In Silico Design of Dimerization Interfaces in Drosophila melanogaster Glutathione Transferase

Abstract Understanding the molecular determinants of protein–protein interface stability remains a central challenge for the rational engineering of oligomeric enzymes. Here, the glutathione transferase GSTE3 from Drosophila melanogaster is used as a model system to decipher the factors governing dimer stability through an integrated structural, computational, and experimental approach. The X–ray crystal structure reveals a canonical homodimeric organization with a conserved Epsilon-class dimerization motif at the subunit interface. Molecular dynamics simulations combined with inter-residue contact network analysis provide a quantitative description of the interface, identifying persistent and transient interactions that contribute to dimer stability. Building on this framework, sequence-space exploration by Monte Carlo simulations is employed to design the dimerization interface and enhance stability. Distinct modeling strategies, differing in mutation schemes and energy evaluation methods, yield multiple classes of variants with diverse interfacial properties. Their stability and interaction patterns are further assessed using molecular dynamics simulations, enabling comparative analysis of structural robustness and contact network organization. Selected variants are subsequently produced in vitro to evaluate their experimental feasibility. Altogether, this study establishes a multiscale framework for the rational engineering of protein–protein interfaces and highlights both the potential and limitations of sequence–based optimization strategies for stabilizing enzyme dimers.