RESUMEN
A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.
Asunto(s)
Nanoestructuras , Proteínas , Cristalografía por Rayos X , Nanoestructuras/química , Proteínas/química , Proteínas/metabolismo , Microscopía Electrónica , Reproducibilidad de los ResultadosRESUMEN
Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.
Asunto(s)
Materiales Biocompatibles , Hidrogeles , Animales , Hidrogeles/química , Biopolímeros , MamíferosRESUMEN
Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
Asunto(s)
Simulación por Computador , Genes Sintéticos/genética , Canales Iónicos/química , Canales Iónicos/genética , Modelos Moleculares , Biología Sintética , Línea Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Conductividad Eléctrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canales Iónicos/metabolismo , Transporte Iónico , Liposomas/metabolismo , Técnicas de Placa-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Solubilidad , Agua/químicaRESUMEN
Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.
Asunto(s)
Nanopartículas , Vacunas , Proteínas , Nanopartículas/químicaRESUMEN
Protein crystallization plays a central role in structural biology. Despite this, the process of crystallization remains poorly understood and highly empirical, with crystal contacts, lattice packing arrangements and space group preferences being largely unpredictable. Programming protein crystallization through precisely engineered side-chain-side-chain interactions across protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach for designing three-dimensional protein crystals with prespecified lattice architectures at atomic accuracy that hierarchically constrains the overall number of degrees of freedom of the system. We design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into >100 µm three-dimensional crystals. The structures of these crystals are nearly identical to the computational design models, closely corresponding in both overall architecture and the specific protein-protein interactions. The dimensions of the crystal unit cell can be systematically redesigned while retaining the space group symmetry and overall architecture, and the crystals are extremely porous and highly stable. Our approach enables the computational design of protein crystals with high accuracy, and the designed protein crystals, which have both structural and assembly information encoded in their primary sequences, provide a powerful platform for biological materials engineering.
Asunto(s)
Proteínas , Proteínas/química , CristalizaciónRESUMEN
Computationally designed multi-subunit assemblies have shown considerable promise for a variety of applications, including a new generation of potent vaccines. One of the major routes to such materials is rigid body sequence-independent docking of cyclic oligomers into architectures with point group or lattice symmetries. Current methods for docking and designing such assemblies are tailored to specific classes of symmetry and are difficult to modify for novel applications. Here we describe RPXDock, a fast, flexible, and modular software package for sequence-independent rigid-body protein docking across a wide range of symmetric architectures that is easily customizable for further development. RPXDock uses an efficient hierarchical search and a residue-pair transform (RPX) scoring method to rapidly search through multidimensional docking space. We describe the structure of the software, provide practical guidelines for its use, and describe the available functionalities including a variety of score functions and filtering tools that can be used to guide and refine docking results towards desired configurations.
Asunto(s)
Algoritmos , Nanoestructuras , Conformación Proteica , Proteínas/química , Programas Informáticos , Unión Proteica , Simulación del Acoplamiento MolecularRESUMEN
Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.
Asunto(s)
Nanoestructuras/química , Ingeniería de Proteínas , Proteínas/química , Repetición de Anquirina , Nanoestructuras/ultraestructura , Conformación Proteica en Hélice alfa , Proteínas/genética , Proteínas/ultraestructuraRESUMEN
In this study, we report the electrophilic cyclization of N,N-dimethyl-o-alkynylanilines with arylsiloxanes in the presence of [Pd(OAc)2] and Ag2O catalytic system, which leads to the efficient synthesis of indoles, similar to the one that is obtained through Larock indole synthesis. A range of aryl(trimethoxy)silanes with EDGs and EWGs were successfully utilized for the synthesis of a diverse variety of substituted indoles via the cleavage of the C-Si bond. This protocol exhibits good functional group tolerance and wide substrate scope to provide 2,3-diaryl-N-methylindoles in 26-88% yields.
RESUMEN
Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biological materials are only beginning to emerge. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as 'enveloped protein nanocages' (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (~100 nm) vesicles containing multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.
Asunto(s)
Materiales Biocompatibles/química , Bioingeniería , Biomimética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas/química , Nanoestructuras/química , Secuencia de Aminoácidos , Membrana Celular/química , Glicoproteínas/genética , Humanos , Vesiculovirus/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Ensamble de Virus , Esparcimiento de VirusRESUMEN
The dodecahedron [corrected] 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 dodecahedron [corrected] 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.
