title: Structure of human POFUT2: insights into thrombospondin type 1 repeat fold and O-fucosylation.
abstract: Protein O-fucosylation is a post-translational modification found on serine/threonine residues of thrombospondin type 1 repeats (TSR). The fucose transfer is catalysed by the protein O-fucosyltransferase 2 (POFUT2) and >40 human proteins contain the TSR consensus sequence for POFUT2-dependent fucosylation. To better understand O-fucosylation on TSR, we carried out a structural and functional analysis of human POFUT2 and its TSR substrate. Crystal structures of POFUT2 reveal a variation of the classical GT-B fold and identify sugar donor and TSR acceptor binding sites. Structural findings are correlated with steady-state kinetic measurements of wild-type and mutant POFUT2 and TSR and give insight into the catalytic mechanism and substrate specificity. By using an artificial mini-TSR substrate, we show that specificity is not primarily encoded in the TSR protein sequence but rather in the unusual 3D structure of a small part of the TSR. Our findings uncover that recognition of distinct conserved 3D fold motifs can be used as a mechanism to achieve substrate specificity by enzymes modifying completely folded proteins of very wide sequence diversity and biological function.
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
authors.forename: Chun-I
authors.initials: CI
authors.lastname: Chen
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Keusch
authors.forename: Dominique
authors.initials: D
authors.lastname: Klein
authors.forename: Daniel
authors.initials: D
authors.lastname: Hess
authors.forename: Jan
authors.initials: J
authors.lastname: Hofsteenge
authors.forename: Heinz
authors.initials: H
authors.lastname: Gut
mesh_terms: D018360:Crystallography, X-Ray; D005643:Fucose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D005643:Fucose; D005647:Fucosyltransferases; C495567:POFUT2 protein, human