Enhancing bezlotoxumab binding to C. difficile toxin B2: insights from computational simulations and mutational...

Clostridioides difficile infection (CDI) is a significant concern caused by widespread antibiotic use, resulting in diarrhea and inflammation from the gram-positive anaerobic bacterium C. difficile. Although bezlotoxumab (Bez), a monoclonal antibody (mAb), was developed to address CDI recurrences, the recurrence rate remains high, partly due to reduced neutralization efficiency against toxin B2. In this study, we aimed to enhance the binding of Bez to C. difficile toxin B2 by combining computational simulations and mutational analyses. We identified specific mutations in Bez, including S28R, S31W/K, Y32R, S56W and G103D/S in the heavy chain (H_c), and S32F/H/R/W/Y in the light chain (L_c), which significantly improved binding to toxin B2 and formed critical protein-protein interactions. Through molecular dynamics simulations, several single mutations, such as H_cS28R, L_cS32H, L_cS32R, L_cS32W and L_cS32Y, exhibited superior binding affinities to toxin B2 compared to Bez wild-type (WT), primarily attributed to Coulombic interactions. Combining the H_cS28R mutation with four different mutations at residue L_cS32 led to even greater binding affinities in double mutants (MTs), particularly H_cS28R/L_cS32H, H_cS28R/L_cS32R and H_cS28R/L_cS32Y, reinforcing protein-protein binding. Analysis of per-residue decomposition free energy highlighted key residues contributing significantly to enhanced binding interactions, emphasizing the role of electrostatic interactions. These findings offer insights into rational Bez MT design for improved toxin B2 binding, providing a foundation for developing more effective antibodies to neutralize toxin B2 and combat-related infections.