Protein Interaction Surfaces Research Articles

Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

Computational completion of the Aurora interaction region of N-Myc in the Aurora a kinase complex

Inhibiting protein–protein interactions of the Myc family is a viable pharmacological strategy for modulation of the levels of Myc oncoproteins in cancer. Aurora A kinase (AurA) and N-Myc interaction is one of the most attractive targets of this strategy because formation of this complex blocks proteasomal degradation of N-Myc in neuroblastoma. Two crystallization studies have captured this complex (PDB IDs: 5g1x, 7ztl), partially resolving the AurA interaction region (AIR) of N-Myc. Prompted by the missing N-Myc fragment in these crystal structures, we modeled the complete structure between AurA and N-Myc, and comprehensively analyzed how the incomplete and complete N-Myc behave in complex by molecular dynamics simulations. Molecular dynamics simulations of the incomplete PDB complex (5g1x) repeatedly showed partial dissociation of the short N-Myc fragment (61–89) from the kinase. The missing N-Myc (19–60) fragment was modeled utilizing the N-terminal lobe of AurA as the protein–protein interaction surface, wherein TPX2, a well-known partner of AurA, also binds. Binding free energy calculations along with flexibility analysis confirmed that the complete AIR of N-Myc stabilizes the complex, accentuating the N-terminal lobe of AurA as a binding site for the missing N-Myc fragment (19–60). We further generated additional models consisting of only the missing N-Myc (19–60), and the fused form of TPX2 (7–43) and N-Myc (61–89). These partners also formed more stable interactions with the N-terminal lobe of AurA than did the incomplete N-Myc fragment (61–89) in the 5g1x complex. Altogether, this study provides structural insights into the involvement of the N-terminus of the AIR of N-Myc and the N-terminal lobe of AurA in formation of a stable complex, reflecting its potential for effective targeting of N-Myc.

Functional contacts for activation of urease from Helicobacter pylori: an integrated approach using evolutionary couplings, in-cell enzymatic assays, and computational docking

Introduction: Urease is an enzyme exploited by many virulent bacteria and fungi to infect the host and exert their virulence. The Gram-negative bacterium Helicobacter pylori relies on the activity of urease to infect the highly acidic human stomach. The activity of urease depends on the presence of a catalytic site containing two Ni(II) ions. In vivo, urease is initially synthesized as an inactive apo-enzyme and requires a post-translational activation process that involves the incorporation of the metal ions into its buried active site. In H. pylori, as well as in other bacteria, this activation process is mediated by four accessory proteins, named UreD, UreF, UreG, and UreE. Targeting the interactions between urease chaperones could potentially inhibit the activation of urease through blocking the Ni(II) ions incorporation, providing a route for the development of antimicrobial strategies against ureolytic pathogens.Methods: In this paper, an evolutionary couplings (EC) approach was adopted to determine the interaction surface between urease and UreD, the first protein that binds the enzyme, preparing it for the subsequent activation steps. Site-directed mutagenesis and an in-cell assay were used to detect urease activity in recombinant bacteria expressing the mutated operon. The obtained data were used to drive a protein-protein docking computational approach.Results and Discussion: The EC prediction retrieved ten pairs of residues lying at the interface between UreD and the urease subunit UreB, likely involved in contacts essential to build the protein complex. These contacts were largely confirmed experimentally, leading to the obtainment of a model for the urease-UreD complex that agrees well with the recently reported experimental cryo-EM structure. This work represents a proof of concept for the calculation of reliable models of protein interaction surfaces in the absence of experimental structures of critical assemblies.

Computational design of N-linked glycans for high throughput epitope profiling.

Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high-resolution low throughput methods like x-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, x-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope.

Inherently disordered regions of axonemal dynein assembly factors.

