Structural basis of substrate specificity of human oligosaccharyl transferase subunit N33/Tusc3 and its role in regulating protein N-glycosylation

Structure. 2014 Apr 8;22(4):590-601. doi: 10.1016/j.str.2014.02.013. Epub 2014 Mar 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Crystallography, X-Ray
  • Disulfides / chemistry*
  • Gene Expression
  • Glycosylation
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides / chemistry*
  • Protein Binding
  • Protein Folding
  • Protein Multimerization
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry*
  • Protein Subunits / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Tumor Suppressor Proteins / chemistry*
  • Tumor Suppressor Proteins / genetics

Substances

  • Disulfides
  • Membrane Proteins
  • N33 protein, human
  • Peptides
  • Protein Subunits
  • Recombinant Proteins
  • Tumor Suppressor Proteins

Associated data

  • PDB/4M8G
  • PDB/4M90
  • PDB/4M91
  • PDB/4M92