Description
The text outlines a five-step de novo design pipeline to create protein binders targeting the RBX1 RING domain while accommodating its essential structural zinc ions.
The Design Pipeline:
Target Preparation: The RBX1 starting structure was prepared by defining specific interaction hotspot residues and retaining three critical zinc ions (Zn²⁺) as fixed contextual elements.
Backbone Generation: RFdiffusion3 was used to build novel protein scaffolds that maximize shape complementarity with the target hotspots while strictly avoiding steric clashes with the zinc ions.
Sequence Design: LigandMPNN assigned specific amino acid sequences to these newly generated backbones. The model was constrained by the zinc ions to ensure the resulting interface was geometrically and electrostatically compatible.
In Silico Validation: OpenFold3 simulated the co-folding of the newly designed binders with the RBX1 target. Only candidates with high-confidence prediction scores (iPTM > 0.80 and pLDDT > 85) were advanced.
Manual Inspection: The surviving top candidates were visually evaluated using VMD. Designs were manually discarded if they showed clashes with the zinc ions, poor hydrogen bonding, suboptimal hydrophobic packing, or excessive interface flexibility.