Enhancing carbohydrate binding module family 40 (CBM40) via in silico sequential mutagenesis approach

The Carbohydrate-Binding Module family 40 (CBM40) is a component of bacterial sialidase that exhibits a high affinity and selectivity for sialic acids. These CBM40s can be independently isolated and modified for use as drug delivery agents against tumours exhibiting hypersialation. Modifying specific amino acid sequences can enhance the sialic acid binding ability of CBM40. This study employed an in silico approach to construct, simulate and evaluate Vibrio cholerae CBM40 mutants via interaction of these mutants with a sialoside ligand via molecular docking and molecular dynamics simulation. Several rounds of mutagenesis were proposed to sequentially improve the CBM40 ligand binding energy. There were discprepancies between the molecular docking and simulation data from the first round of mutagenesis. The CBM40 residue substitution from Arg74 to Trp74 and from Gly196 to Gln196 (R1-Gly-Gln196) had an equal molecular docking binding energy increase. However, the former mutant exhibited weaker binding than non-modified CBM40, while the latter displayed stronger ligand binding. Two mutants identified in the second round of mutagenesis exhibited improved ligand binding energy from the molecular docking analysis. However, both mutants displayed weaker free binding energy than the R1-Gly-Gln196. Molecular dynamics simulation post-analysis suggested R1-Gly-Gln196 formed a more stable interaction with sialoside compared to other CBM40s. The analyses also revealed that mutation of residues can directly or indirectly introduce steric clashes which cause interaction instability and thus reduces ligand binding energy. This study highlighted the significance of validating molecular docking results with simulation analysis, thereby improving the reliability of in silico protein-ligand interaction predictions.