Cellular Synthesis and X‐ray Crystal Structure of a Designed Protein Heterocatenane

Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post‐translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS‐PAGE, LC‐MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X‐ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein‐topology engineering. Protein heterocatenanes could be readily and modularly synthesized in cellulo using a programmed sequence of post‐translational processing events, including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The catenane topology of the designed protein was rigorously proven by X‐ray crystallography.