The dynein-driven beating of cilia is required to move individual cells and to generate fluid flow across surfaces and within cavities. These motor enzymes are highly complex and can contain upwards of 20 different protein components with a total mass approaching 2 MDa. The dynein heavy chains are enormous proteins consisting of ~4500 residues and ribosomes take approximately 15 min to synthesize one. Studies in a broad array of organisms ranging from the green alga Chlamydomonas to humans has identified 19 cytosolic factors (DNAAFs) that are needed to specifically build axonemal dyneins; defects in many of these proteins lead to primary ciliary dyskinesia in mammals which can result in infertility, severe bronchial problems, and situs inversus. How all these factors cooperate in a spatially and temporally regulated manner to promote dynein assembly in cytoplasm remains very uncertain. These DNAAFs contain a variety of well-folded domains many of which provide protein interaction surfaces. However, many also exhibit large regions that are predicted to be inherently disordered. Here I discuss the nature of these unstructured segments, their predicted propensity for driving protein phase separation, and their potential for adopting more defined conformations during the dynein assembly process.

Integrating Protein Interaction Surface Prediction with a Fragment-Based Drug Design: Automatic Design of New Leads with Fragments on Energy Surfaces.

Protein-protein interactions (PPIs) have emerged in the past years as significant pharmacological targets in the development of new therapeutics due to their key roles in determining pathological pathways. Herein, we present fragments on energy surfaces, a simple and general design strategy that integrates the analysis of the dynamic and energetic signatures of proteins to unveil the substructures involved in PPIs, with docking, selection, and combination of drug-like fragments to generate new PPI inhibitor candidates. Specifically, structural representatives of the target protein are used as inputs for the blind physics-based prediction of potential protein interaction surfaces using the matrix of low coupling energy decomposition method. The predicted interaction surfaces are subdivided into overlapping windows that are used as templates to direct the docking and combination of fragments representative of moieties typically found in active drugs. This protocol is then applied and validated using structurally diverse, important PPI targets as test systems. We demonstrate that our approach facilitates the exploration of the molecular diversity space of potential ligands, with no requirement of prior information on the location and properties of interaction surfaces or on the structures of potential lead compounds. Importantly, the hit molecules that emerge from our ab initio design share high chemical similarity with experimentally tested active PPI inhibitors. We propose that the protocol we describe here represents a valuable means of generating initial leads against difficult targets for further development and refinement.

Targeted Degradation of 53BP1 Using Ubiquitin Variant Induced Proximity.

In recent years, researchers have leveraged the ubiquitin-proteasome system (UPS) to induce selective degradation of proteins by E3 ubiquitin ligases, which has great potential as novel therapeutics for human diseases, including cancer and neurodegenerative disorders. However, despite extensive efforts, only a handful of ~600 human E3 ligases were utilized, and numerous protein–protein interaction surfaces on E3 ligases were not explored. To tackle these problems, we leveraged a structure-based protein engineering technology to develop a multi-domain fusion protein bringing functional E3 ligases to the proximity of a target protein to trigger its proteasomal degradation, which we termed Ubiquitin Variant Induced Proximity (UbVIP). We first generated non-inhibitory synthetic UbV binders for a selected group of human E3 ligases. With these UbVs employed as E3 ligase engagers, we designed a library of UbVIPs targeting a DNA damage response protein 53BP1. We observed that two UbVIPs recruiting RFWD3 and NEDD4L could effectively induce proteasome degradation of 53BP1 in human cell lines. This provides a proof-of-principle that UbVs can act as a means of targeted degradation for nucleus-localized proteins. Our work demonstrated that UbV technology is suitable to develop protein-based molecules for targeted degradation and can help identify novel E3 ligases for future therapeutic development.

Are stress granules the RNA analogs of misfolded protein aggregates?

Ribonucleoprotein granules are ubiquitous features of eukaryotic cells. Several observations argue that the formation of at least some RNP granules can be considered analogous to the formation of unfolded protein aggregates. First, unfolded protein aggregates form from the exposure of promiscuous protein interaction surfaces, while some mRNP granules form, at least in part, by promiscuous intermolecular RNA–RNA interactions due to exposed RNA surfaces when mRNAs are not engaged with ribosomes. Second, analogous to the role of protein chaperones in preventing misfolded protein aggregation, cells contain abundant “RNA chaperones” to limit inappropriate RNA–RNA interactions and prevent mRNP granule formation. Third, analogous to the role of protein aggregates in diseases, situations where RNA aggregation exceeds the capacity of RNA chaperones to disaggregate RNAs may contribute to human disease. Understanding that RNP granules can be considered as promiscuous, reversible RNA aggregation events allow insight into their composition and how cells have evolved functions for RNP granules.

