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Structure and mechanism of the ER-based glucosyltransferase ALG6

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 substrate3,4. 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.

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Fig. 1: Functional analysis of ALG6.
Fig. 2: ALG6–6AG9-Fab interactions.
Fig. 3: ALG6 structure and GT-C architecture.
Fig. 4: Donor substrate binding and catalytic site of ALG6.

Data availability

Atomic coordinates of the apo-ALG6 and Dol25-P-Glc-bound ALG6 models were deposited in the RCSB PDB under accession number 6SNI for apo-ALG6 and 6SNH for Dol25-P-Glc-bound ALG6. The 3D cryo-EM maps were deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-10258 for apo-ALG6 and EMD-10257 for Dol25-P-Glc-bound ALG6.

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Acknowledgements

This research was supported by the Swiss National Science Foundation (SNF) Sinergia programmes TransGlyco (CRSII3_147632) and GlycoStart (CRSII5_173709) to M.A., J.-L.R. and K.P.L, SNF grant 310030B_166672 to K.P.L., as well as by the US National Institutes of Health grant GM117372 to A.A.K. Cryo-EM data were collected at the ScopeM facility at ETH Zürich. We thank the staff of ScopeM for technical support; J. Zürcher for technical support with protein expression and purification; J. Kowal and I. Manolaridis for help with EM data collection; and A. Ramirez and A. Alam for helpful discussions.

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Contributions

K.P.L. and J.S.B. conceived the project. J.S.B. cloned, screened, expressed and purified proteins, reconstituted ALG6 into lipid nanodiscs, performed biochemical experiments and produced Dol25-PP-GlcNAc2Man9 using synthetic precursor substrates. G.P. synthesized Dol25-P-Glc and derivatives. J.B. synthesized Dol25-P-Man and Dol25-PP-GlcNAc2. K.N. and J.S.B. performed synthetic antibody generation. J.S.B. prepared the grids. R.N.I. collected cryo-EM data. J.S.B. assisted cryo-EM data collection, processed data, built the ALG6 models and refined the structures. T.D. and J.-L.R. supervised the synthesis of LLO substrates. A.A.K. provided the chaperone-assisted structure determination phage display pipeline and supervised synthetic antibody generation. M.A. analysed the data and contributed to writing the paper. J.S.B. and K.P.L. analysed the data and wrote the paper.

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Correspondence to Kaspar P. Locher.

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Peer review information Nature thanks Xiaochen Bai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 ALG6 activity in distinct lipidic environments and chemo-enzymatic synthesis of LLO intermediates.

a, Tricine gel-based analysis of the ALG6-catalysed reaction using Dol25-PP-GlcNAc2Man9 and Dol25-P-Glc substrates. The reactions were carried out in distinct detergents or with ALG6 reconstituted in lipid nanodiscs, as indicated above the gel lanes. The glycans were analysed as described in Fig. 1b. b, Tricine gel-based analysis of ALG reaction cascade intermediates, with reactions analysed as described in Fig. 1b. The structures of glycopeptides are schematically shown above the lanes and respective glycosyltransferase enzymes are indicated above the arrows.

Extended Data Fig. 2 Data processing and structure determination of the substrate-free ALG6–6AG9-Fab complex in lipid nanodiscs.

a, Overview of the EM data processing and structure determination pipeline using RELION 3.046. b, Representative cryo-EM micrograph. c, Representative 2D classes. d, A representative 2D class with 6AG9-Fab and the nanodisc belt highlighted. e, Spatial distribution of particles in the final iteration of 3D refinement. f, Refined and B-factor-sharpened EM map, coloured by local resolution estimation as calculated in ResMap52. g, Resolution estimation of the final map via Fourier shell correlation (FSC), as calculated in RELION 3.0. h, The FSC between model and map, as calculated in PHENIX mtriage51.

Extended Data Fig. 3 Molecular interactions at the interface of ALG6 and 6AG9-Fab.

a, Surface representation of ALG6 in light brown and cartoon representation of the variable fragment of 6AG9-Fab coloured red (heavy chain) and black (light chain). The five complementarity-determining region (CDR) loops of 6AG9-Fab interacting with ALG6 are shown in sphere representation and make contact mostly with the ER-luminal ‘arch’ formed by EL4 of ALG6. b, Detailed view of the individual CDR:ALG6 interactions, with 6AG9-Fab CDRs and ALG6 in stick representation and coloured as in a. The CDR sequences are indicated above the panels. Note that CDR1 of the light chain (LC) was not randomized. HC, heavy chain.

