The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes.
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