Biology subjects

Stewart, L.

Publications and source records attributed to Stewart, L..

At least 19 recordsLinked to original sources

bioRxiv2025

De novo design of phospho-tyrosine peptide binders

Phosphorylation on tyrosine is a key step in many signaling pathways. Despite recent progress in de novo design of protein binders, there are no current methods for designing binders that recognize phosphorylated proteins and peptides; this is a challenging problem as phosphate groups are highly charged, and phosphorylation often occurs within unstructured regions. Here we introduce RoseTTAFold Diffusion 2 for Molecular Interfaces (RFD2-MI), a deep generative framework for the design of binders for protein, ligand, and covalently modified protein targets. We demonstrate the power and versatility of this method by designing binders for four critical phosphotyrosine sites on three clinically relevant targets: Cluster of Differentiation 3 (CD3{varepsilon}), Epidermal Growth Factor Receptor (EGFR), Insulin Receptor (INSR) and Signal Transducer and Activator of Transcription 5 (STAT5). Experimental characterization shows that the designs bind their phosphotyrosine containing targets with affinities comparable to native binding sites and have negligible binding to non-phosphorylated targets or phosphopeptides with different sequences. X-ray crystal structures of generated binders to CD3{varepsilon} and EGFR are very close to the design models, demonstrating the accuracy of the design approach. A designed binder to an EGFR intracellular region phosphorylated upon EGF activation co-localizes with the receptor following EGF stimulation in single-particle tracking (SPT) experiments, demonstrating pY specific recognition in living cells. RFD2-MI provides a generalizable all-atom diffusion framework for probing and modulating phosphorylation-dependent signaling, and more generally, for developing research tools and targeted therapeutics against post-translationally modified proteins.

bioengineering↗

bioRxiv2025

Accelerating protein design by scaling experimental characterization

Recent advances in de novo protein design have greatly outpaced standard protein biochemistry workflows, and experimental testing has been a bottleneck in the validation of new designs and methodologies. Here, we describe experimental and computational workflows to address the issues of scale, speed and reproducibility of common in vitro protein testing methods, enabling at least an order of magnitude increase in throughput while reducing wetlab time. Semi-Automated Protein Production (SAPP) is a rapid, modular, scalable and cost-effective protocol, enabling up to milligram-scale protein production, and standardized characterization including yield, dispersity, and oligomeric state of hundreds of designs per day, at the cost-equivalent of a few DNA oligos per construct. End-to-end protocol execution takes 48 hours, with about 6 hours spent benchside using mostly standard laboratory equipment. This protocol has become the standard at our institute, providing critical experimental validation for dozens of projects spanning tens of thousands of designs. We showcase the power of the platform by using it to rapidly characterize de novo designed inhibitors of respiratory syncytial virus. Since at least 80% of SAPPs total cost comes from synthetic DNA, we also developed a scalable demultiplexing protocol (DMX) to leverage oligo pools as input DNA, providing a further 5-fold reduction in costs, enabling >1000 designs to be purified and characterized in arrayed, clonal format at a cost of $5 per construct. By reframing standard molecular biology practices and orchestrating wetlab workflows with partial automation instead of complex end-to-end robotics, these protocols should be widely adoptable, accelerating protein design.

biochemistry↗

bioRxiv2025

Metabolic and Population Profiles of Active Subseafloor Autotrophs in Young Oceanic Crust at Deep-Sea Hydrothermal Vents

