6xcu
From Proteopedia
NMR structure of Ost4V23D, a critical mutant of Ost4, in DPC micelles
Structural highlights
FunctionOST4_YEAST Essential subunit of the N-oligosaccharyl transferase (OST) complex which catalyzes the transfer of a high mannose oligosaccharide from a lipid-linked oligosaccharide donor to an asparagine residue within an Asn-X-Ser/Thr consensus motif in nascent polypeptide chains. N-glycosylation occurs cotranslationally and the complex associates with the Sec61 complex at the channel-forming translocon complex that mediates protein translocation across the endoplasmic reticulum (ER). All subunits are required for a maximal enzyme activity. OST4 is required for recruitment of OST3 or OST6 to the OST complex. It is essential for cell growth at 37 but not at 25 degrees Celsius. Publication Abstract from PubMedAsparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multi-subunit enzyme, OST, localized in the endoplasmic reticulum (ER). The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution NMR structures and molecular dynamics simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our molecular dynamics simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix-helix interactions between Ost4V23D and transmembrane (TM)12 and TM13 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. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity. NMR and MD Simulations Reveal the Impact of the V23D Mutation on the Function of Yeast Oligosaccharyltransferase Subunit Ost4.,Chaudhary BP, Zoetewey DL, McCullagh MJ, Mohanty S Glycobiology. 2021 Jan 12. pii: 6090023. doi: 10.1093/glycob/cwab002. PMID:33442744[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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