Technical Report: Computational Design of De Novo Protein Binders for RBX-1 Project Title: Strategic Truncation and Multi-Model Design of RBX-1 Binders Submission Date: March 15, 2026 Target Protein: RBX-1 (E3 Ubiquitin Ligase) Research Team: Protein Design for Africa (PDFA) proteindesignforafrica@gmail.com Oghenejabor Laurence Jaboro (Co-founder and President of PDFA) laurencejaboro858@gmail.com Mcgregory Nosakhare Ogbomo (Co-founder and Vice President of PDFA) mcgregorygregory0902@gmail.com

1. Abstract This report details the computational pipeline used to design high-affinity protein binders against RBX-1, a component of the SCF E3 ubiquitin ligase complex. RBX-1 presents challenges due to significant intrinsically disordered regions (IDRs). To address this, we implemented a targeted truncation strategy focused on the structured N-terminal domain of PDB ID: 3DQV. A focused design approach was employed in which three N-terminal binders were generated using BindCraft with soluble MPNN optimization. Additionally, hotspot residues spanning positions A1–17 were incorporated to guide binding toward key interface regions. All designs were computationally evaluated for structural stability, interface quality, and experimental feasibility. This workflow produced structurally diverse, high-confidence binders optimized for the N-terminal interface of RBX-1.
2. Target Analysis and Preparation 2.1 Intrinsically Disordered Region (IDR) Truncation RBX-1 contains highly flexible regions that can confound de novo design algorithms. To maximize the likelihood of successful binding, we truncated these non-structured segments, focusing the design pipelines on stable surface epitopes suitable for protein–protein interactions. 2.2 Domain Specification Target A (N-Terminal): Structured N-terminal domain of PDB ID 3DQV, Chain A.
3. Design Methodology 3.1 N-Terminal Design: BindCraft and Soluble MPNN For the N-terminal domain, three de novo binders were generated using BindCraft via the Taramind Bio platform. Scaffold Generation: Binders were constrained to 100–250 amino acids to balance interaction surface area with folding stability. Hotspot Integration: Residues A1–17 were defined as binding hotspots to guide interface formation toward biologically relevant contact regions. Interface Optimization: Soluble MPNN was employed to prioritize solubility and reduce the risk of hydrophobic aggregation at the binding interface.
4. Design Selection and Validation All candidates were evaluated using computational metrics: Structural Integrity: High pLDDT scores across scaffold regions. Interface Confidence: Favorable iPAE and pTM scores for stable predicted interactions. Experimental Feasibility: Solubility profiles optimized via MPNN. (Note: Detailed pLDDT, pAE, and pTM scores for each design are included in the accompanying CSV file.)
5. Conclusion By integrating IDR truncation with a focused design strategy, we produced three high-confidence de novo binders targeting the N-terminal domain of RBX-1. These binders are structurally diverse, computationally optimized for stable interface formation, and demonstrate strong alignment to the target surface. This work illustrates a streamlined, efficient strategy for generating robust protein binders against challenging targets, enabling potential modulation of E3 ubiquitin ligase activity via high-affinity protein–protein interactions.