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

Area of Science:

• Biochemistry
• Protein Engineering
• Synthetic Biology

Background:

• Genetically encoded protein entangling motifs are essential for creating complex protein topologies.
• Heterogeneous motifs are particularly important for achieving structural complexity and functional relevance.

Purpose of the Study:

• To systematically discover heterodimeric entangling motifs.
• To analyze their formation mechanisms and biological significance.
• To provide a modular platform for protein topology engineering.

Main Methods:

• Discovery of heterodimeric entangling motifs using criteria like Gauss linking number, buried surface area, and terminal distances.
• Analysis of motif formation mechanisms (precursor cleavage, synergistic folding, segment piercing/wrapping).
• Experimental validation of six selected motifs via ring closure using orthogonal split inteins.

Main Results:

• Successful discovery and analysis of heterodimeric entangling motifs.
• Identification of formation mechanisms and biological significance, including stability enhancement.
• Experimental validation yielding protein hetero[2]catenanes from six motifs.
• Demonstration of versatile templates for mechanically interlocked protein conjugates.

Conclusions:

• The study provides a modular platform for protein topology engineering.
• The discovered motifs enable the synthesis of complex protein structures like hetero[2]catenanes.
• This work enriches the toolbox for creating mechanically interlocked protein conjugates for applications such as FRET pairs and bispecific binders.