Crystal structure of a β1,3-Glucosyltransferase reveals an unusual substrate recognition by a two-domain GT-A fold glycosyltransferase.

β1,3-Glucosyltransferase (B3GLCT) adds a glucose onto O-linked fucose on thrombospondin type 1 repeats (TSRs). Protein O-fucosyltransferase 2 (POFUT2) first transfers a fucose to properly folded TSRs containing a consensus sequence for O-fucosylation. This uncommon O-fucose modification is then extended to a glucose-fucose disaccharide by B3GLCT. B3GLCT is a GT-A fold glycosyltransferase and pathogenic variants cause Peters Plus Syndrome (PTRPLS, OMIM #261540), a Congenital Disorder of Glycosylation (CDG). Most GT-A fold family members have a single GT-A domain, but B3GLCT contains an additional GT-A domain. To assess the function of the additional GT-A domain and binding of TSR substrates, we determined the crystal structure of an O-fucosylated TSR (Fuc-O-TSR3) from Thrombospondin-1 bound to B3GLCT. The additional GT-A domain is essential for substrate binding but is catalytically inactive: it does not bind UDP or Mn2+ and is not a β1,3-glucosyltransferase. It also creates a deep pocket with a vestigial active site located on the opposite face of the Fuc-O-TSR3 substrate binding site. The Fuc-O-TSR3 acceptor substrate binds in a cleft between the two GT-A domains, each of which has evolved hypervariable regions for Fuc-O-TSR recognition. This structure is an unusual example of a glycosyltransferase with a catalytically inactive extra GT-A domain, providing insight into the binding of B3GLCT's diverse Fuc-O-TSR substrates. We also discuss how B3GLCT mimics the two-domain structure of GT-B fold glycosyltransferases. All GT-B fold glycosyltransferases contain two bilobal Rossmann-like fold domains, like B3GLCT. Our data also explain how PTRPLS-associated variants and predicted pathogenic mutations disrupt B3GLCT function.