A Generalizable Interface-Seeded Framework for De Novo Design of Functional Oligomers 1. The paper introduces an “interface-seeded” generative design strategy: instead of docking existing protein building blocks into a target symmetry, it uses a previously validated protein–protein interface (PPI) as the seed and generates new symmetry-compatible protomers around it. 2. Key implementation detail: a new symmetric motif-scaffolding module in RFdiffusion that preserves the interface motifs while sampling seed orientations and radial placements, then “drags” the motif radially during denoising to bias toward compact, well-packed cyclic oligomers. 3. This directly addresses a practical bottleneck in symmetric assembly design: dock-and-design success is often limited by geometric incompatibility between chosen protomers and target symmetries (C3/C4/C5), producing strong biases and low hit rates. 4. Benchmarking with LHD heterodimer seeds (LHD101, LHD29), the framework produced many expressible designs (63/64 expressed), with a substantial fraction matching the intended oligomeric states by SEC (33/63) and SEC-MALS (18/63). 5. Structural accuracy was validated by crystallography for multiple designs: C3 trimers (PI25, PI31) and a C4 tetramer (PI57) closely matched design models (Cα RMSD < 1.6 Å), including accurate interface side-chain placement; the resulting folds were also “new-to-nature” by Foldseek comparisons. 6. The approach generalizes to chemical triggers by reusing ligand/metal-dependent PPIs as seeds (often fragmented into many discontinuous segments), enabling conditional C3 assembly for Cu2+ (MC11), cholic acid (CHD04), and venetoclax (LBM10). Measured effective assembly affinities were in the ~100 nM range (e.g., MC11 Kd,eff 137 nM; LBM10 Kd,eff 130 nM). 7. Crystal structures of CHD04 and LBM10 matched their design models and showed ligand occupancy in the engineered pockets, demonstrating that the seeded responsive interface can be transplanted into entirely new oligomer topologies while retaining chemical control. 8. A major functional advance is reversible phosphorylation-controlled oligomerization using a dynamic “phosphoswitch” interface seed: phosphorylation by PKA shifts monomer→oligomer, and λ-phosphatase reverses it back. Single interface-weakening mutations reduced unwanted basal oligomerization while preserving phospho-dependent assembly (PO5s, PO18s). 9. The work also demonstrates multi-input control: introducing a disulfide lock created a system that requires both reducing conditions and phosphorylation to oligomerize, enabling dual-gated assembly behavior in a de novo designed oligomer. 10. Applications go beyond in vitro biophysics: (i) ligand-triggered membrane binding via MinD membrane-targeting sequence fusions (Cu2+ or CHD induces oligomerization-driven avidity on supported lipid bilayers), and (ii) phosphorylation-inducible transcription in HEK293T cells by coupling PO18s trimerization to an HSF1-based reporter, yielding ~6-fold induction (and >14-fold with combined endogenous + engineered PKA activation). 💻Code: github.com/Khmelinskaia-Lab/… ; github.com/Khmelinskaia-Lab/… 📜Paper: biorxiv.org/content/10.64898… #ProteinDesign #RFdiffusion #AlphaFold3 #SyntheticBiology #ComputationalBiology #ProteinEngineering #DeNovoDesign #SignalTransduction #Phosphorylation #Nanobiotechnology