PDBsum entry 6snh

Membrane protein

PDB id: 6snh

Contents

Protein chains

479 a.a.

225 a.a.

214 a.a.

Ligands

LMH

Waters ×1

References listed in PDB file

Key reference

• Title: Structure and mechanism of the er-Based glucosyltransferase alg6.
• Authors: J.S.Bloch, G.Pesciullesi, J.Boilevin, K.Nosol, R.N.Irobalieva, T.Darbre, M.Aebi, A.A.Kossiakoff, J.L.Reymond, K.P.Locher.
• Ref.: Nature, 2020, 579, 443-447. [DOI no: 10.1038/s41586-020-2044-z]
• PubMed id: 32103179

Abstract

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins¹. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates². The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate^3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.