Oligosaccharyltransferase (OST) is the central enzyme of N-linked protein glycosylation. It catalyzes the transfer of a pre-assembled glycan, GlcNAc₂Man₉Glc₃, from a dolichyl-pyrophosphate donor to acceptor sites in secretory proteins in the lumen of the endoplasmic reticulum. Precise recognition of the fully assembled glycan by OST is essential for the subsequent quality control steps of glycoprotein biosynthesis. However, the molecular basis of the OST-donor glycan interaction is unknown. Here we present cryo-EM structures of S. cerevisiae OST in distinct functional states. Our findings reveal that the terminal glucoses (Glc₃) of a chemo-enzymatically generated donor glycan analog bind to a pocket formed by the non-catalytic subunits WBP1 and OST2. We further find that binding either donor or acceptor substrate leads to distinct primed states of OST, where subsequent binding of the other substrate triggers conformational changes required for catalysis. This alternate priming allows OST to efficiently process closely spaced N-glycosylation sites.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Ramírez AS, de Capitani M, Pesciullesi G, Kowal J, Bloch JS, Irobalieva RN, Reymond JL, Aebi M, Locher KP

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