Structure and mechanism of the ER-based glucosyltransferase ALG6

Article abstract of DOI:10.1038/s41586-020-2044-z

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?? 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?? 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.

Full text of DOI:10.1038/s41586-020-2044-z

Article
Structure and mechanism of the ER-based
glucosyltransferase ALG6
1
2
2
1,3
1
https://doi.org/10.1038/s41586-020-2044-z
Received: 26 August 2019
Joël S. Bloch , Giorgio Pesciullesi , Jérémy Boilevin , Kamil Nosol , Rossitza N. Irobalieva ,
2 4 3 2
Tamis Darbre , Markus Aebi , Anthony A. Kossiakoff , Jean-Louis Reymond &
ꢀ✉
1
Kaspar P. Locher
Accepted: 6 January 2020
Published online: xx xx xxxx
Check for updates
In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the
biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer
1
onto acceptor proteins . The ꢀnal seven steps occur in the lumen of the endoplasmic
reticulum (ER) and require dolichylphosphate-activated mannose and glucose as
2
donor substrates . The responsible enzymes—ALG3, ALG9, ALG12, ALG6, ALG8 and
ALG10—are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely
deꢀned as containing membrane-spanning helices and processing an isoprenoid-
3,4
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
puriꢀed 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 identiꢀed a catalytic aspartate residue that
probably acts as a general base. This residue is conserved in the GT-C superfamily. Our
results deꢀne the architecture of ER-luminal GT-C enzymes and provide a structural
basis for understanding their catalytic mechanisms.
In the eukaryotic cell, N-glycosylation of secretory proteins is an
essential process that involves the transfer of a high-mannose gly-
In vitro activity of purified yeast ALG6
can (GlcNAc Man Glc ) from a dolichylpyrophosphate (Dol-PP) car- Neither the donor nor the acceptor substrate of ALG6 is commercially
2
9
3
1
rier, catalysed by oligosaccharyltransferase . The biosynthesis of available. To generate the acceptor substrate, we started from our previ-
this donor substrate is a sequential process that is initiated in the ously developed method for chemo-enzymatic synthesis and analysis
cytoplasm, requires the flipping of the GlcNAc Man -containing of Dol25-PP-GlcNAc Man , where Dol25 refers to a citronellyl-farnesyl
10
2
5
2
5
intermediate, and is completed on the luminal side of the ER, where moiety. The lipid-linked glycans were analysed by tricine gel analy-
2
the transfer of the final seven hexoses occurs (Fig. 1a). For the cyto- sis following transfer onto a fluorescent acceptor peptide catalysed
plasmic reactions, the donor substrates are soluble, nucleotide- by the eukaryotic, single-subunit oligosaccharyltransferase STT3A
10
activated sugars. By contrast, the ER-luminal reactions depend on the from Trypanosoma brucei (Fig. 1b). To append mannose moieties to
membrane-embedded donor substrates dolichylphosphate-mannose Dol25-PP-GlcNAc Man , we heterologously expressed and purified the
2
5
2
(
Dol-P-Man) and dolichylphosphate-glucose (Dol-P-Glc) . ALG6 membrane-integral enzymes ALG3, ALG9 and ALG12 (Supplementary
transfers the first of three glucose moieties onto the pre-assembled Fig. 1a) and synthesized the required mannose donor analogue Dol25-
5
6
GlcNAc Man glycan. Following the identification and cloning of the P-Man (Fig. 1c). Using a stepwise procedure, we extended Dol25-PP-
2
9
ALG6 locus, it was shown that deficiencies in ALG6 are a frequent cause GlcNAc Man to the ALG6 acceptor substrate Dol25-PP-GlcNAc Man
2
5
2
9
7
of congenital disorders of glycosylation (CDGs) , in which patients (Fig. 1b). We also synthesized the required donor substrate Dol25-P-Glc
8,9
generally have hypo-glycosylated serum glycoproteins . To under- de novo (Fig. 1c). Incubation of ALG6 with Dol25-P-Glc and Dol25-PP-
stand the mechanism of ALG6, we sought to obtain high-resolution GlcNAc Man led to complete conversion to Dol25-PP-GlcNAc Man Glc
2
9
2
9
structural insight.