At deep-sea hydrothermal vents, magmatically driven rock-water reactions in the crust generate gases and other reduced compounds that subseafloor microorganisms use for chemolithoautotrophy. In this study, microbial autotrophs from three diffuse flow hydrothermal vents at Axial Seamount in 2013 and 2014 were isotopically labeled using RNA Stable Isotope Probing (RNA-SIP), targeting subseafloor autotrophic mesophiles (30{degrees}C), thermophiles (55{degrees}C), and hyperthermophiles (80{degrees}C). We constructed taxonomic and functional profiles of active chemolithoautotrophs, examined population distributions across sites, and linked primary producers to their specific metabolic strategies within the subseafloor community. Dominant autotrophs exhibited hydrogen-dependent dissimilatory metabolisms such as sulfur and nitrate reduction and methanogenesis, as well as microaerophilic sulfide oxidation even at 80{degrees}C, consistent with fluid chemistries at each site. While hydrogenotrophic methanogenic archaea such as Methanothermococcus were restricted in their distribution and activity, hydrogenotrophic sulfur and nitrate reducers from the Aquificota (Thermovibrio), Campylobacterota (Nautiliaceae, Hydrogenimonas, and Desulfurobacteriaceae) were consistently active and present at all sites and years at both the population and community levels. Hydrogenase transcripts were significantly differentially expressed, and diverse hydrogenases were found in metagenome-assembled genomes of Aquificota members, highlighting the importance and versatility of their hydrogen utilization strategies which likely contribute to their cosmopolitan distribution across geochemically disparate subseafloor sites. Together, this study provides new insights into the functional dynamics and distribution of key subseafloor autotrophic microbial communities in young oceanic crust at deep-sea hydrothermal vents. IMPORTANCEDeep-sea hydrothermal vents are hotspots for life in the dark ocean, where rich animal ecosystems are supported by microbial primary producers utilizing the abundant chemical energy supplied by high-temperature water-rock reactions. Despite increasing knowledge about the geochemistry and microbiology of deep-sea hydrothermal vents, there is still a gap in our understanding of the key microbial players who fix much of the carbon at these sites, especially in the productive subseafloor. In this study, stable isotope probing was used to label active microbial autotrophs in diffuse flow venting fluids from three sites over two years and was combined with metatranscriptomic sequencing to identify their specific metabolic strategies. This research highlights the microbial community composition, function, gene regulation, and population dynamics that enable hydrothermal ecosystems to persist.

microbiology↗

bioRxiv2025

Designed NGF mimetics with reduced nociceptive signatures in neurons

The clinical use of Nerve Growth Factor (NGF) for neuronal regeneration has been hampered by pain sensitization side effects. NGF signals through the receptor tyrosine kinase TrkA and the co-receptor p75NTR; pain sensitization is thought to involve p75NTR. We sought to overcome this limitation by de novo design of a TrkA agonist that does not bind p75NTR. We designed homodimeric TrkA engaging constructs that dimerize TrkA subunits in a variety of geometries, and identified those eliciting the strongest signaling. The resulting designed agonists are able to stimulate transdifferentiated neurons and neuroblastoma cell lines, leading to neurite outgrowth and neuronal differentiation, with considerably reduced transcription of inflammation and pain related genes. These agonists are promising candidates for promoting neuronal regeneration without adverse side effects. HighlightsO_LIDe novo designed TrkA agonists activate MAPK and PI3K-AKT signaling C_LIO_LIRigid fusions allow for highly tunable signaling signatures C_LIO_LITrkA agonists lead to neurite outgrowth in neuroblastoma cells comparable to retinoic acid C_LIO_LIModulation of the TrkA pathway without co-stimulating p75NTR leads to a downregulation of inflammatory and nociceptive signature in neurons. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/648806v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c48066org.highwire.dtl.DTLVardef@c9c5a7org.highwire.dtl.DTLVardef@cf8ebdorg.highwire.dtl.DTLVardef@a4345a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

De novo design of miniprotein agonists and antagonists targeting G protein-coupled receptors

G protein-coupled receptors (GPCRs) play key roles in physiology and are central targets for drug discovery and development, but the design of protein agonists and antagonists has been challenging as GPCRs are integral membrane proteins and conformationally dynamic. Here we describe computational de novo design methods and a high throughput "receptor diversion" microscopy-based screen for generating GPCR binding miniproteins with high affinity, potency and selectivity, and the use of these methods to generate agonists for MRGPRX1, NK1R and CCR5, as well as antagonists for CXCR4, CCR5, OXTR, GLP1R, GIPR, GCGR, PTH1R and CGRPR.. Cryo-electron microscopy data reveals atomic-level agreement between designed and experimentally determined structures for CGRPR- and CXCR4-bound antagonists and MRGPRX1-bound agonists. Our de novo design and screening approach opens new frontiers in GPCR drug discovery and development.