Asunto(s)
Diseño de Fármacos , Multimerización de Proteína , Subunidades de Proteína/química , Simulación por Computador , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Modelos Moleculares , Nanoestructuras/química , Nanoestructuras/ultraestructura , Estabilidad Proteica/efectos de los fármacos , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Computational protein design provides the tools to expand the diversity of protein complexes beyond those found in nature. Understanding the rules that drive proteins to interact with each other enables the design of protein-protein interactions to generate specific protein assemblies. In this work, we designed protein-protein interfaces between dimers and trimers to generate dodecameric protein assemblies with dihedral point group symmetry. We subsequently analyzed the designed protein complexes by native MS. We show that the use of ion mobility MS in combination with surface-induced dissociation (SID) allows for the rapid determination of the stoichiometry and topology of designed complexes. The information collected along with the speed of data acquisition and processing make SID ion mobility MS well-suited to determine key structural features of designed protein complexes, thereby circumventing the requirement for more time- and sample-consuming structural biology approaches.
Asunto(s)
Espectrometría de Masas/métodos , Complejos Multiproteicos/química , Avidina/química , Lactoglobulinas/química , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Prealbúmina/química , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/químicaRESUMEN
Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications.
Asunto(s)
Cisteína/síntesis química , Fibroínas/síntesis química , Seda/síntesis química , Secuencia de Aminoácidos , Animales , Araña Viuda Negra , Cisteína/análisis , Fibroínas/análisis , Fibroínas/genética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Seda/análisis , Seda/genéticaRESUMEN
Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.
Asunto(s)
Araña Viuda Negra/química , Fibroínas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Araña Viuda Negra/genética , Fibroínas/genética , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment and molecular dynamics (MD) simulation, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in non-Newtonian biomaterials exhibiting fluid-like properties under rest and low shear, but shear-stiffening solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly, in correlation with matching formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.
RESUMEN
Discrete protein assemblies ranging from hundreds of kilodaltons to hundreds of megadaltons in size are a ubiquitous feature of biological systems and perform highly specialized functions 1-3. Despite remarkable recent progress in accurately designing new self-assembling proteins, the size and complexity of these assemblies has been limited by a reliance on strict symmetry 4,5. Inspired by the pseudosymmetry observed in bacterial microcompartments and viral capsids, we developed a hierarchical computational method for designing large pseudosymmetric self-assembling protein nanomaterials. We computationally designed pseudosymmetric heterooligomeric components and used them to create discrete, cage-like protein assemblies with icosahedral symmetry containing 240, 540, and 960 subunits. At 49, 71, and 96 nm diameter, these nanoparticles are the largest bounded computationally designed protein assemblies generated to date. More broadly, by moving beyond strict symmetry, our work represents an important step towards the accurate design of arbitrary self-assembling nanoscale protein objects.
RESUMEN
Discrete protein assemblies ranging from hundreds of kilodaltons to hundreds of megadaltons in size are a ubiquitous feature of biological systems and perform highly specialized functions1-3. Despite remarkable recent progress in accurately designing new self-assembling proteins, the size and complexity of these assemblies has been limited by a reliance on strict symmetry4,5. Inspired by the pseudosymmetry observed in bacterial microcompartments and viral capsids, we developed a hierarchical computational method for designing large pseudosymmetric self-assembling protein nanomaterials. We computationally designed pseudosymmetric heterooligomeric components and used them to create discrete, cage-like protein assemblies with icosahedral symmetry containing 240, 540, and 960 subunits. At 49, 71, and 96 nm diameter, these nanoparticles are the largest bounded computationally designed protein assemblies generated to date. More broadly, by moving beyond strict symmetry, our work represents an important step towards the accurate design of arbitrary self-assembling nanoscale protein objects.
RESUMEN
A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures 1 . Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.
RESUMEN
Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.
Asunto(s)
Fibroínas/química , Proteínas Recombinantes de Fusión/química , Seda/biosíntesis , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , ADN Complementario/química , ADN Complementario/genética , Elasticidad , Escherichia coli , Fibroínas/genética , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/ultraestructura , Homología de Secuencia de Aminoácido , Seda/ultraestructura , Arañas , Resistencia a la Tracción , Difracción de Rayos XRESUMEN
The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling.