Illuminating the function of the orphan transporter, SLC22A10, in humans and other primates.

SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17ß-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Multi-scale photocatalytic proximity labeling reveals cell surface neighbors on and between cells.

The cell membrane proteome is the primary biohub for cell communication, yet we are only beginning to understand the dynamic protein neighborhoods that form on the cell surface and between cells. Proximity labeling proteomics (PLP) strategies using chemically reactive probes are powerful approaches to yield snapshots of protein neighborhoods but are currently limited to one single resolution based on the probe labeling radius. Here, we describe a multi-scale PLP method with tunable resolution using a commercially available histological dye, Eosin Y, which upon visible light illumination, activates three different photo-probes with labeling radii ranging from ∼100 to 3000 Å. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor (EGFR) and orthogonally validated >20 neighbors using immuno-assays and AlphaFold-Multimer prediction that generated plausible binary interaction models. We further profiled the protein neighborhoods of cell-cell synapses induced by bi-specific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR)T cells at longer length scales. This integrated multi-scale PLP platform maps local and distal protein networks on cell surfaces and between cells. We believe this information will aid in the systematic construction of the cell surface interactome and reveal new opportunities for immunotherapeutics.

RCSB Protein Data bank: Tools for visualizing and understanding biological macromolecules in 3D.

Now in its 52nd year of continuous operations, the Protein Data Bank (PDB) is the premiere open-access global archive housing three-dimensional (3D) biomolecular structure data. It is jointly managed by the Worldwide Protein Data Bank (wwPDB) partnership. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) is funded by the National Science Foundation, National Institutes of Health, and US Department of Energy and serves as the US data center for the wwPDB. RCSB PDB is also responsible for the security of PDB data in its role as wwPDB-designated Archive Keeper. Every year, RCSB PDB serves tens of thousands of depositors of 3D macromolecular structure data (coming from macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro-electron diffraction). The RCSB PDB research-focused web portal (RCSB.org) makes PDB data available at no charge and without usage restrictions to many millions of PDB data consumers around the world. The RCSB PDB training, outreach, and education web portal (PDB101.RCSB.org) serves nearly 700 K educators, students, and members of the public worldwide. This invited Tools Issue contribution describes how RCSB PDB (i) is organized; (ii) works with wwPDB partners to process new depositions; (iii) serves as the wwPDB-designated Archive Keeper; (iv) enables exploration and 3D visualization of PDB data via RCSB.org; and (v) supports training, outreach, and education via PDB101.RCSB.org. New tools and features at RCSB.org are presented using examples drawn from high-resolution structural studies of proteins relevant to treatment of human cancers by targeting immune checkpoints.

Characterization of an A3G-VifHIV-1-CRL5-CBFß Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host-Pathogen Complexes.

Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFß) to determine the structure of the (A3G-Vif-CRL5-CBFß) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFß complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes.

Global Protease Activity Profiling Identifies HER2-Driven Proteolysis in Breast Cancer.

Differential expression of extracellular proteases and endogenous protease inhibitors has been associated with distinct molecular subtypes of breast cancer. However, due to the tight post-translational regulation of protease activity, protease expression-level data alone are not sufficient to understand the role of proteases in malignant transformation. Therefore, we hypothesized that global profiles of extracellular protease activity could more completely reflect differences observed at the transcriptional level in breast cancer and that subtype-associated protease activity may be leveraged to identify specific proteases that play a functional role in cancer signaling. Here, we used a global peptide library-based approach to profile the activities of proteases within distinct breast cancer subtypes. Analysis of 3651 total peptide cleavages from a panel of well-characterized breast cancer cell lines demonstrated differences in proteolytic signatures between cell lines. Cell line clustering based on protease cleavages within the peptide library expanded upon the expected classification derived from transcriptional profiling. An isogenic cell line model developed to further interrogate proteolysis in the HER2 subtype revealed a proteolytic signature consistent with activation of TGF-ß signaling. Specifically, we determined that a metalloprotease involved in TGF-ß signaling, BMP1, was upregulated at both the protein (2-fold, P = 0.001) and activity (P = 0.0599) levels. Inhibition of BMP1 and HER2 suppressed invasion of HER2-expressing cells by 35% (P < 0.0001), compared to 15% (P = 0.0086) observed in cells where only HER2 was inhibited. In summary, through global identification of extracellular proteolysis in breast cancer cell lines, we demonstrate subtype-specific differences in protease activity and elucidate proteolysis associated with HER2-mediated signaling.

Enhancer Reprogramming within Pre-existing Topologically Associated Domains Promotes TGF-ß-Induced EMT and Cancer Metastasis.

Transcription growth factor ß (TGF-ß) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-ß-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-ß-induced EMT process. Enhancers gained upon TGF-ß treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-ß stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-ß signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-ß signaling-associated cancer metastasis.

Integrative structure and function of the yeast exocyst complex.

Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative-stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride cross-links from chemical-crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo-EM structure, cross-links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo-EM structure, except for Sec5. While not observed in the cryo-EM map, the integrative model localizes the N-terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low-resolution structural data can inform biologically relevant hypotheses, even in the absence of high-resolution data.

SSEThread: Integrative threading of the DNA-PKcs sequence based on data from chemical cross-linking and hydrogen deuterium exchange.

X-ray crystallography and electron microscopy maps resolved to 3-8 Šare generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.

Predicting CD4 T-cell epitopes based on antigen cleavage, MHCII presentation, and TCR recognition.

Accurate predictions of T-cell epitopes would be useful for designing vaccines, immunotherapies for cancer and autoimmune diseases, and improved protein therapies. The humoral immune response involves uptake of antigens by antigen presenting cells (APCs), APC processing and presentation of peptides on MHC class II (pMHCII), and T-cell receptor (TCR) recognition of pMHCII complexes. Most in silico methods predict only peptide-MHCII binding, resulting in significant over-prediction of CD4 T-cell epitopes. We present a method, ITCell, for prediction of T-cell epitopes within an input protein antigen sequence for given MHCII and TCR sequences. The method integrates information about three stages of the immune response pathway: antigen cleavage, MHCII presentation, and TCR recognition. First, antigen cleavage sites are predicted based on the cleavage profiles of cathepsins S, B, and H. Second, for each 12-mer peptide in the antigen sequence we predict whether it will bind to a given MHCII, based on the scores of modeled peptide-MHCII complexes. Third, we predict whether or not any of the top scoring peptide-MHCII complexes can bind to a given TCR, based on the scores of modeled ternary peptide-MHCII-TCR complexes and the distribution of predicted cleavage sites. Our benchmarks consist of epitope predictions generated by this algorithm, checked against 20 peptide-MHCII-TCR crystal structures, as well as epitope predictions for four peptide-MHCII-TCR complexes with known epitopes and TCR sequences but without crystal structures. ITCell successfully identified the correct epitopes as one of the 20 top scoring peptides for 22 of 24 benchmark cases. To validate the method using a clinically relevant application, we utilized five factor VIII-specific TCR sequences from hemophilia A subjects who developed an immune response to factor VIII replacement therapy. The known HLA-DR1-restricted factor VIII epitope was among the six top-scoring factor VIII peptides predicted by ITCall to bind HLA-DR1 and all five TCRs. Our integrative approach is more accurate than current single-stage epitope prediction algorithms applied to the same benchmarks. It is freely available as a web server (http://salilab.org/itcell).

EpCAM homo-oligomerization is not the basis for its role in cell-cell adhesion.

Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays. Second, we also show that EpCAM forms stable dimers on the surface of a cell with pre-formed cell-cell contacts using FLIM-FRET; however, no inter-cellular homo-oligomers were detectable. Thus, our study provides clear evidence that EpCAM indeed does not function as a homophilic cell adhesion molecule and therefore calls for a significant revision of its role in both normal and cancerous tissues. In the light of this, we strongly support the previously suggested name Epithelial Cell Activating Molecule instead of the Epithelial Cell Adhesion Molecule.

Atlas of the Radical SAM Superfamily: Divergent Evolution of Function Using a "Plug and Play" Domain.

The radical SAM superfamily contains over 100,000 homologous enzymes that catalyze a remarkably broad range of reactions required for life, including metabolism, nucleic acid modification, and biogenesis of cofactors. While the highly conserved SAM-binding motif responsible for formation of the key 5'-deoxyadenosyl radical intermediate is a key structural feature that simplifies identification of superfamily members, our understanding of their structure-function relationships is complicated by the modular nature of their structures, which exhibit varied and complex domain architectures. To gain new insight about these relationships, we classified the entire set of sequences into similarity-based subgroups that could be visualized using sequence similarity networks. This superfamily-wide analysis reveals important features that had not previously been appreciated from studies focused on one or a few members. Functional information mapped to the networks indicates which members have been experimentally or structurally characterized, their known reaction types, and their phylogenetic distribution. Despite the biological importance of radical SAM chemistry, the vast majority of superfamily members have never been experimentally characterized in any way, suggesting that many new reactions remain to be discovered. In addition to 20 subgroups with at least one known function, we identified additional subgroups made up entirely of sequences of unknown function. Importantly, our results indicate that even general reaction types fail to track well with our sequence similarity-based subgroupings, raising major challenges for function prediction for currently identified and new members that continue to be discovered. Interactive similarity networks and other data from this analysis are available from the Structure-Function Linkage Database.

Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites.

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.

Immunoproteasome functions explained by divergence in cleavage specificity and regulation.

The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and cancer. However, divergence in function between the iP and the constitutive proteasome (cP) has been unclear. A global peptide library-based screening strategy revealed that the proteasomes have overlapping but distinct substrate specificities. Differing iP specificity alters the quantity of production of certain MHC I epitopes but does not appear to be preferentially suited for antigen presentation. Furthermore, iP specificity was found to have likely arisen through genetic drift from the ancestral cP. Specificity differences were exploited to develop isoform-selective substrates. Cellular profiling using these substrates revealed that divergence in regulation of the iP balances its relative contribution to proteasome capacity in immune cells, resulting in selective recovery from inhibition. These findings have implications for iP-targeted therapeutic development.

