Active Template Synthesis of Protein Heterocatenanes

Abstract

Covalent-bond-forming protein domains can be versatile tools for creating unconventional protein topologies. In this study,t hrough rewiring the SpyTag-SpyCatcher complex to induce rationally designed chain entanglement, we developed ab iologically enabled active template for the concise,m odular,a nd programmable synthesis of protein heterocatenanes both in vitro and in vivo.I ti sageneral and good-yielding reaction for forming heterocatenanes with precisely controlled ring sizes and broad structural diversity. More importantly,such heterocatenation not only provides an efficient means of bioconjugation for integrating multiple native functions,b ut also enhances the stability of the component proteins against proteolytic digestion, thermal unfolding, and freeze/thaw-induced mechanical denaturation, thus opening up aversatile path in the nascent field of proteintopology engineering.Scheme 1. Biologicallye nabled, active template approach for the concise synthesis of protein heterocatenanes.

Dynamic covalent self-assembly of mechanically interlocked molecules solely made from peptides

“…Although various attempts 16 – 20 have been made, applicable synthesis strategies for wholly peptidic MIMs similar in size to synthetic MIMs, 21 have not been found yet. Elegant synthetic strategies that leverage the bisecting U motif of the p53 tetramerization domain have been developed to generate protein homocatenanes 22 , 23 , heterocatenanes 24 , 25 , and other entangled structures 26 . However, these mechanically interlocked proteins are much larger (~9 kDa and up) and bear only a passing resemblance to synthetic MIMs.…”

Protein Conjugation via SpyStapler‐Mediated SpyTag/BDTag Coupling

“…It has facilitated the synthesis of various topological proteins (e.g., cyclic proteins and branched proteins) by site‐specific ligation via isopeptide bonding, with excellent yields (Wu et al., 2018). Moreover, by rational introduction of artificial entanglement into the SpyTag/BDTag/SpyStapler complex by rewiring, we successfully developed the active template synthesis of protein heterocatenanes, representing a completely new mode of bioconjugation by mechanical bonding (Da & Zhang, 2019). There is no covalent linkage between the two components, but they cannot be separated without breaking covalent bonds.…”

“…1). Not only does this method facilitate the synthesis of various nonlinear proteins, but it also enables the "active-template" synthesis of protein heterocatenanes with a new mode of bioconjugation via mechanical interlocking (Da & Zhang, 2019; Fig. 2).…”

“…The final conjugate is labeled as cat ‐POI1‐POI2. Reprinted and adapted with permission from Da and Zhang (2019). Copyright (2019) John Wiley and Sons.…”

Mechano-bioconjugation Strategy Empowering Fusion Protein Therapeutics with Aggregation Resistance, Prolonged Circulation, and Enhanced Antitumor Efficacy

“…In supramolecular chemistry, mechanical bonds are commonly encountered in the making of molecular machines. − Their emergence in macromolecules is a recent event, presenting a novel mode for bioconjugation. In nature, proteins containing mechanical bonds have also been identified and found to exhibit enhanced stabilities. − Cultivating artificial mechanically interlocked proteins has received considerable success in the past decades as demonstrated in the successful synthesis of deeply knotted protein polymers and tightly linked protein catenanes. − Various synthetic strategies, such as passive templates, − active templates, streamlined synthesis, and topological transformation, − have been developed. Indeed, protein catenation has been proven to bring in enhanced stability without compromising activity. ,,, We envision that bioconjugation via mechanical bonds may be utilized in therapeutic protein engineering to enable simultaneous functional integration and stability enhancement, which shall open a new avenue beyond simple fusion.…”