These results establish motif scaffolding of surface-complementing seeds as an effective strategy for overcoming current limitations of de novo generative models, enabling the design of proteins that can engage challenging interface sites.
Abstract
De novo design of protein binders targeting extended, multi-site interaction surfaces remains difficult for current generative methods, which often produce limited structural diversity and predominantly helical topologies. Here, we advance diffusion-based binder design by guiding protein backbone generation through “seeds,” which are PDB-derived fragments selected for geometric complementarity to the target surface. To test this approach, we computationally generated seed-guided binders of the bacterial toxin RelE. RelB, the native antitoxin of RelE, engages two distinct interfaces with high surface complementarity, making it an appropriate test case. Seed-guided RFdiffusion produced backbones with substantially higher structural diversity and more target contacts than RFdiffusion alone. Experimental screening of 1,402 designs in a high-throughput bacterial survival assay identified multiple functional binders, including variants with nanomolar to low-micromolar affinity and one design with RelE neutralization comparable to RelBpep. Computational structure prediction and mutational analyses support that the designed interfaces rely on seed-derived contacts and adopt binding modes distinct from RelB. Molecular dynamics simulations and hydrogen-deuterium exchange experiments further suggest that one high-affinity design undergoes a conformational change upon binding. Notably, successful designs exhibited reduced cross-reactivity to RelE orthologs compared with RelBpep, suggesting that the extensive interfaces generated through seed-guided design can enable enhanced selectivity. These results establish motif scaffolding of surface-complementing seeds as an effective strategy for overcoming current limitations of de novo generative models, enabling the design of proteins that can engage challenging interface sites.
HighPlay2 is presented as a feasible framework for the early-stage design and screening of cyclic peptide candidates containing ncAAs, while further affinity maturation and experimental structural validation remain necessary.
Huitian Lin, Wentong Wang, Ning Zhu et al.· European journal of medicina...· 0 citations
Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract
EvoBind-multimer is presented, a deep learning framework for the de novo design of molecular glues directly from protein sequences that generates small macrocyclic peptides that bridge user-defined protein pairs without requiring prior interface knowledge or existing ligands.
Andrä Brunner, Krzysztof Wierbiłowicz, Diandra Daumiller et al.· bioRxiv· 0 citations
Single-domain antibodies (sdAbs) derived from naive phage display libraries offer a time-efficient alternative to animal immunization but often exhibit suboptimal affinity, particularly for small-molecule haptens, where restricted binding interfaces limit the efficacy of traditional saturation mutagenesis. For instance, the wild-type sdAb (B1)-targeting ethoxyquin (EQ) exhibits a moderate equilibrium dissociation constant (KD) at the submicromolar level (within the 10-7 M range), restricting its practical sensitivity. To overcome the structural and energetic barriers inherent in hapten recognition, a "structure-guided directed modification" strategy was developed that focuses on conformational tuning rather than simple side-chain replacement. Integrating AlphaFold2 modeling and AutoDock mechanistic analysis identified key interactions (e.g., the ASP19-EQ-N8 hydrogen bond). Subsequently, a random single-amino acid insertion strategy was implemented within the CDR3 loop (residues 77-90) to fine-tune local loop geometry. Top candidates were screened via MM/GBSA binding free energy calculations and experimentally validated using biolayer interferometry. This approach yielded three high-affinity mutants-77A, 79C, and 85H-with calculated ΔΔG values of -10.56, -4.35, and -6.69 kcal/mol, respectively. The mutants achieved enhanced affinities in the tens-of-nanomolar range (down to 35.1 nM), representing up to approximately 3 times overall improvement. Importantly, orthogonal surface plasmon resonance analysis using nonconjugated, free EQ successfully verified this affinity maturation trend, showing that mutant 79C bound free EQ with a KD of 1.30 × 10-5 M (a 2.3 times improvement over wild-type) through simultaneously accelerated association and slowed dissociation. Mechanistically, this substantial increase in affinity is attributed not only to reinforced noncovalent networks that significantly stabilize the complex but also to favorable CDR3 geometric outward flips that alleviate steric hindrance, leading to an approximate 3.8 times acceleration in the kon. Crucially, these mutants demonstrated notable anti-interference tolerance in complex aquatic food matrices (e.g., sea bass extracts). Ultimately, this work provides a practical and efficient computational framework for assisting the rapid evolution of low-affinity hapten sdAbs into high-performing biorecognition elements with potential for next-generation biosensing architectures.
Zenglin Yang, Guoqiang Li, Yuanyuan Ge et al.· ACS Biomaterials Science & E...· 0 citations
Targeted protein degradation (TPD) represents a promising approach for eliminating disease-causing proteins beyond traditional inhibition. However, the reliance on a limited number of E3 ligases remains a major bottleneck. FEM1B, an E3 ligase substrate receptor with multiple substrate-recognition modes, represents an attractive but underexplored TPD platform. In this study, through a structure-guided approach exploiting the spatial proximity between a druggable C-degron-binding pocket and a second binding site containing a reactive cysteine, we developed FL47, a dual-site ligand that combines extensive noncovalent interactions with targeted covalent engagement. FL47 exhibits submicromolar affinity, robust cellular target engagement, and markedly reduced cytotoxicity relative to previously reported covalent recruiters. We further applied FL47 in the development of FEM1B-based PROTACs and incorporated a chemical endocytic prodrug strategy that markedly enhanced degradation activity. This work introduces a novel dual-site binding strategy for E3 ligase ligand discovery and broadens the potential toolbox for TPD applications.
Ling-xiang Xu, Ruyi Huang, Yuying Ma et al.· Journal of Medicinal Chemist...· 0 citations
NACraft, a training-free and programmatic framework for all-atom nucleic-acid aptamer design based on backpropagation through structure-model feedback, is presented, demonstrating the effectiveness and versatility of NACraft and extending structure-model hallucination toward programmatic nucleic-acid aptamer design.