(Fig. 1b). To confirm the physiologically relevant glycan structure, the
1
2
3
Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland. Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland. Department of Biochemistry
4
✉
and Molecular Biology, University of Chicago, Chicago, IL, USA. Institute of Microbiology, ETH Zürich, Zürich, Switzerland. e-mail: locher@mol.biol.ethz.ch
Nature | www.nature.com | 1

Article
a
b
ALG3
ALG9
ALG8
ALG10
ALG12
ALG9
ALG6
P
OST
CWH8
P
P
P
RFT1?
P
P
P
P
P
P
P
P
NXT/S
4×P
2× P
ER lumen
P
P
5× GDP
P
P
P
P
P
Dolichol
Glc
Fluorescent
peptide
ALG7
ALG13/14
(recycling)
P
Phosphate
GlcNAc
Man
ALG1
ALG2
ALG11
One pot
Hydrolysed
Man
α-1,2-mannosidase
c
1
. H , PtO2
2
R1
1. ClPO(OPh)2 AcO
R1
2. R3-OH
CCl3CN
HO
R1
AcO
R
3=
O
DMAP
O
O
AcO
AcO
OPh
HO
HO
O
O
O
R3
AcO
AcO
OH
P
P
R2
R2
OPh
3. MeOH
NH4OH
R2
O– NH4+
O
O
1
2
R1 = OAc, R2 = H (Man)
R1 = H, R2 = OAc (Glc)
3
4
R1 = OAc, R2 = H
R1 = H, R2 = OAc
5
6
R1 = OH, R2 = H
R1 = H, R2 = OH
Fig. 1 | Functional analysis of ALG6. a, Schematic of eukaryotic LLO
biosynthesis and glycan transfer by oligosaccharyltransferase (OST)
above the lanes. Note that α-1,2-mannosidase is unable to remove mannoses
from the A-branch of GlcNAc Man Glc, demonstrating the presence of a
2
,36
.
2
9
Enzyme names are indicated above the reaction arrows. The curved arrows
across the ER membrane indicate flipping reactions. b, Tricine gel-based
glucose moiety. c, Synthesis scheme of Dol-P-hexose analogues including
Dol25-P-Man (5) and Dol25-P-Glc (6). Note that the citronellyl-farnesyl moiety
of Dol25 faithfully reflects the stereochemistry of native dolichol. DMAP,
4-dimethylaminopyridine.
analysis of chemo-enzymatic LLO extension and of ALG6-catalysed glucose
transfer. Glycans were transferred onto a fluorescent peptide using purified
1
1
T. brucei STT3A enzyme . The glycan structures are indicated schematically
peptide-attached glycans were incubated with α-1,2-mannosidase, Fig. 2) aided particle alignment, and the electron microscopy (EM)
which showed that ALG6-catalysed addition of glucose protected the reconstruction of nanodisc-reconstituted apo-ALG6 bound to 6AG9-
A-branch of the Man -containing glycan from mannosidase activity Fab had an overall resolution of 3.0 Å (Fig. 2c, Extended Data Fig. 2,
9
(Fig. 1b). ALG6 was found to be active in different detergents as well as Extended Data Table 1, Supplementary Fig. 3). This allowed us to build
reconstituted in lipid nanodiscs (Extended Data Fig. 1a). an atomic model of ALG6 that lacked only 37 residues at the presum-
Notably, our chemo-enzymatic approach enabled us to synthesize ably flexible N terminus and 28 residues in the cytoplasmic loop that
every lipid-linked oligosaccharide (LLO) intermediate of the biosyn- connects transmembrane helix 12 (TM12) and TM13, a segment that
thetic pathway (Extended Data Fig. 1b). Our LLO analogues are readily contains several short linear motifs including a di-arginine ER-retention
11
14
accepted by both the single-subunit OST enzyme from T. brucei and motif . 6AG9-Fab bound to a conformational, ER-luminal epitope of
1
2
the octameric yeast OST enzyme .
ALG6 and all interacting complementarity-determining region loops
of the Fab fragment were well resolved (Extended Data Fig. 3). The EM
maps revealed bound lipids and cholesterol hemisuccinate molecules
in contact with transmembrane helices of ALG6 (Extended Data Fig. 4a).
