Design of a chimeric probe specific to K48-K63-linked heterotypic ubiquitin chains

Ubiquitin signaling regulates many cellular functions, but studying complex heterotypic ubiquitin chains is challenging due to a lack of specific detection tools. Here, we have developed chimeric polypeptide probes by combining ubiquitin-interacting motifs (UIM) from two proteins and optimizing the linker between UIMs. The initial probe, RA20, selectively binds to K48- and K63-linked tri-ubiquitin chains, with approximately 10 µM affinity for both mixed and branched linkages, and distinguishes the linkage order in mixed chains. Guided by the insights from mutagenesis, SPR, NMR, and structural modeling, we shortened the inter-UIM linker to develop an improved probe, △ 4 RA20, which exhibits higher affinity (1.4 µM) and tenfold selectivity for branched K48/K63 chains. This engineered polypeptide probe specifically recognizes branched heterotypic polyubiquitin chains, providing a foundation for future tools to study diverse polyubiquitin architectures.