High‐Throughput Quality Control Assay for the Solid‐Phase Synthesis of DNA‐Encoded Libraries of Macrocycles**

AbstractThere is considerable interest in the development of libraries of scaffold‐diverse macrocycles as a source of ligands for difficult targets, such as protein–protein interaction surfaces. A classic problem in the synthesis of high‐quality macrocyclic libraries is that some linear precursors will cyclize efficiently while some will not, depending on their conformational preferences. We report here a powerful quality control method that can be employed to readily distinguish between scaffolds that do and do not cyclize efficiently during solid‐phase synthesis of thioether macrocycles without the need for tedious liquid chromatography/mass spectrometry analysis. We demonstrate that this assay can be employed to identify linear impurities in a DNA‐encoded library of macrocycles. We also use the method to establish a useful quality control protocol for re‐synthesis of putative macrocyclic screening hits.

Diversification of CD1 Molecules Shapes Lipid Antigen Selectivity.

Molecular studies of host–pathogen evolution have largely focused on the consequences of variation at protein–protein interaction surfaces. The potential for other microbe-associated macromolecules to promote arms race dynamics with host factors remains unclear. The cluster of differentiation 1 (CD1) family of vertebrate cell surface receptors plays a crucial role in adaptive immunity through binding and presentation of lipid antigens to T-cells. Although CD1 proteins present a variety of endogenous and microbial lipids to various T-cell types, they are less diverse within vertebrate populations than the related major histocompatibility complex (MHC) molecules. We discovered that CD1 genes exhibit a high level of divergence between simian primate species, altering predicted lipid-binding properties and T-cell receptor interactions. These findings suggest that lipid–protein conflicts have shaped CD1 genetic variation during primate evolution. Consistent with this hypothesis, multiple primate CD1 family proteins exhibit signatures of repeated positive selection at surfaces impacting antigen presentation, binding pocket morphology, and T-cell receptor accessibility. Using a molecular modeling approach, we observe that interspecies variation as well as single mutations at rapidly-evolving sites in CD1a drastically alter predicted lipid binding and structural features of the T-cell recognition surface. We further show that alterations in both endogenous and microbial lipid-binding affinities influence the ability of CD1a to undergo antigen swapping required for T-cell activation. Together these findings establish lipid–protein interactions as a critical force of host–pathogen conflict and inform potential strategies for lipid-based vaccine development.

Exploring the treatment of COVID-19 with Yinqiao powder based on network pharmacology.

Coronavirus disease 2019 (COVID‐19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). In China, Yinqiao powder is widely used to prevent and treat COVID‐19 patients with Weifen syndrome. In this study, the screening and verification of active ingredients, target selection and DisGeNET scoring, drug–ingredient–gene network construction, protein–protein interaction network construction, molecular docking and surface plasmon resonance (SPR) analysis, gene ontology (GO) functional analysis, gene tissue analysis, and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were used to explore the active ingredients, targets, and potential mechanisms of Yinqiao powder in the treatment of COVID‐19. We also predicted the therapeutic effect of Yinqiao powder using TCM anti‐COVID‐19 (TCMATCOV). Yinqiao powder has a certain therapeutic effect on COVID‐19, with an intervention score of 20.16. Hesperetin, eriodictyol, luteolin, quercetin, and naringenin were the potentially effective active ingredients against COVID‐19. The hub‐proteins were interleukin‐6 (IL‐6), mitogen‐activated protein kinase 3 (MAPK3), tumor necrosis factor (TNF), and tumor protein P53 (TP53). The potential mechanisms of Yinqiao powder in the treatment of COVID‐19 are the TNF signaling pathway, T‐cell receptor signaling pathway, Toll‐like receptor signaling pathway, and MAPK signaling pathway. This study provides a new perspective for discovering potential drugs and mechanisms of COVID‐19.

