRAP protein from G. stearothermophilus from its natural 11-fold rotational symmetry form to one with the equivalent of 12 rather than 11 monomers (i.e., 12-fold rotational symmetry) this was achieved by producing tandem gene fusions of the TRAP proteins connected by unstructured amino acid linkers. When three or four such monomers were fused together they were unable to form the equivalent of an 11-membered ring (11 being a prime number). Instead the closest equivalent was 3×4mers or 4×3mers, which assembled to form the equivalent of a 12-monomer ring. These structures were confirmed by X-ray crystallography (55). Artificial protein nanotubes are another area of interest (59, 60) and nanotubes in general, including carbon nanotubes, peptide nanotubes and lipid nanotubes may be useful in electronics, and for delivery of materials (such as therapeutics) that are filled into their internal cavity (59, 61). Protein rings lend themselves to nanotube construction by virtue of the fact that a tube can simply be formed by the stacking of rings on top of each other, with the hole in the ring acting as the central cavity. We stacked TRAP rings in this way as outlined in Figure 2: One residue per monomer on the surface of each of the two flat faces of the ring was changed to a cysteine, resulting in 11 cysteines per face per ring (TRAP has no other cysteine residues). The residues were chosen so that the resulting cysteines on opposing faces would each be approximately the same radial distance from the center of the ring. It was originally envisaged that this would allow cysteines to align and form disulfide bonds: A similar approach was previously used by Ballister et al. utilizing the ring protein hcp (62). We did find that our modified TRAP proteins could form nanotubes with the overall shape resembling bamboo due to the fact that one end of TRAP is narrower then the other and the interface between TRAP rings was always between like faces (i.e., narrow face-narrow face and wide face-wide face) most likely because the cysteines on non-identical faces do not precisely align. Surprisingly we found that these tubes could only form in the presence of reducing agents such as dithiothreitol (DTT) or dimercaptopropanol (DMP) (Figure 2). This was at first counterintuitive as DTT, in particular is used in protein biochemistry as a reducing agent to specifically ensure that disulfide bonds do not form. However, DTT (and DMP) are essentially short carbon chains containing two –SH groups, one at either end of the chain. It is known that adducts can be formed between these –SH groups and those in cysteine side chains and we proposed that in the case of TRAP rings this was occurring at both SH groups of the 8 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


reducing agent, essentially allowing them to act as cross linkers to link together TRAP rings into the tube form (56). More recent work (63) has allowed us to confirm the details of ring formation and structure in more detail and showed that the wide face-wide face interface absolutely requires the presence of a cross linker due to the fact that other nearby bulky groups prevent the opposing cysteines from approaching close enough for direct disulfide bonds to form. This is not the case at the narrow face-narrow face interface where direct disulfides can form and in fact cysteines can be removed altogether and tubes can still result as long as the cysteines are replaced with residues that promote protein-protein interactions. The protein nanotube work highlights the need for careful use of structural information in designing protein assemblies, particularly at the protein-protein interface.

Figure 2. Formation of a nanotube from modified TRAP. a) Shows crystal structures of four TRAP rings (pdb 1qaw (40)) with residues E50 and V69 shown as black and dark grey spheres respectively. Rings are arranged with like faces opposing each other and double-headed arrows represent interactions between residues of opposing faces. b) Shows molecules of DTT and DMP which are known to be able to promote tube formation by acting as cross-linkers between cysteine groups at position 69 and possibly, at position 50.

We have also used TRAP to build protein cages: This work used a novel method of cage production which, while still under investigation, appears to utilize gold nanoparticles in promoting interactions between rings, resulting in formation of highly stable “spheres”. The discovery of TRAP-cage, complete with low resolution structural data was first reported in 2012 (57): It is constructed from a mutant of TRAP containing cysteine at position 35 in place of the wild type lysine, with the resulting mutant being referred to as TRAP-K35C (Figure 1). We found that upon incubation with 1.4 nm diameter gold nanoparticles (GNP), TRAP-K35C assembled into structures which, under transmission electron microscopy (TEM) resemble spherical virus capsids approximately 21 nm in diameter (Figure 3). The spherical and hollow nature of the assembly was confirmed using cryo-electron tomography (Figure 3). Furthermore if the concentration of GNP was increased, a second, smaller diameter (~15 nm) cage was observed. 9 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. a) Cartoon showing that TRAP rings (left) in the presence of gold nanoparticles (“Au”) form large cage like “spheres” (right). b) Shows a cryo-EM image of individual cages in vitreous ice. Scale bars in a and b = 40 nm. c) shows two views of five reconstructed individual TRAP-cages. d) Shows tomographic subvolumes of the particles shown in b, demonstrating their hollow nature. e) Shows AFM image of TRAP-cages. f) Shows the AFM height profile taken along the dotted line in d. Panels b-f are reproduced with permission from ref. (57). Copyright 2002 American Chemical Society.