bioengineering↗

bioRxiv2025

Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Library screening and selection methods can determine the binding activities of individual members of large protein libraries given a physical link between protein and nucleotide sequence, which enables identification of functional molecules by DNA sequencing. However, the solution properties of individual protein molecules cannot be probed using such approaches because they are completely altered by DNA attachment. Mass spectrometry enables parallel evaluation of protein properties amenable to physical fractionation such as solubility and oligomeric state, but current approaches are limited to libraries of 1,000 or fewer proteins. Here, we improved mass spectrometry barcoding by co-synthesizing proteins with barcodes optimized to be highly multiplexable and minimally perturbative, scaling to libraries of >5,000 proteins. We use these barcodes together with mass spectrometry to assay the solution behavior of libraries of de novo-designed monomeric scaffolds, oligomers, binding proteins and nanocages, rapidly identifying design failure modes and successes.

biochemistry↗

bioRxiv2024

Controlling semiconductor growth with structured de novo protein interfaces

Protein design now enables the precise arrangement of atoms on the nanometer length scales of inorganic crystal nuclei, opening up the possibility of templating the growth of metal oxides including semiconductors. We designed proteins presenting regularly repeating interfaces containing functional groups that organize ions and water molecules, and characterized their ability to bind to and template metal oxides. Two interfaces promoted the growth of hematite under conditions that otherwise resulted in the formation of magnetite. Three interfaces promoted ZnO nucleation under conditions where traditional ZnO-binding peptides and control proteins were ineffective. Designed cyclic assemblies with these ZnO nucleating interfaces lining interior cavities promoted ZnO growth within the cavity. CryoEM analysis of a designed octahedral nanocage revealed atomic density likely corresponding to the growing ZnO directly adjacent to the designed nucleation promoting interfaces. These findings demonstrate that designed proteins can direct the formation of metal oxides not observed in biological systems, opening the door to protein-semiconductor hybrid materials. One Sentence SummaryWe describe the design of structured protein interfaces that bind to, promote, and localize the growth of zinc oxide and hematite, inorganic materials which are not found in biological systems.

bioengineering↗

bioRxiv2024

Design of High Affinity Binders to Convex Protein Target Sites

While there has been progress in the de novo design of small globular miniproteins (50-65 residues) to bind to primarily concave regions of a target protein surface, computational design of minibinders to convex binding sites remains an outstanding challenge due to low level of overall shape complementarity. Here, we describe a general approach to generate computationally designed proteins which bind to convex target sites that employ geometrically matching concave scaffolds. We used this approach to design proteins binding to TGF{beta}RII, CTLA-4 and PD-L1 which following experimental optimization have low nanomolar to picomolar affinities and potent biological activity. Co-crystal structures of the TGF{beta}RII and CTLA-4 binders in complex with the receptors are in close agreement with the design models. Our approach provides a general route to generating very high affinity binders to convex protein target sites.

biochemistry↗

bioRxiv2024

An artificial intelligence accelerated virtual screening platform for drug discovery

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we developed a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screened multi-billion compound libraries against two unrelated targets, a novel ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. On both targets, we discover hits, including seven novel hits (14% hit rate) to KLHDC2 and four novel hits (44% hit rate) to NaV1.7 with single digit micromolar binding affinities. Screening in both cases was completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

biochemistry↗

bioRxiv2023

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation and amplify signaling

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

synthetic biology↗

bioRxiv2023

De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8

The RGD (Arg-Gly-Asp)-binding integrins v{beta}6 and v{beta}8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of v{beta}6 and v{beta}8 with high selectivity. The v{beta}6 and v{beta}8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7[A] root-mean-square deviation (RMSD) to the computational design models; the designed v{beta}6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-v{beta}6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the v{beta}8 inhibitor maintains the constitutively fixed extended-closed v{beta}8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the v{beta}6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

biochemistry↗

bioRxiv2023

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways. HighlightsO_LIDe novo designed cyclic oligomers with tunable geometric properties C_LIO_LICyclic, homo-oligomeric FGFR binding modules induce geometry- and valency-dependent activity of isoform-specific FGF signaling C_LIO_LIModulation of FGFR isoform activity controls bifurcation of endothelial and mesenchymal fate during vascular development C_LIO_LIC-isoform activation favors arterial endothelial cell formation while B-isoform induces pericyte differentiation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/532666v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@151f6eforg.highwire.dtl.DTLVardef@2ae877org.highwire.dtl.DTLVardef@103e0d1org.highwire.dtl.DTLVardef@19cd695_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

