Binders to the Rbx1 IDR that induce folding upon binding.

General information: The resulting binders are mostly beta-structured (or mixed alpha-beta) and incorporate the target epitope (or some parts of it) as a beta strand within their beta sheet. For the target epitope, we selected the region at positions 17-35: it is located in the unstructured N‑terminal tail, undergoes disorder-to-order transition in the Rbx1-Cul1 complex, does not include probable PTMs (e.g., phosphorylation at positions 9, 13, 15), and is not located too close to the globular part.

Pipeline details: Binders were generated using BoltzGen. The number of generations was set to 30,000 with a budget of 150 and an alpha parameter of 0.1 to obtain a more diverse range of structures. The peptide-anything protocol was used. The tail region with the sequence AGKKRFEVKKWNAVALWAW was used as the target fragment for generation. Proteins with lengths ranging from 100 to 160 were generated (similar to the range of successful designs from Liu et al., Nature 2025). To ensure that the target IDR fragment is not deeply buried or topologically entangled with the binder, we applied an additional target exposure filter (removed the designs that contained a 5-mer with a mean relative SASA below 0.45). After generation, we refolded the binders with the full-length Rbx1 using AlphaFold2 to check the consistency of the generated and predicted structures (using a Cα-RMSD threshold of 3 Å).  Binders were ranked according to the BoltzGen quality score.