Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase [Protein Structure and Folding]

March 20th, 2014 by Liu, C., Sheng, J., Krahn, J. M., Perera, L., Xu, Y., Hsieh, P.-H., Dou, W., Liu, J., Pedersen, L. C.

Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3-phosphoadenosine 5-phosphate, and a heptasaccharide. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg80, Lys350, and Arg190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.