Molecular Dynamics Investigation of Human Endosulfatase HSulf-2 Reveals the Structural Roles of Intrinsically Disordered Regions, Catalytic Residues, and N-Glycosylation

Abstract Human endosulfatases HSulf-1 and HSulf-2 are the only extracellular sulfatases that selectively remove 6-O-sulfate groups from heparan sulfate chains, thereby regulating numerous signaling pathways involved in development, tissue homeostasis, and cancer. In particular, HSulf-2 is frequently overexpressed in human cancers, making it an attractive therapeutic target. However, its three-dimensional structure has not been resolved experimentally, mainly because of its multidomain organization, extensive post-translational modifications, and large intrinsically disordered hydrophilic domain (HD). Here, molecular dynamics simulations based on the AlphaFold2 model of HSulf-2 were used to investigate the structural and dynamic contributions of its domains and selected post-translational modifications. The simulations identified several intrinsically disordered regions, confirming that the HD constitutes the principal source of conformational disorder within the enzyme. They further demonstrate that the CAT–Cter assembly reproduces the canonical catalytic architecture conserved throughout the sulfatase family, whereas the HD induces only minor changes in the average structure while significantly destabilizing the conformational dynamics of the catalytic framework. In contrast, maturation of the catalytic residue modulates the conformational landscape of HSulf-2 without altering its overall architecture, whereas the conserved chitobiose cores of N-glycans exert only limited structural effects. These results support a modular structural organization in which the CAT–Cter assembly provides the conserved catalytic scaffold, while the HD confers the dynamic properties required for recognition of polymeric heparan sulfate substrates. This work provides a structural framework for understanding HSulf specificity and opens new perspectives for the rational design of selective HSulf inhibitors. In particular, the modular organization suggests that future dual-site inhibitors that simultaneously target the conserved catalytic pocket and the unique hydrophilic domain could combine high binding affinity with selectivity for HSulf enzymes.