Structure-based identification of a new IAP-targeting compound that induces cancer cell death inducing NF-κB pathway

Inhibitors of apoptosis proteins (IAPs) are validated onco-targets, as their overexpression correlates with cancer onset, progression, diffusion and chemoresistance. IAPs regulate cell death survival pathways, inflammation, and immunity. Targeting IAPs, by impairing their protein–protein interaction surfaces, can affect events occurring at different stages of cancer development.To this purpose, we employed a rational virtual screening approach to identify compounds predicted to interfere with the assembly of pro-survival macromolecular complexes. One of the candidates, FC2, was shown to bind in vitro the BIR1 domains of both XIAP and cIAP2. Moreover, we demonstrated that FC2 can induce cancer cell death as a single agent and, more potently, in combination with the Smac-mimetic SM83 or with the cytokine TNF. FC2 determined a prolonged activation of the NF-κB pathway, accompanied to a stabilization of XIAP-TAB1 complex. This candidate molecule represents a valuable lead compound for the development of a new class of IAP-antagonists for cancer treatment.

Simplified geometric representations of protein structures identify complementary interaction interfaces.

Protein‐protein interactions are critical to protein function, but three‐dimensional (3D) arrangements of interacting proteins have proven hard to predict, even given the identities and 3D structures of the interacting partners. Specifically, identifying the relevant pairwise interaction surfaces remains difficult, often relying on shape complementarity with molecular docking while accounting for molecular motions to optimize rigid 3D translations and rotations. However, such approaches can be computationally expensive, and faster, less accurate approximations may prove useful for large‐scale prediction and assembly of 3D structures of multi‐protein complexes. We asked if a reduced representation of protein geometry retains enough information about molecular properties to predict pairwise protein interaction interfaces that are tolerant of limited structural rearrangements. Here, we describe a reduced representation of 3D protein accessible surfaces on which molecular properties such as charge, hydrophobicity, and evolutionary rate can be easily mapped, implemented in the MorphProt package. Pairs of surfaces are compared to rapidly assess partner‐specific potential surface complementarity. On two available benchmarks of 185 overall known protein complexes, we observe predictions comparable to other structure‐based tools at correctly identifying protein interaction surfaces. Furthermore, we examined the effect of molecular motion through normal mode simulation on a benchmark receptor‐ligand pair and observed no marked loss of predictive accuracy for distortions of up to 6 Å Cα‐RMSD. Thus, a shape reduction of protein surfaces retains considerable information about surface complementarity, offers enhanced speed of comparison relative to more complex geometric representations, and exhibits tolerance to conformational changes.

Structural and functional modelling of SARS-CoV-2 entry in animal models

SARS-CoV-2 is the novel coronavirus responsible for the outbreak of COVID-19, a disease that has spread to over 100 countries and, as of the 26th July 2020, has infected over 16 million people. Despite the urgent need to find effective therapeutics, research on SARS-CoV-2 has been affected by a lack of suitable animal models. To facilitate the development of medical approaches and novel treatments, we compared the ACE2 receptor, and TMPRSS2 and Furin proteases usage of the SARS-CoV-2 Spike glycoprotein in human and in a panel of animal models, i.e. guinea pig, dog, cat, rat, rabbit, ferret, mouse, hamster and macaque. Here we showed that ACE2, but not TMPRSS2 or Furin, has a higher level of sequence variability in the Spike protein interaction surface, which greatly influences Spike protein binding mode. Using molecular docking simulations we compared the SARS-CoV and SARS-CoV-2 Spike proteins in complex with the ACE2 receptor and showed that the SARS-CoV-2 Spike glycoprotein is compatible to bind the human ACE2 with high specificity. In contrast, TMPRSS2 and Furin are sufficiently similar in the considered hosts not to drive susceptibility differences. Computational analysis of binding modes and protein contacts indicates that macaque, ferrets and hamster are the most suitable models for the study of inhibitory antibodies and small molecules targeting the SARS-CoV-2 Spike protein interaction with ACE2. Since TMPRSS2 and Furin are similar across species, our data also suggest that transgenic animal models expressing human ACE2, such as the hACE2 transgenic mouse, are also likely to be useful models for studies investigating viral entry.

Characterization of protein interaction surface on fatty acyl selectivity of starter condensation domain in lipopeptide biosynthesis.

