Neil P. King

Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy

Design and Build Self-assembling biomolecules are attractive building blocks in the development of functional materials. Sophisticated DNA-based materials have been developed; however, progress in designing protein-based materials has been slower. King et al. (p. 1171) describe a general computational method in which protein building blocks are first symmetrically docked onto a target architecture, and then binding interfaces that drive self-assembly of the building blocks are designed. As a proof of principle, trimeric building blocks were used to design self-assembling 12-subunit complexes with tetrahedral symmetry and 24-subunit complexes with octahedral symmetry. Lai et al. (p. 1129) were able to build a 12-subunit tetrahedral protein cage from fused oligomeric protein domains. A general computational method is used to design protein building blocks that self-assemble into target architectures. We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

Accurate design of co-assembling multi-component protein nanomaterials.

The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.

Accurate design of megadalton-scale two-component icosahedral protein complexes.

Designed to assemble Symmetric macromolecular structures that form cages, such as viral capsids, have inspired protein engineering. Bale et al. used pairwise combinations of dimeric, trimeric, or pentameric building blocks to design two-component, 120-subunit protein complexes with three distinct icosahedral architectures. The capsid-like nanostructures are large enough to hold nucleic acids or other proteins, and because they have two components, the assembly of cargoes such as drugs and vaccines can be done in a controlled way. Science, this issue p. 389 A computational approach helped in the design of 120-subunit icosahedral protein cages capable of packaging macromolecular cargo. Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

Principles for designing ordered protein assemblies

In nature, many proteins have evolved to have self-complementary shapes. This drives them to assemble into supramolecular structures, sometimes of great complexity, and often carrying out sophisticated cellular functions. Designing novel proteins that can self-assemble into similarly complex structures is a longstanding goal in bioengineering. New ideas, combined with continually improving computer algorithms, are making it possible to advance on that goal, bringing wide-ranging applications in synthetic biology within reach. Prospective applications range from vaccine design to molecular delivery to bioactive materials. Recent strategies and examples of successfully designed protein cages, layers, and crystals are reviewed.

Structure and folding of a designed knotted protein

A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy—connecting two monomers of an intertwined homodimer into a single protein chain—supports a model for evolution of knotted structures via gene duplication.

Design of a hyperstable 60-subunit protein icosahedron

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

Practical approaches to designing novel protein assemblies.

Molecular self-assembly offers a means by which sophisticated materials can be constructed with unparalleled precision. Designing self-assembling protein structures is of particular interest as a result of the unique functional capabilities of proteins. Custom-designed protein materials could lead to new possibilities in therapeutics, bioenergy, and materials science. Although the field was long hampered by the challenges involved in designing such complex molecules, novel approaches and computational tools have recently led to remarkable progress. Here we review recent design studies in the context of three fundamental aspects of self-assembling materials: subunit organization, subunit interactions, and regulation of assembly.

Protein stabilization in a highly knotted protein polymer

The polypeptide backbones of a few proteins are tied in a knot. The biophysical effects and potential biological roles of knots are not well understood. Here, we test the consequences of protein knotting by taking a monomeric protein, carbonic anhydrase II, whose native structure contains a shallow knot, and polymerizing it end-to-end to form a deeply and multiply knotted polymeric filament. Thermal stability experiments show that the polymer is stabilized against loss of structure and aggregation by the presence of deep knots.

Structures and functional implications of an AMP-binding cystathionine β-synthase domain protein from a hyperthermophilic archaeon

Cystathionine beta-synthase domains are found in a myriad of proteins from organisms across the tree of life and have been hypothesized to function as regulatory modules that sense the energy charge of cells. Here we characterize the structure and stability of PAE2072, a dimeric tandem cystathionine beta-synthase domain protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Crystal structures of the protein in unliganded and AMP-bound forms, determined at resolutions of 2.10 and 2.35 A, respectively, reveal remarkable conservation of key functional features seen in the gamma subunit of the eukaryotic AMP-activated protein kinase. The structures also confirm the presence of a suspected intermolecular disulfide bond between the two subunits that is shown to stabilize the protein. Our AMP-bound structure represents a first step in investigating the function of a large class of uncharacterized prokaryotic proteins. In addition, this work extends previous studies that have suggested that, in certain thermophilic microbes, disulfide bonds play a key role in stabilizing intracellular proteins and protein-protein complexes.

Evolution of a designed protein assembly encapsulating its own RNA genome

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.