Covalent bicyclization of protein complexes yields durable quaternary structures

George H Hutchins; Sebastian Kiehstaller; Pascal Poc; Abigail H Lewis; Jisun Oh; Raya Sadighi; Nicholas M Pearce; Mohamed Ibrahim; Ivana Drienovská; Anouk M Rijs; Saskia Neubacher; Sven Hennig; Tom N Grossmann

doi:10.1016/j.chempr.2023.10.003

Covalent bicyclization of protein complexes yields durable quaternary structures

Chem. 2024 Feb 8;10(2):615-627. doi: 10.1016/j.chempr.2023.10.003.

Authors

George H Hutchins^{1

2}, Sebastian Kiehstaller³, Pascal Poc¹, Abigail H Lewis¹, Jisun Oh¹, Raya Sadighi^{1

2}, Nicholas M Pearce^{1

2

4}, Mohamed Ibrahim¹, Ivana Drienovská^{1

2}, Anouk M Rijs^{1

2}, Saskia Neubacher³, Sven Hennig^{1

2}, Tom N Grossmann^{1

2}

Affiliations

¹ Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, the Netherlands.
² Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, the Netherlands.
³ Incircular B.V., 1081 HZ Amsterdam, the Netherlands.
⁴ Department of Physics, Chemistry and Biology, SciLifeLab, Linköping University, 581 83 Linköping, Sweden.

Abstract

Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.

Keywords: Aggregation; INCYPRO; biocatalysis; bioconjugation; chemical biology; cross-linking; crystallography; enzymes; protein engineering; structural stabilization.