Determination of the structure of ALG6
We used phage display and a synthetic library of antigen-binding
1
3
Fab fragments to identify a conformational binder (6AG9-Fab) that
increased the size and thermostability of detergent-solubilized ALG6
ALG6 topology and GT-C architecture
(Fig. 2a, Supplementary Fig. 2a, b) but did not interfere with its catalytic ALG6 has 14 transmembrane helices and two long loops (EL1 and EL4)
activity (Fig. 2b). The increased particle mass and size (Extended Data that form helices in the ER lumen (Fig. 3a–c). A disulfide bridge cova-
lently links the ER-luminal end of TM14 with the external loop EL4 that
connects TM7 and TM8 (Fig. 3b, c). While the overall fold and membrane
topology of ALG6 are unlike those of previously described structures,
we noticed that the transmembrane arrangement and topology of
the first half of the protein resemble partial structures of previously
reported GT-C enzymes. We superimposed the structure of ALG6, which
a
b
c
ALG6
ALG6 +
6AG9-Fab
6AG9-
Fab
4
2
0
0
0
1
5
belongs to the GT57 subfamily according to the CAZy database , onto
Void
16
17
12,18
19
ALG6
those of PglB (GT 66), AglB (GT66), STT3
(GT66), ArnT (GT83)
20
and PMT1/PMT2 (GT39). We found that the first seven transmembrane
helices (including the luminal helices EL1-h1 and EL1-h2) are struc-
turally similar, whereas the remainder of the proteins differ in fold,
topology and number of transmembrane helices (Fig. 3d, Extended
Data Fig. 5). This suggests that GT-C enzymes contain a structurally
conserved, N-terminal module consisting of TM1–TM7, and a structur-
ally variable C-terminal module (Fig. 3b–d). The structural similarity
of the conserved module is not reflected in sequence conservation,
10
15
Retention time (min)
Fig. 2 | ALG6–6AG9-Fab interactions. a, Size-exclusion chromatography of
purified ALG6 in detergent (64 kDa) in the presence and absence of 6AG9-
Fab (49 kDa). b, Analysis of ALG6 activity as described in Fig. 1b but in the
presence of 6AG9-Fab. 6AG9-Fab was incubated with ALG6 at a 5-fold or 15-fold
molar excess to test inhibition. The lane labelled Man9-LLO is a control sample
for size comparison. c, Refined and B-factor-sharpened EM map of nanodisc-
reconstituted ALG6 bound to 6AG9-Fab coloured by proximity to protein
chains of ALG6 (light brown), 6AG9-Fab heavy chain (red) and 6AG9-Fab light
chain (grey).
2
1
as the primary sequences of the compared proteins are dissimilar .
However, the first external loop contains the catalytically essential
aspartate residue Asp69 (see below).
2
| Nature | www.nature.com

Fig. 3 | ALG6 structure and GT-C architecture. a, Cartoon representation of
ALG6 in rainbow colouring (blue at the N terminus and red at the C terminus).
b, Schematic representation of ALG6 topology, with transmembrane and
external loop helices numbered and coloured as in a. The pink dot indicates the
proposed catalytic residue Asp69. The yellow bar depicts a disulfide bond. aa,
amino acids. c, Top view of the schematic representation of ALG6 coloured as in
b, with numbered circles depicting transmembrane helices and cylinders
depicting helices in the ER-luminal loops. d, Comparison of the topologies of
ALG6 and other selected GT-C enzymes of known structure. Ribbon diagrams
of the top views of the structures are shown as in c. The constant GT-C modules
are shaded grey, whereas the variable GT-C modules are shaded in different
colours for each structure. Transmembrane helices are numbered. Note that
GT39 and GT66 contain similar variable modules (shaded green). The pink
asterisks indicate the locations of the catalytic sites. The red dashed arrows
indicate the entry site for the dolichol-linked donor substrates, which are
depicted using standard glycobiology symbols.
