title: Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase.
abstract: 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 substrate. 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 Arg-80, Lys-350, and Arg-190 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.
authors.affiliation: From the Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Chunhui
authors.initials: C
authors.lastname: Liu
authors.forename: Juzheng
authors.initials: J
authors.lastname: Sheng
authors.forename: Juno M
authors.initials: JM
authors.lastname: Krahn
authors.forename: Lalith
authors.initials: L
authors.lastname: Perera
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Xu
authors.forename: Po-Hung
authors.initials: PH
authors.lastname: Hsieh
authors.forename: Wenfang
authors.initials: W
authors.lastname: Dou
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Lars C
authors.initials: LC
authors.lastname: Pedersen
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002645:Chickens; D018360:Crystallography, X-Ray; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D015238:Sulfotransferases; C021350:heparitin sulfotransferase
keywords: Heparan Sulfate; Heparin; Heparin-binding Protein; Oligosaccharide; Sulfotransferase