Extended Data Fig. 4 Substrate and lipid interactions of ALG6.

a, Distinct lipid binding sites were identified in the structure of nanodisc-reconstituted ALG6, shown in light brown surface representation. Ordered phospholipids and cholesteryl-hemisuccinate molecules are shown in stick representation (green), and the EM density map was contoured at 5 r.m.s.d. and carved to 1.6 Å. b, Electrostatic surface potential of ALG6, with negative charges coloured in red, neutral charges in white, positive charges in blue and bound Dol25-P-Glc depicted in stick representation with carbons in green. The inset shows a zoomed-in view of the kink induced in the dolichol moiety. c, Electrostatic surface potential of ALG6 shown from four angles to indicate potential binding sites for lipid-linked substrates of ALG6 based on the presence of suitable surface cavities. The red solid arrow indicates the binding site of the observed donor substrate Dol25-P-Glc. The red dashed arrows indicate potential binding sites of the acceptor substrate Dol-PP-GlcNAc2Man9. These sites were chosen based on groove-like features in the transmembrane region that might bind dolichol and positively charged patches at the ER-luminal membrane boundary that could help to bind the pyrophosphate moiety of the acceptor LLO. Binding to these sites would allow the terminal mannose of the A-branch of the acceptor substrate to reach the active site. d, Membrane deformation by ALG6 shown from distinct views. The EM map of the apo-ALG6 complex with the 6AG9-Fab complex in a lipid nanodisc is coloured yellow for ALG6, red (heavy chain) and black (light chain) for 6AG9-Fab, and transparent grey for density indicating the lipid nanodisc. Note that the lipid bilayer of the nanodisc surrounding the ALG6 protein is twisted and thinner in the region opposite the arch formed by EL4.

Extended Data Fig. 5 Structural similarity of the conserved module of GT-C enzymes.

a, Cα traces of the first seven transmembrane helices and the first external loop EL1 of selected structures are shown following superposition. These following GT-C family members of known structure were used, with the GT family number indicated: S. cerevisiae (S.c.) PMT1 (GT39, PDB ID 6P25), S.c. ALG6 (GT57, this study), C. lari (C.l.) PglB (GT66, PDB ID 3RCE), A. fulgidus (A.f.) AglB (GT66, PDB ID 3WAJ), S.c. STT3 (GT66, PDB ID 6EZN) and C. metallidurans ArnT (GT83, PDB ID 5F15). b, Close-up view of EL-h1, EL-h2 and the connecting loop of GT-Cs where the catalytic role of the conserved aspartate, shown in stick representation, was experimentally demonstrated.

Extended Data Fig. 6 Residues involved in CDGs caused by ALG6 mutations.

The structure of yeast ALG6 is depicted as a Cα trace and as a transparent surface representation coloured in light brown. The surface is coloured green for residues conserved between yeast and human ALG6. Cα atoms of residues that have been shown to cause CDGs in the human homologue of ALG6 are shown in sphere representation. Residue numbers in brackets indicate the corresponding residues of human ALG6 according to pairwise protein sequence alignment with EMBOSS needle58.

Extended Data Fig. 7 Sequence alignment of ALG6 homologues and orthologues.

a, Alignment of amino acid sequences of yeast and human ALG6 and ALG8 proteins, generated with Clustal Omega57 (Uniprot identifiers: Q12001, Q9Y672, P40351 and Q9BVK2). Secondary structure elements of yeast ALG6 are depicted and labelled above the sequence. The dashed lines indicate regions that are disordered in the ALG6 structures. Cytosolic regions are labelled ‘cyto’ and ER-luminal regions are labelled ‘ER-lumen’. The red dot indicates the catalytic base Asp69, which is a structurally conserved aspartate residue that has been observed or proposed to be the catalytic base in all GT-C enzymes of known structures. The black dots indicate every tenth amino acid in the sequence of S. cerevisiae ALG6. b, Surface representation of yeast ALG6 (light brown), with residues identical to human ALG6 highlighted in green (left) and residues identical to yeast ALG8 highlighted in blue (right).

Extended Data Fig. 8 Data processing and structure determination of the substrate-bound ALG6–6AG9-Fab complex in detergent solution.

a, Overview of the EM data processing and structure determination pipeline using RELION 3.046. b, A representative cryo-EM micrograph. c, Representative 2D classes. d, Spatial distribution of particles in the final iteration of 3D refinement. e, Resolution estimation of the final map via FSC, as calculated in RELION 3.0. f, The final refined and B-factor-sharpened map, coloured by local resolution estimation, as calculated in ResMap52. g, The FSC between model and map, as calculated in PHENIX mtriage51.

Extended Data Fig. 9 Mechanism of ALG6-catalysed glucosyl transfer.

a, Chemical structures of synthetic donor substrate analogues (left) and their functional analysis (right). Compound numbers are indicated in bold and in parentheses. Analysis of ALG6 activity as described in Fig. 1b but in the presence of different substrate analogues as indicated above the lanes. The lane labelled Man9-LLO is a control sample for size comparison. In one of the ALG6 reactions, a pre-incubation of ALG6 with 10 mM EDTA was used to remove any divalent ions from the solution. b, Proposed three-state mechanism of ALG6; intermediate states are numbered and encircled. State 1 represents the apo state of ALG6 based on our EM structure, with ALG6 in surface representation (brown) and clipped for better visualization of the substrate-binding pocket. State 2 represents ALG6 bound to a donor substrate based on our EM structure, with Dol25-P-Glc shown in sphere representation with carbon atoms coloured green and oxygen atoms coloured red. State 3 represents a putative ternary complex based on the apo structure of ALG6, with substrates drawn manually. The inset depicts the proposed reaction mechanism, with Asp69 acting as a general base that deprotonates the C3 hydroxyl group of the attacking mannose moiety.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statisticsa as defined in PHENIX49

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Bloch, J.S., Pesciullesi, G., Boilevin, J. et al. Structure and mechanism of the ER-based glucosyltransferase ALG6. Nature 579, 443–447 (2020). https://doi.org/10.1038/s41586-020-2044-z

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