Heterodimeric protein entangling motifs: systematic discovery, feature analysis, and topology engineering

Synthesis of nontrivial protein topologies calls for genetically encoded protein entangling motifs, especially those of heterogeneous nature, to achieve structural complexity and functional relevance. Herein, we report the systematic discovery of heterodimeric entangling motifs using criteria like Gauss linking number, buried surface area and terminal distances. These motifs were analyzed to reveal their formation mechanisms (i.e., precursor cleavage, synergistic folding and segment piercing/wrapping) and biological significance (i.e., stability enhancement crucial for executing functions like regulation and catalysis). Six premium motifs were selected for experimental validation. Upon ring closure mediated by orthogonal split inteins, all six motifs led to protein hetero[2]catenanes with varying efficiency, providing versatile templates for making mechanically interlocked protein conjugates, such as Förster resonance energy transfer pairs and bispecific binders. The study not only helps untangle the influence of chain entanglements on protein properties but also provides a modular platform to enrich the toolbox of protein topology engineering.