Structural and dynamical insights revealed the disaccharide glycosylation in vitamin D-binding protein (VDBP) enhances the vitamin D-binding stability

The difference in the levels of glycosylation in Vitamin D--Binding Protein (VDBP) has a significant effect on its biological function and role in immune modulation and cancer treatments. This study reports the impact of differential glycosylation on the structural dynamics and binding efficiency of Vitamin D-Binding Protein (VDBP) with Vitamin D3 (VitD3). The focus is to understand how glycosylation affects protein conformation and then associated immune modulation based therapeutic applications specially in cancer treatment. Molecular Modelling and Molecular Dynamics Simulations were used to investigate the conformational dynamics of the dimer in both forms, un-glycosylated (VitD3-VDBP) with no sugar moiety attached (VitD3-VDBP) and with disaccharide linkage (VitD3-dgVDBP). Vitamin D-binding protein dimer, (VitD3-dgVDBP) was identified in the samples through SDS-PAGE followed by UV/VIS and CD/ORD analysis to confirm protein and vitamin D3 dimer. The molecular Structure of the protein with disaccharide sugars (VitD3-dgVDBP) was modelled and aligned with the reported sequence and structure of the protein. RMSD and RMSF analysis helped to estimate the effect of differential glycosidic linkage on the conformational integrity of protein. Dynamical Cross-Correlation Movement (DCCM) further revealed that (VitD3-dgVDBP) got opposite movements upon disaccharide linkage. Principal Component Analysis (PCA) Investigations showed that both forms of the protein, (VitD3-VDBP) and (VitD3-dgVDBP) have different dominant movements across the protein surface and solvent-accessible surface SASA analysis in addition showed the disaccharide linkage to have a larger accessible surface area comparatively. Binding energy estimation of vitamin D and protein dimer using MMPBSA method along with hydrogen bond analysis revealed higher binding energy in the case of (VitD3-dgVDBP), – 116.09 ± 16.04, thus suggesting the disaccharide linkage enhances the binding of protein with VitD3 and its availability in the blood cells.