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

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 (~2.0 Å) 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 ~9.10 Å. Rather than relying on static structural approximations, we rationally matched this spatial requirement with a specific bifunctional cross-linker, bis-maleimidoethane (BMOE, 8.06 Å), 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.