6SNI

Cryo-EM structure of nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab

Summary for 6SNI

• Entry DOI: 10.2210/pdb6sni/pdb
• EMDB information: 10258
• Descriptor: Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase, 6AG9-Fab heavy chain, 6AG9-Fab light chain, ... (6 entities in total)
• Functional Keywords: glycosyltransferase, glucosyltransferase, gt-c, n-glycosylation, membrane protein
• Biological source: Saccharomyces cerevisiae (Baker's yeast); More
• Total number of polymer chains: 3
• Total formula weight: 118157.51

Authors

Bloch, J.S.,Pesciullesi, G.,Boilevin, J.,Nosol, K.,Irobalieva, R.N.,Darbre, T.,Aebi, M.,Kossiakoff, A.A.,Reymond, J.L.,Locher, K.P. (deposition date: 2019-08-24, release date: 2020-03-11, Last modification date: 2024-10-16)

Primary citation

Bloch, J.S.,Pesciullesi, G.,Boilevin, J.,Nosol, K.,Irobalieva, R.N.,Darbre, T.,Aebi, M.,Kossiakoff, A.A.,Reymond, J.L.,Locher, K.P.
Structure and mechanism of the ER-based glucosyltransferase ALG6.
Nature, 579:443-447, 2020

PubMed Abstract: In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates. 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. 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.

PubMed: 32103179
DOI: 10.1038/s41586-020-2044-z

Experimental method

ELECTRON MICROSCOPY (3 Å)