title: Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.
abstract: Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly2H,15N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in1H,15N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[13C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic.
authors.affiliation: Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.
authors.forename: Lena Lisbeth
authors.initials: LL
authors.lastname: Grimm
authors.affiliation: Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.
authors.forename: Sophie
authors.initials: S
authors.lastname: Weissbach
authors.affiliation: Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Flügge
authors.affiliation: Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.
authors.forename: Nora
authors.initials: N
authors.lastname: Begemann
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, P. O. Box 3800, STN CSC, Victoria, BC, V8W 3P6, Canada.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
authors.affiliation: Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.
authors.forename: Thomas
authors.identifier: 0000-0002-7570-8260
authors.initials: T
authors.lastname: Peters
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014530:Uridine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014531:Uridine Diphosphate Galactose; D014530:Uridine Diphosphate; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase; C023155:blood-group-substance alpha-D-galactosyltransferase
keywords: NMR spectroscopy; blood group antigens; enzyme catalysis; glycosyltransferases; transferases