Computationally guided cross-linking overcomes interfacial mismatch in protein dimerization: Creating a long-acting
Xingyu Deng1, Zhiguo Wang2, Yuxin Hou3
1School of Pharmacy, East China University of Science and Technology, Shanghai, 200237, China.
International Journal of Biological Macromolecules
|July 6, 2026
Summary
Engineered cocaine esterase (CocE) homodimers (CocE-HD) show enhanced stability and extended plasma half-life. This protein stabilization strategy uses adaptive cross-linking for improved therapeutic potential.
Area of Science:
- Biochemistry
- Protein Engineering
- Computational Biology
Background:
- Conventional disulfide bond engineering for protein stabilization faces limitations due to topological mismatches with dynamic protein interfaces.
- Engineered cocaine esterase (CocE) mutants often exhibit incomplete dimerization and reduced in vivo persistence.
Purpose of the Study:
- To develop a computationally guided, topologically adaptive cross-linking strategy for enhanced protein stabilization.
- To create a site-specific homodimer of CocE (CocE-HD) with improved stability and therapeutic efficacy.
Main Methods:
- Molecular dynamics simulations to map interfacial plasticity and identify optimal cross-linking distances.
- Site-specific cross-linking using bis-maleimidoethane (BMOE) and ablation of off-target cysteines.
- Assessment of CocE-HD stability (thermal, chemical, serum) and in vivo pharmacokinetics in a rat model.
Main Results:
- Identified a converged C196-C301 distance of ~9.10 Å in CocE subunits, matched with BMOE (8.06 Å) for robust cross-linking.
- CocE-HD demonstrated significantly enhanced thermal, chemical, and serum stability compared to the monomer.
- CocE-HD exhibited a 3.6-fold longer plasma half-life (103.94 min) in rats and maintained rapid cocaine clearance.
Conclusions:
- The developed adaptive cross-linking strategy effectively stabilizes protein dimers by aligning cross-linker chemistry with interfacial topology.
- CocE-HD represents a promising therapeutic candidate for cocaine abuse treatment due to its enhanced stability and pharmacokinetics.
- This approach provides a rational framework for stabilizing other protein therapeutics.
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