To guide interface placement on the RBX1 RING domain, we leveraged prior knowledge that glomulin is a natural RBX1 inhibitor that blocks E2 recruitment by binding the RING surface. In particular, we used reported RBX1 interface “hotspot” residues W33, W35, I44, R46, W87, and R91 as reference positions when defining the targeted binding region and when prioritizing designs that make favorable contacts near these sites.