Figure 4. Analysis of TRAP-cage structure using AFM. ai) Closely packed TRAP-cages (image area 100 nm x 100 nm). aii) Histogram showing height of 65 TRAP-cages, mean = 19.5 nm ± 0.9. bi) unprocessed high speed AFM image of TRAP-cage; bii) 3D reconstruction image of Gauss filtered (3 Å~ 3 pixel) image of bi) with discernible rings numbered; biii) same image as bi) after the application of Gauss filter (3 Å~ 3 pixel) and high-pass FFT frequency filter (10 nm); biv) same image as biii) with a number assigned to each identifiable TRAP ring. Image area 40 nm x 25 nm. Brightness scales on the left show height information in nanometers. Figure is reproduced with permission from ref. (57). Copyright 2002 American Chemical Society



We have subsequently focused research efforts on understanding the larger cage. Intriguingly a convex polyhedron cannot be formed from an 11-sided shape so we initially assumed that the TRAP-cage was formed from TRAP subunits rather than TRAP rings. However, atomic force microscopy (AFM) analysis seemed to suggest that the cage was formed from in tact rings, indicating unusual geometry (Figure 4) (58). It was hypothesized that the cages were held together by disulfide bonds between constituent rings, possibly catalyzed by the presence of the gold nanoparticles which are known to be oxidatively catalytically active (64). The presence of some form of reducible bond (such as a disulfide bond) gained support from spectacular high-speed AFM images which showed the “bursting” of TRAP-cages upon addition of reducing agent (58). These results are interesting – as the interiors of cells are reducing, a stable, inducible cage protein could be envisaged as a potentially useful cell delivery system. Loading of the cage could potentially be achieved simply by cage formation being “switched on” in the presence of cargo molecules (e.g., therapeutics) by the addition of gold particles. These results have raised a number of interesting questions. Firstly the role of gold nanoparticles is intriguing. It was in the 1980s that the discovery was made that small GNPs were highly chemically active (64) a surprising finding given the well-known noble nature of the metal. Initial reactions observed were the catalysis of the oxidation of carbon monoxide to carbon dioxide. The ability of these small gold particles to act as catalysts is now well established although the exact mechanism whereby this is achieved remains the subject of some debate (65). The ability of GNPs to interact with proteins had previously been noted and as the thioaurate bond is well known it is no surprise that some form of interaction with a cysteine residue at the easily accessible position 35 on TRAP could occur. However, typically interactions between GNPs and proteins are non–specific and result in simple aggregation of the protein or formation of a protein “corona” around the GNP (66). The TRAP-cage work represented, to our knowledge, the first example of GNP interaction with proteins in a highly specific manner, resulting in production of a limited variety of highly ordered products with an apparent high yield. The size, geometry and ligand on the gold particle may all be contributing factors to this unique reaction and are currently under investigation. The requirement of the gold nanoparticle in TRAP-cage assembly also offers a challenge to future research and development: As gold particles are not required as part of the final cage structure, then any added particles are likely to be present dispersed throughout the solution or non-specifically interacting with the TRAPcage. Indeed AFM work seems to suggest a preference for binding of the GNPs to the central hole of the TRAP ring presumably through electrostatic interactions (58). For high-resolution X-ray crystal studies of TRAP-cage this could be an obstacle to crystallization of the protein. Furthermore if the TRAP-cage was to be developed therapeutically the presence of these gold particles could be problematic as small (~1.4 nm) GNPs have been shown to be cytotoxic (67, 68). Therefore a way of utilizing gold particles in a different phase from the in-solution TRAP-cage would be advantageous and current experiments are directed towards using GNPs that are tethered by a linker molecules to a surface, over which a solution of cage12 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

forming TRAP is flowed. It is envisaged that this will allow cage to form and be collected free of GNPs which remain tethered to the surface. Overall, TRAP has proved a very versatile protein building block with the ability to interact with GNPs to form unusual cage structures being the latest manifestation. A deeper understanding of the TRAP-cage assembly mechanism and structure may allow us to advance capabilities for new protein chemistries and assemblies.


Acknowledgments The author was funded by the Malopolska Centre of Biotechnology and by the National Science Centre (NCN, Poland) grant No. 2016/20/W/NZ1/00095 (Symfonia-4).

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