文献类型：期刊文献

英文题名：Computationally guided cross-linking overcomes interfacial mismatch in protein dimerization: Creating a long-acting cocaine esterase

作者：Deng, Xingyu[1];Wang, Zhiguo[3];Hou, Yuxin[2];Xu, Nuo[2];Li, Zhenzhen[2];Hu, Qi[2];Mo, Nan[2];Ren, Ziying[2];Chen, Cong[2];Hou, Shurong[2];Chen, Xiabin[2];Ma, Lei[1]

机构：[1]East China Univ Sci & Technol, Sch Pharm, Shanghai 200237, Peoples R China;[2]Hangzhou Normal Univ, Sch Pharm, Hangzhou 311121, Zhejiang, Peoples R China;[3]Hangzhou Normal Univ, Inst Aging Res, Sch Basic Med Sci, Hangzhou 311121, Zhejiang, Peoples R China

年份：2026

卷号：375

外文期刊名：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES

收录：;EI(收录号：20262821081992);Scopus(收录号：2-s2.0-105044228986);WOS:【SCI-EXPANDED(收录号:WOS:001824914600001)】；

基金：This work was supported by the STI2030-Major Projects (2022ZD0214600), the Interdisciplinary Research Project of Hangzhou Normal University (2025JCXK02 to X. Chen) and Central Guidance on the Development of Local Science and Technology (2025ZY01109 to J. Wang). This study was supported by Advanced Computation Center of Hangzhou Normal University.

语种：英文

外文关键词：Cocaine esterase; Protein stability; BMOE cross-linking; Pharmacokinetics; Protein therapy; Protein engineering

摘要：Engineering intermolecular disulfide bonds to stabilize protein dimers is a conventional strategy, yet its efficacy is frequently constrained by the topological mismatch between rigid disulfide linkages (similar to 2.0 & Aring;) and the dynamic fluctuations of protein interfaces. This structural rigidity often leads to incomplete dimerization, conformational strain, and suboptimal in vivo persistence, as exemplified by the engineered cocaine esterase (CocE) mutant E196-301. In response to this challenge, we propose a computationally guided, topologically adaptive cross-linking strategy that harnesses the dynamic conformational ensemble of protein interfaces. Firstly, utilizing molecular dynamics simulation, we mapped the interfacial plasticity of the CocE subunit, identifying a converged C196-C301 distance of similar to 9.10 & Aring;. Rather than relying on static structural approximations, we rationally matched this spatial requirement with a specific bifunctional cross-linker, bis-maleimidoethane (BMOE, 8.06 & Aring;), achieving a robust interfacial bridge that accommodates natural structural fluctuations was achieved. Next, the off-target surface cysteines (C107S and C551S) were strategically ablated, yielding a precisely controlled, site-specific homodimer of CocE (CocE-HD). CocE-HD exhibited significant improvements in thermal, chemical, and serum stability relative to the monomer. In a rat model, CocE-HD (10 mg/kg) demonstrated a plasma half-life of 103.94 +/- 43.62 min, representing a 3.6-fold extension compared to the monomer. Functional assays confirmed that CocE-HD maintains rapid cocaine clearance even 2 h post-administration, completely abolishing cocaine-induced hyperlocomotion. This work establishes CocE-HD as a promising therapeutic candidate and provides a rational approach for protein stabilization by aligning cross-linker chemistry with interfacial topology.