Biology subjects

Woo, E.-J.

Publications and source records attributed to Woo, E.-J..

5 recordsLinked to original sources

bioRxiv2026

Rational Redesign of an Fc-Binding Peptide for Multivalent Antibody Assembly

Multivalent antibody assemblies offer opportunities to enhance avidity, organize immune complexes, and modulate higher-order protein interactions, but constructing such architectures from existing immunoglobulin G (IgG) molecules without redesigning the antibody scaffold remains challenging. Here, we report the rational redesign of a Protein A-derived Fc-binding peptide into ADP1, a stable dimeric Fc-binding peptide that directs Fc-mediated antibody assembly. ADP1 was designed from the parent Fc-binding peptide Z34C by preserving the Fc-recognition surface while redesigning the opposite helical surface to promote peptide-peptide association. Biophysical characterization showed that ADP1 retained nanomolar Fc-binding affinity while exhibiting markedly enhanced chemical and proteolytic stability compared with the parent peptide. Structural analyses of ADP1-Fc complexes revealed that ADP1 bridges neighboring Fc regions through a combined ADP1-Fc and ADP1-ADP1 interface, generating spiral higher-order Fc assemblies. This assembly principle was further extended to full-length IgG, where ADP1 promoted higher-order antibody association in a concentration-sensitive manner. In addition, covalent ADP1 functionalization enabled Fc-directed modification of full-length IgG while retaining Fab-mediated antigen recognition, demonstrating the utility of ADP1 as an antibody assembly and functionalization module. Finally, competitive addition of the parent Z34C peptide modulated ADP1-driven antibody assembly, suggesting a potential route for tuning Fc-mediated assembly propagation. Together, this work establishes a redesigned Fc-binding peptide platform for directing multivalent antibody assembly and functionalization without genetic reengineering of the IgG scaffold. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/737354v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@57cadeorg.highwire.dtl.DTLVardef@1c7d8e6org.highwire.dtl.DTLVardef@170fb85org.highwire.dtl.DTLVardef@90e6d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

AI-guided Protein Inhibitor Design for Modulating FAD-dependent Glucose Dehydrogenase Redox Output

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) is a redox enzyme widely used in glucose monitoring, bioelectronic devices, and enzymatic biofuel cells because of its oxygen-independent catalysis and compatibility with electron-transfer processes. However, protein-based regulators that directly bind GDH and modulate its redox output remain underdeveloped. Here, we present an AI-guided strategy for developing a de novo protein inhibitor targeting FAD-GDH. GDH-targeting candidates generated through structure-based computational design were evaluated by yeast surface display and fluorescence-activated cell sorting, leading to the identification of FAD-GDH inhibitor-1 (FGI-1) as a GDH-targeting inhibitory scaffold. Purified His-MBP-FGI-1 reduced GDH-mediated DCIP reduction, demonstrating attenuation of GDH-derived redox output. Random mutagenesis followed by secondary FACS screening yielded evolved variants with increased GDH-binding signals and enhanced redox-output suppression, showing that the de novo inhibitory scaffold could be functionally tuned through experimental evolution. In addition, an FGI-1-based construct fused to a larger protein module retained GDH-output suppressive activity, and electrode-based measurements showed reduced GDH-derived current output. Because electrode-associated measurements may be influenced by protein-mediated surface shielding and altered electron-transfer accessibility, this decrease was interpreted conservatively as attenuation of GDH-derived electrochemical output rather than direct evidence of active-site inhibition. Together, this work establishes an AI-guided design-validation workflow for developing protein inhibitors that modulate FAD-GDH redox output and provides a foundation for protein-level control of enzyme output in biosensing and bioelectronic applications.

biochemistry↗

bioRxiv2026

AI-Driven Design of Nanobinders Targeting the TSLPR Heterodimer Interface to Suppress Type 2 Inflammatory Signaling