Lipopeptides are important non-ribosomal peptide synthetases (NRPSs) products with broad therapeutic potential in biotechnology and biopharmaceutical applications. Fatty acyl modifications in N-terminal of lipopeptides have attracted wide interest in the engineering processes of altered fatty acyl selectivity. In this study, we focused on the starter condensation domain of antibiotic A54145 (lptC1) and its indiscriminate selectivity of fatty acyl substrates, which results in multi-component products. Using in silico analysis, five site-directed mutations at protein-protein interface were identified with altered activity and selectivity towards wildtype lptC1. The variants Y149W and A330T exhibited changed substrate selectivity to prefer longer branched chain fatty acyl substrate, while T16A and A350D showed improved selectivity for shorter linear chain fatty acyl substrates. Subsequently, molecular dynamics (MD) simulations were performed to analyze the impact of these residues on the changes of catalytic activity and conformation. Through in silico analysis, the altered binding free energy were coincident with the corresponding activity performance of the variants, and surface forces indicated that other factors or processes may influence the activity and selectivity. Moreover, the MD results revealed even altered active center conformations, implying the importance of these interface residues affected on distant active center thus reflected to catalysis activity. Based on the biochemistry and computational results, our work provides detailed insights from molecular and dynamics aspects into the role of C1's interface residues during complex NRPS biosynthesis machinery, prompting further rational engineering for lipopeptide catalysis.

Modified Potential Functions Result in Enhanced Predictions of a Protein Complex by All-Atom Molecular Dynamics Simulations, Confirming a Stepwise Association Process for Native Protein-Protein Interactions.

The relative prevalence of native protein-protein interactions (PPIs) are the cornerstone for understanding the structure, dynamics and mechanisms of function of protein complexes. In this study, we develop a scheme for scaling the protein-water interaction in the CHARMM36 force field, in order to better fit the solvation free energy of amino acids side-chain analogues. We find that the molecular dynamics simulation with the scaled force field, CHARMM36s, as well as a recently released version, CHARMM36m, effectively improve on the overly sticky association of proteins, such as ubiquitin. We investigate the formation of a heterodimer protein complex between the SAM domains of the EphA2 receptor and the SHIP2 enzyme by performing a combined total of 48 μs simulations with the different potential functions. While the native SAM heterodimer is only predicted at a low rate of 6.7% with the original CHARMM36 force field, the yield is increased to 16.7% with CHARMM36s, and to 18.3% with CHARMM36m. By analyzing the 25 native SAM complexes formed in the simulations, we find that their formation involves a preorientation guided by Coulomb interactions, consistent with an electrostatic steering mechanism. In 12 cases, the complex could directly transform to the native protein interaction surfaces with only small adjustments in domain orientation. In the other 13 cases, orientational and/or translational adjustments are needed to reach the native complex. Although the tendency for non-native complexes to dissociate has nearly doubled with the modified potential functions, a dissociation followed by a reassociation to the correct complex structure is still rare. Instead, the remaining non-native complexes undergo configurational changes/surface searching, which, however, rarely leads to native structures on a time scale of 250 ns. These observations provide a rich picture of the mechanisms of protein-protein complex formation and suggest that computational predictions of native complex PPIs could be improved further.

Epigenetics and transcription regulation during eukaryotic diversification: the saga of TFIID.

The basal transcription factor TFIID is central for RNA polymerase II-dependent transcription. Human TFIID is endowed with chromatin reader and DNA-binding domains and protein interaction surfaces. Fourteen TFIID TATA-binding protein (TBP)-associated factor (TAF) subunits assemble into the holocomplex, which shares subunits with the Spt-Ada-Gcn5-acetyltransferase (SAGA) coactivator. Here, we discuss the structural and functional evolution of TFIID and its divergence from SAGA. Our orthologous tree and domain analyses reveal dynamic gains and losses of epigenetic readers, plant-specific functions of TAF1 and TAF4, the HEAT2-like repeat in TAF2, and, importantly, the pre-LECA origin of TFIID and SAGA. TFIID evolution exemplifies the dynamic plasticity in transcription complexes in the eukaryotic lineage.

MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis.

SummaryIn mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.

FragLites-Minimal, Halogenated Fragments Displaying Pharmacophore Doublets. An Efficient Approach to Druggability Assessment and Hit Generation.

Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.

Directed Assembly of Homopentameric Cholera Toxin B-Subunit Proteins into Higher-Order Structures Using Coiled-Coil Appendages.

The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein–protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are prescreened for “designability”, limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multisubunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. Analysis of a tubular structure determined by X-ray crystallography has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight “native-state” forced protein–protein interfaces, which may prove useful as starting conformations for future computational design.