Professor Oklahoma State University Stillwater, Oklahoma, United States
Smita Mohanty (Oklahoma State University)| Bharat Chaudhary (Oklahoma State University)| David Zoetewey (Georgia College and University)| Martin McCullagh (Oklahoma State University)
Oligosaccharyl transferase (OST) is a multi-subunit enzyme that catalyzes the co-translational N-glycosylation of nascent polypeptides in the endoplasmic reticulum (ER). In the case of Saccharomyces cerevisiae, OST is composed of nine non-identical transmembrane protein subunits. In the central step of N-glycosylation, a preassembled oligosaccharide moiety is transferred to the asparagine side chain located in the Asn-X-Ser/Thr consensus sequence of the newly synthesized polypeptide chain in the lumen of the endoplasmic reticulum (ER). Defects in N-glycosylation pathway can cause disorders known as congenital disorders of glycosylation (CDG) that includes but not limited to mental retardation, developmental delay, dysmorphic features, etc. Complete loss of N-glycosylation is lethal in organisms. Due to the inherent difficulties associated with integral membrane protein studies, the enzymatic mechanism of OST function remains obscure, although prior mutagenesis results indicate that the smallest subunit, Ost4 stabilizes the catalytic subunit, Stt3. The structural and functional characterization of Ost4 subunit and its functionally important mutant, Ost4V23D using NMR and molecular dynamics simulations will be presented. Our data demonstrate that although Ost4 and Ost4V23D have very similar 3D structures, however, the mutation disrupted most of the hydrophobic helix-helix interactions between the mutant protein and TM 12 and TM 13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions.
1. Amit Kumar*, Priscilla Ward, Uma Katre and Smita Mohanty, A Novel Method of Production and Biophysical Characterization of a Mini-Membrane protein, Ost4p: A Subunit of Yeast Oligosaccharyl Transferase, Biopolymers, 97, 499-507, (2012).
2. Bharat P Chaudhary, David L Zoetewey, Martin J McCullagh, and Smita Mohanty, NMR and MD simulations Reveal the Impact of the V23D mutation on the Function of the Yeast Oligosaccharyltransferase Subunit Ost4, 2021, DOI: 10.1093/glycob/cwab002.