Cross-activating c-Met/ß1 integrin complex drives metastasis and invasive resistance in cancer.

The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/ß1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/ß1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/ß1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and ß1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/ß1 complex to maintain the high-affinity ß1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/ß1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from ß1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5ß1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/ß1 complex whose ligand-independent cross-activation and robust affinity for FN drive invasive oncologic processes.

Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer.

Many anticancer and antiviral drugs are purine or pyrimidine analogues, which use membrane transporters to cross cellular membranes. Concentrative nucleoside transporters (CNTs) mediate the salvage of nucleosides and the transport of therapeutic nucleoside analogues across plasma membranes by coupling the transport of ligands to the sodium gradient. Of the three members of the human CNT family, CNT3 has the broadest selectivity and the widest expression profile. However, the molecular mechanisms of the transporter, including how it interacts with and translocates structurally diverse nucleosides and nucleoside analogues, are unclear. Recently, the crystal structure of vcCNT showed that the prokaryotic homologue of CNT3 forms a homotrimer. In this study, we successfully expressed and purified the wild type human homologue, hCNT3, demonstrating the homotrimer by size exclusion profiles and glutaraldehyde cross-linking. Further, by creating a series of cysteine mutants at highly conserved positions guided by comparative structure models, we cross-linked hCNT3 protomers in a cell-based assay, thus showing the existence of hCNT3 homotrimers in human cells. The presence and absence of cross-links at specific locations along TM9 informs us of important structural differences between vcCNT and hCNT3. Comparative modeling of the trimerization domain and sequence coevolution analysis both indicate that oligomerization is critical to the stability and function of hCNT3. In particular, trimerization appears to shorten the translocation path for nucleosides across the plasma membrane and may allow modulation of the transport function via allostery.

Computational Discovery and Experimental Validation of Inhibitors of the Human Intestinal Transporter OATP2B1.

Human organic anion transporters (OATPs) are vital for the uptake and efflux of drugs and endogenous compounds. Current identification of inhibitors of these transporters is based on experimental screening. Virtual screening remains a challenge due to a lack of experimental three-dimensional protein structures. Here, we describe a workflow to identify inhibitors of the OATP2B1 transporter in the DrugBank library of over 5,000 drugs and druglike molecules. OATP member 2B1 transporter is highly expressed in the intestine, where it participates in oral absorption of drugs. Predictions from a Random forest classifier, prioritized by docking against multiple comparative protein structure models of OATP2B1, indicated that 33 of the 5,000 compounds were putative inhibitors of OATP2B1. Ten predicted inhibitors that are prescription drugs were tested experimentally in cells overexpressing the OATP2B1 transporter. Three of these ten were validated as potent inhibitors of estrone-3-sulfate uptake (defined as more than 50% inhibition at 20 µM) and tested in multiple concentrations to determine exact IC50. The IC50 values of bicalutamide, ticagrelor, and meloxicam suggest that they might inhibit intestinal OATP2B1 at clinically relevant concentrations and therefore modulate the absorption of other concomitantly administered drugs.

Reconstruction of 3D structures of MET antibodies from electron microscopy 2D class averages.

Dynamics of three MET antibody constructs (IgG1, IgG2, and IgG4) and the IgG4-MET antigen complex was investigated by creating their atomic models with an integrative experimental and computational approach. In particular, we used two-dimensional (2D) Electron Microscopy (EM) images, image class averaging, homology modeling, Rapidly exploring Random Tree (RRT) structure sampling, and fitting of models to images, to find the relative orientations of antibody domains that are consistent with the EM images. We revealed that the conformational preferences of the constructs depend on the extent of the hinge flexibility. We also quantified how the MET antigen impacts on the conformational dynamics of IgG4. These observations allow to create testable hypothesis to investigate MET biology. Our protocol may also help describe structural diversity of other antigen systems at approximately 5 Å precision, as quantified by Root-Mean-Square Deviation (RMSD) among good-scoring models.

Clustering of disulfide-rich peptides provides scaffolds for hit discovery by phage display: application to interleukin-23.

BACKGROUND: Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity. RESULTS: We developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the representatives were structurally distinct from one another, but similar to other DRPs in their respective clusters. To demonstrate the utility of the clusters, phage libraries were constructed for three of the representative scaffolds and panned against interleukin-23. One library produced a peptide that bound to this target with an IC50 of 3.3 µM. CONCLUSIONS: Most DRP clusters contained members that were diverse in sequence, host organism, and interacting proteins, indicating that cluster members were functionally diverse despite having similar structure. Only 20 peptide scaffolds accounted for most of the natural DRP structural diversity, providing suitable starting points for seeding phage display experiments. Through selection of the scaffold surface to vary in phage display, libraries can be designed that present sequence diversity in architecturally distinct, biologically relevant combinations of secondary structures. We supported this hypothesis with a proof-of-concept experiment in which three phage libraries were constructed and panned against the IL-23 target, resulting in a single-digit µM hit and suggesting that a collection of libraries based on the full set of 20 scaffolds increases the potential to identify efficiently peptide binders to a protein target in a drug discovery program.