Our interpretation of a modular architecture is supported by the these cavities are not only highly conserved among ALG6 homologues
2,26
observation that in all published structures of GT-C enzymes, the active but are also in the functionally related glucosyltrasferase ALG8 , sug-
site and the substrate-binding cavities are located at the interface of the gesting that they are involved in substrate binding or form the active site
conserved and variable modules (pink asterisks and red dashed arrows of the enzyme (Extended Data Fig. 7). To investigate the roles of these
in Fig. 3d). The architectural similarity further extends to how GT-Cs cavities, we collected a second cryo-EM data set for the ALG6–6AG9-
bind to their dolichyl(pyro)phosphate-linked donor substrates: the Fab complex (Extended Data Fig. 8, Extended Data Table 1). Here, ALG6
dolichol moiety invariably interacts with TM6 of the conserved GT-C was solubilized in digitonin and pre-incubated with the synthetic donor
module, whereas the distinct attached carbohydrates, which include substrate Dol25-P-Glc. Although the overall resolution of the structure
single sugars or complex glycans, interact with the variable module. The is 3.9 Å and thus lower than that of nanodisc-reconstituted apo-ALG6,
donor substrates reach their respective active sites by ‘diving under’ there is a well-resolved density for bound donor substrate in a lipid-
arch-like structures formed by the external (or ER-luminal) loops that exposed groove formed by TM6, TM7 and TM8 (Fig. 4a, b, Extended
either link the conserved to the variable modules, or are part of the Data Fig. 4b). The shape of the density can clearly accommodate a
variable modules (Fig. 3d). These loops contain, at the minimum, a Dol25-P-Glc molecule. The citronellyl-farnesyl moiety is located in the
helix running parallel to the membrane but may contain entire domains conserved, lipid-facing groove and interacts mostly with hydrophobic
(
Fig. 3d). We conclude that the conserved module of GT-Cs probably residues from TM6. The loop EL4 forms an arch above the groove on
served as a platform that allowed distinct functionalities to evolve, the ER-luminal side, resulting in a funnel-like entrance, and the Dol25
and that the modularity has allowed GT-C enzymes to accommodate moiety bends under this arch at the level of the membrane boundary.
different donor and acceptor substrates and to generate diverse active This causes the phosphate group, which represents the leaving group
sites for distinct catalytic mechanisms during glycan transfer reactions. of the glucose transfer reaction, to be lodged in a slightly positively
An analysis of residues that have been reported to be implicated in charged surface region of ALG6 (Extended Data Fig. 4b).
7,22–25
ALG6-associated human CDGs
revealed that none of them point
The EM density revealed a feature large enough to accommodate
directly at the presumed active site. Rather, they are second shell residues the glucose moiety of Dol25-P-Glc, but the quality of the map does not
with respect to the active site or are located even further away (Extended allow exact positioning. This is in part due to the fact that this density
Data Fig. 6). Such mutations do not fully abolish ALG6 function, but rather also covers the adjacent side chain of the conserved histidine residue
22
reduce the enzymatic activity to a level that is not yet lethal .
His378 (Fig. 4a, b). We could nevertheless assign the orientation of the
glucose moiety, with one face packing against the surface of ALG6 and
the other pointing towards the solvent. We built the model such that
the α-anomeric position of the C1 carbon is accessible for a nucleophilic
Structure of Dol25-P-Glc-bound ALG6
ALG6 forms a large, hydrophilic cavity facing the ER lumen and a attack by the mannose C3 hydroxyl group of the acceptor glycan. This
groove-shaped cavity facing the lipid bilayer. The residues that line binding mode can ensure the specificity of ALG6 for Dol-P-Glc over
Nature | www.nature.com | 3

Article
Dol-P-Man, as the axial C2 hydroxyl group of a mannose would cause
a steric clash with the enzyme surface (Fig. 4a). The specificity of ALG6
can be demonstrated in vitro, where only glucose, but not mannose, is
transferred from a Dol25-P carrier (Extended Data Fig. 9a).