Aberrant thymic stromal lymphopoietin (TSLP) signaling is a central driver of type 2 inflammatory diseases, yet the only approved TSLP-targeted therapy is a 150 kDa monoclonal antibody whose bulky format limits tissue penetration and precludes inhaled delivery. Here, we report an AI-driven framework for designing ultra-compact de novo nanobinders that suppress TSLP signaling by sterically disrupting assembly of the TSLPR-IL-7R heterodimer. We compare two structure-guided strategies, namely purely de novo helical bundle generation and interface-mimetic grafting of native binding motifs onto designed scaffolds. Although both yield nanomolar binders, only orthosteric mimicry of the native cytokine geometry blocks receptor heterodimerization, showing that functional antagonism is governed by precise epitope geometry rather than affinity alone. After library-based maturation, the lead nanobinder TRB5.1 is a hyper-stable monomer (Tm [~]97.4 {degrees}C) with single-digit nanomolar affinity (KD = 9.7 nM) and strict selectivity over related -chain interleukin receptors. TRB5.1 suppresses TSLP-induced JAK1 and STAT5 phosphorylation across multiple cellular models and drives a transcriptome-wide reversal of the pathogenic type 2 program. This work delivers a developable, potentially inhalable non-antibody lead and a scalable blueprint for antagonizing heterodimeric cytokine receptors.

biochemistry↗

bioRxiv2026

Cyclic-Phe-Pro Binds the ToxRS Interface to Promote Signal Transduction in Vibrio vulnificus

In Vibrio vulnificus, the quorum-sensing signal cyclo-(L-phenylalanine-L-proline) (cFP) binds the membrane receptor ToxRS to activate genes linked to oxidative-stress resistance and virulence. ToxR is a transmembrane transcription factor that pairs with ToxS to sense periplasmic signals, yet how V. vulnificus ToxRS recognizes cFP has remained undefined. AI-guided structure prediction revealed preferential ToxR/S heterodimer formation and a fold conserved with the V. cholerae crystal structure. Molecular docking placed cFP in a hydrophobic pocket at the ToxR-ToxS interface, where Phe279 stacked with its phenyl ring and Arg277 hydrogen-bonded its carbonyl oxygen. Introducing R277L and F279A substitutions lowered basal leuO expression and abolished cFP-dependent induction in a lacZ fusion assay. ChIP showed that cFP enhanced wild-type ToxR binding to the leuO promoter, whereas the mutant bound weakly and did not respond. Thus cFP bridges ToxR and ToxS to stabilize the heterodimer, facilitating ToxR recruitment to promoters and transcriptional activation in V. vulnificus.

biochemistry↗

bioRxiv2025

De Novo design of a potent Wnt Surrogate specific for the frizzled7 subtype members

In humans, 19 Wnt ligands interact with 10 Frizzled (Fzd) receptors and the co-receptors LRP5/6 to initiate signaling. Wnts and Fzds are highly promiscuous, making it challenging to dissect the specific outcomes of individual Wnt-Fzd interactions. Developing Wnt surrogates with specificity for individual Fzd subtypes could be pivotal. We present a modular, potent, and Fzd7-specific Wnt surrogate that consists of three de novo designed modules, a Fzd7 binder, an LRP6 binder and a homodimeric protein. The Fzd7-specific module was designed by targeting two less conserved surface patches on the cysteine-rich domain (CRD) of Fzds to achieve both selectivity and affinity. It exhibits a strong binding affinity (KD < 2.3 nM) for the very closely related Fzd7 subtype members (Fzd7, Fzd1, Fzd2) with no measurable binding to the CRDs of the other seven Fzd receptors. This Wnt surrogate induced spheroid organoid formation from intestinal stem cells at subnanomolar concentration, and promoted full hair follicle regeneration and robust hair growth in mice. These results suggest that our strategy could be extended to design modular Wnt surrogates capable of selectively activating individual Fzd receptors, providing a valuable tool kit for development and differentiation, organoid cultures and targeted regeneration.

bioengineering↗