title: Structural basis of substrate specificity of human oligosaccharyl transferase subunit N33/Tusc3 and its role in regulating protein N-glycosylation.
abstract: N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is essential in eukaryotes and catalyzed by oligosaccharyl transferase (OST). Human OST is a hetero-oligomer of seven subunits. The subunit N33/Tusc3 is a tumor suppressor candidate, and defects in the subunit N33/Tusc3 are linked with nonsyndromic mental retardation. Here, we show that N33/Tusc3 possesses a membrane-anchored N-terminal thioredoxin domain located in the ER lumen that may form transient mixed disulfide complexes with OST substrates. X-ray structures of complexes between N33/Tusc3 and two different peptides as model substrates reveal a defined peptide-binding groove adjacent to the active site that can accommodate peptides in opposite orientations. Structural and biochemical data show that N33/Tusc3 prefers peptides bearing a hydrophobic residue two residues away from the cysteine forming the mixed disulfide with N33/Tusc3. Our results support a model in which N33/Tusc3 increases glycosylation efficiency for a subset of human glycoproteins by slowing glycoprotein folding.
authors.affiliation: ETH Zürich, Department of Biology, Institute of Molecular Biology and Biophysics, Otto-Stern-Weg 5, 8093 Zurich, Switzerland.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mohorko
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK.
authors.forename: Robin L
authors.initials: RL
authors.lastname: Owen
authors.affiliation: ETH Zürich, Department of Biology, Institute of Molecular Biology and Biophysics, Otto-Stern-Weg 5, 8093 Zurich, Switzerland.
authors.forename: Goran
authors.initials: G
authors.lastname: Malojčić
authors.affiliation: ETH Zürich, Department of Biology, Institute of Molecular Biology and Biophysics, Otto-Stern-Weg 5, 8093 Zurich, Switzerland.
authors.forename: Maurice S
authors.initials: MS
authors.lastname: Brozzo
authors.affiliation: ETH Zürich, Department of Biology, Institute of Microbiology, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.affiliation: ETH Zürich, Department of Biology, Institute of Molecular Biology and Biophysics, Otto-Stern-Weg 5, 8093 Zurich, Switzerland. Electronic address: rudi@mol.biol.ethz.ch.
authors.forename: Rudi
authors.initials: R
authors.lastname: Glockshuber
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D004220:Disulfides; D008565:Membrane Proteins; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; C524338:TUSC3 protein, human; D025521:Tumor Suppressor Proteins