a
b
TM11
E379
TM9
TM8
We could not unambiguously identify a second dolichol-binding
groove in the ALG6 structure, and multiple smaller grooves are poten-
tial candidates for binding the acceptor substrate Dol-PP-GlcNAc Man
ER
D307
Dol25-P-Glc
E306
2
9
TM10
(
Extended Data Fig. 4c). Because several of the transmembrane helices
TM3b
H378
of ALG6 are not perpendicular to the lipid bilayer, the protein appears
to induce a distortion of the lipid bilayer of the nanodisc, with the lipid
bilayer appearing to thin out at the side of the cavity opposite to the
TM6
EL4
D69
TM4
D99
‘arch’ generated by EL4 (Extended Data Fig. 4d). Such membrane thin-
TM5
ning might facilitate the binding of the dolichol-linked donor substrate
from the membrane. Deformation of a membrane caused by a protein
has been reported previously in the TMEM16 scramblase, where it has
4
0°
2
0°
c
27
been proposed to facilitate or assist in lipid flipping .
P
E
Catalytic mechanism
GiventhatALG6isaninvertingglycosyltransferase,itsreactionisthought
E =
P =
C1
2
8
to occur via an S 2 mechanism . This implies that the C3 hydroxyl
N
Fig. 4 | Donor substrate binding and catalytic site of ALG6. a, Surface
group of the terminal A-branch mannose of Dol-PP-GlcNAc Man9
2
representation of substrate-bound ALG6 in light brown with an EM density map
for Dol25-P-Glc in the blue mesh carved to 3.0 Å, with bound Dol25-P-Glc in
stick representation. The front of ALG6 was clipped for clarity. The inset shows
a zoomed-in view. The red dashed arrow indicates the anomeric C1 of the
glucose moiety. An ordered water molecule close to the C1 carbon is shown as a
red sphere. b, Structure of the active site, with ALG6 shown in light brown
ribbon representation with EL4 coloured in light blue. Acidic residues in
the active site are shown in stick representation and labelled, and bound
Dol25-P-Glc is depicted in stick representation, with carbon atoms coloured
green. c, Analysis of substrate conversion by ALG6 variants, with mutations
has to be activated for a nucleophilic attack on the anomeric C1 of
the glucose moiety to form an α-1-3-glycosidic bond. This is gener-
ally achieved by deprotonation of the attacking hydroxyl group by
an aspartate or glutamate side chain acting as a general base, either
directly or by forming catalytic dyads or triads with suitable groups
such as imidazole rings (histidine), hydroxyl groups of protein side
2
9–31
chains or ordered water molecules
. In our Dol25-P-Glc-bound
ALG6 structure, we identified five acidic residues in external loops
that could act as a general base (Fig. 4b): Asp69 and Asp99 from EL1,
Glu306 and Asp307 from EL4 and Glu379 from EL5. We mutated all indicated above the lanes. WT, wild type.
five residues to alanine and, in the case of aspartates or glutamates,
to asparagine or glutamine. We also mutated His378, which is in direct
contact with the glucose moiety, to investigate its potential function moiety carried distinct substituents that were originally designed for
in substrate binding. We expressed and purified the resulting ALG6 labelling studies (Extended Data Fig. 9a). We found that substituting
variants (Supplementary Fig. 1b, c) and tested their in vitro activity the C4 hydroxyl group with a 2-azido-N-ethylacetamido group abol-
using our coupled assay (Fig. 4c). Activity was unaffected by muta- ished glucose transfer, which can be rationalized by the structural data
tion of Glu306, Asp307 and Glu379. Mutating Asp99 to an alanine as this would lead to a clash with the side chain of His378 (Fig. 4b). Sub-
abolished ALG6 function, whereas mutation to an asparagine did not stituting the C6 hydroxyl group of glucose with an azido group did not
reduce enzymatic activity. This suggests that Asp99 has a functional impair glucosyl transfer, which is in line with the structural data given
role, possibly in acceptor substrate binding, but is not the essential that this hydroxyl group is facing the solvent and can probably rotate
catalytic base. Finally, mutating Asp69 to an alanine abolished ALG6 freely (Fig. 4a). ALG6 activity was abolished when a larger substituent
function, and mutating it to an asparagine strongly reduced activity. (tetraethylene glycol (PEG )-linked rhodamine 110) replaced the C6
4
We conclude that the structurally conserved Asp69 is the catalytic hydroxyl group (Extended Data Fig. 9a), where it probably hinders
base in ALG6. The corresponding aspartates (Extended Data Fig. 5b) access of the acceptor substrate to the active site (Fig. 4a, Extended
have been demonstrated to be catalytically essential in oligosacchar- Data Fig. 4b).
