Crystal enigma: Understanding diverse protein conformational dynamics, ligand selectivity and interaction in multi-space group crystals using computational modelling

Empirical structural methods have been instrumental in drug design, but they often fall short of capturing protein dynamics. To address this limitation, computer-aided drug design (CADD) is essential. Given that Schistosoma bovis 28 kDa-Glutathione S -Transferase ( Sb 28GST) is a promising anti-schistosome drug target, a careful selection of crystal structure space groups used for CADD is emphasized in this study. Sb 28GST was successfully overexpressed and purified to grow protein crystals with a resolution of 2.4 Å in an orthorhombic space group system. High-throughput virtual screening (HTVS) of a flavonoid compound was performed on monoclinic 8ALS and orthorhombic 8BHZ Sb28GST to identify potential ligands. The results showed a diverse selection of hit compounds with apigenin 7-O-(2G-rhamnosyl)gentiobioside (apigenin) being the common ligand. However, quercetin-3-O-Beta- d -Glucose-7-O-Beta-D-Gentiobioside (quercetin) showed the highest affinity to 8ALS with a Glide gscore of −15.66 kcal/mol. Also, 500-ns molecular dynamics simulations (MDS) of the 8ALS- Sb 28GST and 8BHZ- Sb 28GST apo systems, as well as their corresponding apigenin and quercetin complexes, show two distinct trajectories which reveal significant differences in their dynamic behaviour and also showed varying interactions. This highlights the need for collective assessment of protein polymorphs to comprehensively understand protein dynamics. The inhibitory potency of apigenin and quercetin via a GSH-CDNB conjugation assay confirmed that they significantly reduced the specific enzyme activity of Sb 28GST with both ligands having half-maximal inhibitory concentration (IC 50 ) of 0.13 mM. Extrinsic fluorescence studies and thermal shift assay indicated that these compounds bind to the hydrophobic H-site and allosteric L-site at the dimer interface and have a minimal effect on the protein's thermal stability. • Assessing polymorphic Sb28GST (Schistosoma bovis) for conformational dynamics and ligand selectivity • Using virtual screening, molecular dynamics, and experiments to identify potential anti-schistosome drugs. • Apigenin and quercetin were identified as potential anti-schistosome drugs. • Apigenin and quercetin showed stable binding and excellent inhibitory ability towards Sb 28GST.