De novo design of high-affinity protein binders to bioactive helical peptides

Many peptide hormones form an alpha-helix upon binding their receptors1-4, and sensitive detection methods for them could contribute to better clinical management. De novo protein design can now generate binders with high affinity and specificity to structured proteins5,6. However, the design of interactions between proteins and short helical peptides is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that with the RFdiffusion generative model, picomolar affinity binders can be generated to helical peptide targets either by noising and then denoising lower affinity designs generated with other methods, or completely de novo starting from random noise distributions; to our knowledge these are the highest affinity designed binding proteins against any protein or small molecule target generated directly by computation without any experimental optimization. The RFdiffusion designs enable the enrichment of parathyroid hormone or other bioactive peptides in human plasma and subsequent detection by mass spectrometry, and bioluminescence-based protein biosensors. Capture reagents for bioactive helical peptides generated using the methods described here could aid in the improved diagnosis and therapeutic management of human diseases.7,8

bioengineering↗

bioRxiv2022

De novo design of modular peptide binding proteins by superhelical matching

General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptides backbone polar groups (1-3). Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity (4-11), we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide (12); the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW--in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein sidechain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occuring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

biochemistry↗

bioRxiv2022

Improving de novo Protein Binder Design with Deep Learning

We explore the improvement of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency.

bioengineering↗

bioRxiv2022

The salivary, metal-binding peptide histatin-5 buffers extracellular copper availability

Antimicrobial peptides (AMPs) are key components of diverse host innate immune systems. The family of human salivary AMPs known as histatins bind Zn and Cu. Fluctuations in Zn and Cu availability play significant roles in the host innate immune response (so-called "nutritional immunity"). Thus, we hypothesised that histatins contribute to nutritional immunity by influencing host Zn and/or Cu availability. We posited that histatins limit Zn availability (promote bacterial Zn starvation) and/or raise Cu availability (promote bacterial Cu poisoning). To test this hypothesis, we examined the interactions between histatin-5 (Hst5) and Group A Streptococcus (GAS), which colonises the human oropharynx. Our results showed that Hst5 does not strongly influence Zn availability. Hst5 did not induce expression of Zn-responsive genes in GAS, nor did it suppress growth of mutant strains that are impaired in Zn transport. Biochemical examination of purified peptides confirmed that Hst5 binds Zn only weakly. By contrast, Hst5 bound Cu tightly and it strongly influenced Cu availability. However, Hst5 did not promote Cu toxicity. Instead, Hst5 suppressed expression of Cu-inducible genes, stopped intracellular accumulation of Cu, and rescued growth of a {Delta}copA mutant strain that is impaired in Cu efflux. We thus proposed a new role for salivary histatins as major Cu buffers in saliva that contribute to microbial homeostasis in the oral cavity and oropharynx by reducing the potential negative effects of Cu exposure (e.g. from food) to microbes. Our results raise broad questions regarding the physiological roles of diverse metal-binding AMPs and the management of host metal availability during host-microbe interactions.

microbiology↗

bioRxiv2021

Robust de novo design of protein binding proteins from target structural information alone

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains an outstanding challenge. We describe a general solution to this problem which starts with a broad exploration of the very large space of possible binding modes and interactions, and then intensifies the search in the most promising regions. We demonstrate its very broad applicability by de novo design of binding proteins to 12 diverse protein targets with very different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of four of the binder-target complexes, and all four are very close to the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvement of both. Our approach now enables targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

synthetic biology↗

bioRxiv2021

Multivalent designed proteins protect against SARS-CoV-2 variants of concern

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

synthetic biology↗