Structural analysis of the sulfotransferase (3-o-sulfotransferase isoform 3) involved in the biosynthesis of an entry receptor for herpes simplex virus 1.

Heparan sulfate (HS) plays essential roles in assisting herpes simplex virus infection and other biological processes. The biosynthesis of HS includes numerous specialized sulfotransferases that generate a variety of sulfated saccharide sequences, conferring the selectivity of biological functions of HS. We report a structural study of human HS 3-O-sulfotransferase isoform 3 (3-OST-3), a key sulfotransferase that transfers a sulfuryl group to a specific glucosamine in HS generating an entry receptor for herpes simplex virus 1. We have obtained the crystal structure of 3-OST-3 at 1.95 A in a ternary complex with 3'-phosphoadenosine 5'-phosphate and a tetrasaccharide substrate. Mutational analyses were also performed on the residues involved in the binding of the substrate. Residues Gln255 and Lys368 are essential for the sulfotransferase activity and lie within hydrogen bonding distances to the carboxyl and sulfo groups of the uronic acid unit. These residues participate in the substrate recognition of 3-OST-3. This structure provides atomic level evidence for delineating the substrate recognition and catalytic mechanism for 3-OST-3.

Crystal structure of an alpha 1,4-N-acetylhexosaminyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis.

EXTL2, an alpha1,4-N-acetylhexosaminyltransferase, catalyzes the transfer reaction of N-acetylglucosamine and N-acetylgalactosamine from the respective UDP-sugars to the non-reducing end of [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol, an acceptor substrate analog of the natural common linker of various glycosylaminoglycans. We have solved the x-ray crystal structure of the catalytic domain of mouse EXTL2 in the apo-form and with donor substrates UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, a structure of the ternary complex with UDP and the acceptor substrate analog [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol has been determined. These structures reveal three highly conserved residues, Asn-243, Asp-246, and Arg-293, located at the active site. Mutation of these residues greatly decreases the activity. In the ternary complex, an interaction exists between the beta-phosphate of the UDP leaving group and the acceptor hydroxyl of the substrate that may play a functional role in catalysis. These structures represent the first structures from the exostosin gene family and provide important insight into the mechanisms of alpha1,4-N-acetylhexosaminyl transfer in heparan biosynthesis.