Structural insights into hyaluronic acid recognition by human HYAL2: A cross-species comparative study

Hyaluronidases are crucial for remodelling the extracellular matrix by breaking down hyaluronic acid (HA); however, the structural factors that govern HA recognition and size-dependent processing remain poorly understood. In this study, we examined the interaction between HA oligomers and human hyaluronidase 2 (hHYAL2) using both computational and experimental methods. Molecular docking results showed that binding affinity increased with the length of HA oligomers. Longer oligomers gradually filled the catalytic cleft and interacted with conserved catalytic residues. Structural analysis of the docking models indicated that HA adopts a partially curled yet elongated conformation within the catalytic groove, allowing it to bind to multiple sites simultaneously. Molecular dynamics simulations confirmed that HA binding preserves the overall stability of hHYAL2 while triggering synchronised loop movements that facilitate substrate accommodation within the catalytic channel. Comparisons among human HYAL1, bovine, and bee hyaluronidases revealed that, despite similarities in catalytic residues, differences in cleft shape and electrostatic environment lead to distinct substrate-binding modes. Bovine hyaluronidase tends to stabilise a more curled HA conformation due to its shallower catalytic pocket. Circular dichroism and fluorescence spectroscopy demonstrated that HA binding causes subtle structural changes in hHYAL2 while maintaining the protein's overall stability. Overall, these findings offer insights into HA recognition mechanisms and highlight species-specific variations in HA degradation, emphasising structural factors that influence hyaluronidase substrate specificity.