Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability

Abstract

Recombinant proteins are traditionally limited to linear configuration. Herein, we report in vivo protein topology engineering using highly efficient, mechanically interlocking SpyX modules named AXB and BXA. SpyX modules are protein domains composed of p53dim (X), SpyTag (A), and SpyCatcher (B). The p53dim guides the intertwining of the two nascent protein chains followed by autocatalytic isopeptide bond formation between SpyTag and SpyCatcher to fulfill the interlocking, leading to a variety of backbone topologies. Direct expression of AXB or BXA produces protein catenanes with distinct ring sizes. Recombinant proteins containing SpyX modules are obtained either as mechanically interlocked obligate dimers if the protein of interest is fused to the N- or C-terminus of SpyX modules, or as star proteins if the protein is fused to both N- and C-termini. As examples, cellular syntheses of dimers of (GB1)2 (where GB1 stands for immunoglobulin-binding domain B1 of streptococcal protein G) and of four-arm elastin-like star proteins were demonstrated. Comparison of the catenation efficiencies in different constructs reveals that BXA is generally much more effective than AXB, which is rationalized by the arrangement of three domains in space. Mechanical interlocking induces considerable stability enhancement. Both AXB and BXA have a melting point ∼20 °C higher than the linear controls and the BXA catenane has a melting point ~2 °C higher than the cyclic control BX’A. Notably, four-arm elastin-like star proteins demonstrate remarkable tolerance against trypsin digestion. The SpyX modules provide a convenient and versatile approach to construct unconventional protein topologies via the “assembly-reaction” synergy, which opens a new horizon in protein science for stability enhancement and function reinforcement via topology engineering.

Protein Catenation Enhances Both the Stability and Activity of Folded Structural Domains

“…Inspired by this approach, we combined it with SpyTag/SpyCatcher chemistry to achieve the first cellular synthesis of artificial protein catenanes based on unstructured elastin‐like protein . The method was later adapted to enable in vivo protein topology engineering to give obligate dimers and star proteins . Nevertheless, no recombinant folded protein catenanes have been reported to date, and it remains a challenge to illustrate how, and in which aspects, catenation would influence protein properties.…”

Lasso Proteins: Modular Design, Cellular Synthesis, and Topological Transformation

“…Ion mobility‐mass spectrometry (IM‐MS) is a powerful method for investigating the structures and conformations of protein molecules and their assemblies . IM‐MS separates protein ions by collision cross sections (CCSs) which provides the size and shape information related to protein conformation .…”

Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization

“…It is speculative, but the enzyme might have underdone partial degradation during the 12 h digestion at room temperature by EK. Thus, it is of considerable interest to investigate the effects and mechanisms of molecular cyclization on stability against crowded adverse conditions such as the presence of trypsin and metal ions …”