1 H, 13 C, 15 N resonance assignments and secondary structure of yeast oligosaccharyltransferase subunit Ost4 and its fu
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H, 13C, 15N resonance assignments and secondary structure of yeast oligosaccharyltransferase subunit Ost4 and its functionally important mutant Ost4V23D Bharat P. Chaudhary1 · David Zoetewey2 · Smita Mohanty1 Received: 28 January 2020 / Accepted: 16 April 2020 © Springer Nature B.V. 2020
Abstract Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high-mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an –N–X–T/S– sequence (where X is any residue except proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as Congenital Disorders of Glycosylation. Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3–Ost4–Ost3 (or Ost6) sub-complex. Mutation of any residue from M18I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C, and 15N resonance assignments for Ost4 and Ost4V23D in dodecylphosphocholine micelles. Keywords N-linked glycosylation · Oligosaccharyltransferase · Solution NMR · Ost4 · Congenital Disorders of Glycosylation · Membrane proteins
Biological context The oligosaccharyltransferase (OST) enzyme complex is localized next to the ribosome in the endoplasmic reticulum (ER) and carries out the post-translational asparaginelinked or N-linked glycosylation in a newly synthesized protein (Kornfeld and Kornfeld 1985). This process is an essential, ubiquitous, and highly conserved protein modification in eukaryotes and some prokaryotes. In the central * Smita Mohanty [email protected] 1
Department of Chemistry, Oklahoma State University, 74078 Stillwater, OK, USA
Present Address: Department of Chemistry, Physics and Astronomy, Georgia College and State University, 31061 Milledgeville, GA, USA
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step of this reaction, a carbohydrate molecule is covalently attached to the side-chain nitrogen of an asparagine residue in the –N–X–T/S– (X≠P) sequence in a nascent protein (Marshall 1972). N-linked glycosylation of proteins is extremely important for many critical biologic
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