Extracellular endosulfatases, such as human SULF2 (HSULF2), play central roles in modulating heparan sulfate (HS) sulfation patterns ; yet, despite being linked to numerous physiological and pathological processes, the molecular features of HSULF2 remain only partially understood. Here, we present a high-resolution cryo-EM structure of the HSULF2 structured domain supported by complementary biochemical, biophysical, and functional assays. The structure reveals the architecture of HSULF2’s catalytic domain, which adopts a conserved sulfatase α/β-fold. It also illustrates the spatial arrangement of the active site, featuring an essential formylglycine residue, a Ca²⁺ ion, and key substrate-recognition residues commonly found in S1-sulfatases. Complementary NMR and activity assays highlight the hydrophilic domain (HD) as a structured but flexible module required for HS binding and processive 6-O-desulfation. We show that HSULF2 is organized into two functional domains that work together, one securing the HS substrate and the other carrying out the desulfation reaction. Our functional analyses further demonstrate that HSULF2 modulates TGF-β1 binding in a manner dependent on both 6-O-sulfation and GAG chain length. In parallel, we characterized SoS1_27, a bacterial endosulfatase from Prevotella oris, which lacks an HD yet retains internal activity, pointing to distinct strategies for substrate engagement. Together, these results provide the first high-resolution framework for a mammalian extracellular sulfatase, establish mechanistic principles for HSULF2 function, and uncover structural parallels with bacterial endosulfatases, raising new questions about how endo-activity is defined and diversified across systems. Ultimately, understanding HSULF2’s mode of substrate recognition and catalysis could open the way to the development of selective inhibitors with potential applications in cancer and inflammatory disease therapies.​
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