1
2,16,17
19
yltransferases
and the arabinosyltransferase ArnT and proposed
On the basis of our structural and functional data, we propose a
2
0
to be essential in the mannosyltransferase PMT1–PMT2 . Hence, an three-state mechanism for ALG6 function (Extended Data Fig. 9b).
aspartate or glutamate at the N-terminal end of the helix EL-h1 may The structure of substrate-bound ALG6 suggests that Dol-P-Glc binds
be catalytically essential in all GT-C enzymes. Mutating His378, which before the Man -containing acceptor substrate, because the glucose
9
is in the immediate vicinity of the glucose moiety of Dol25-P-Glc, to moiety is at the bottom of the active site cavity. We therefore propose
alanine, asparagine or glutamine, strongly reduced the activity of that donor and acceptor substrates bind sequentially and that Asp69
ALG6 but did not fully abolish it. Given the location of this residue, acts as a general base that abstracts the proton of the 3-hydroxyl group
it is most likely to be involved in the binding and orientation of the of the terminal A-branch mannose of the acceptor substrate to activate
donor substrate. Although it has been shown that at least one of the ER- it for a nucleophilic attack. This may require a conformational change
luminal mannose transfer reactions of the LLO biosynthesis pathway during which the glucose moiety of the donor substrate moves closer
32
is metal-dependent , our EM map showed no evidence for a bound to the acceptor substrate and to the catalytic residue Asp69, akin to
2
+
divalent metal ion (M ), nor does ALG6 contain a DXD sequence motif induced fit. An analogous conformational change has been described
2
+
33
similar to those shown to be essential for M binding in oligosaccharyl- in another GT-C enzyme: the bacterial oligosaccharyltransferase PglB .
1
2,16,20
transferase
of EDTA (Extended Data Fig. 9a), demonstrating that the reaction is futile donor substrate hydrolysis. Upon formation of a ternary complex,
metal ion-independent. the glucose moiety may not only become better ordered but might also
To probe the reaction mechanism further, we tested whether ALG6 deviate from the chair conformation, as has been previously observed
. Purified ALG6 indeed retains activity in the presence In ALG6, an induced fit mechanism would allow the enzyme to prevent
3
4,35
could process synthetic Dol25-P-Glc analogues in which the glucose in other glycosyltransferases and glycosyl hydrolases
.
4
| Nature | www.nature.com

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© The Author(s), under exclusive licence to Springer Nature Limited 2020
Nature | www.nature.com | 5

Article
Methods
NaCl and 40 mM HEPES pH 7.4). To remove empty nanodiscs, the recon-
stitution mixture was incubated with anti-Flag antibody-coupled resin
for 1 h. After washing the resin with 2 × 10 cv. washing buffer, the sample
Overexpression and purification of ALG3
A synthetic gene (GenScript) of full-length ALG3-PreScission-EYFP- was eluted by incubation with 3C-protease.
D4 from Saccharomyces cerevisiae (UniProtKB: P38179) was opti-
1
mized for expression in Spodoptera frugiperda (GeneArt, Thermo Expression and purification of EYFP
Fisher). The gene was cloned into a pOET1 vector (Oxford Expres- His6-EYFP-1D4 was cloned into a modified pET19b vector (Novagen) and
sion Technologies) and was expressed in S. frugiperda (Sf9) cells overexpressed in BL21 gold cells (DE3) (Stratagene). Cells were grown
transfected with baculovirus that was generated with FlashBAC DNA in Terrific Broth medium at 37ꢁ°C to an OD600 of 3.0 before induction
(
Oxford Expression Technologies). Cells were cultured in serum- with 1 mM isopropyl β-ꢂ-1-thiogalactopyranoside (IPTG). For protein
free SF-4 Baculo Express ICM medium (AMIMED) at 27ꢁ°C. Cells were expression, cells were then grown overnight at 25ꢁ°C and harvested by
6
transfected at a density of 1 × 10 cells per ml and were harvested centrifugation at 10,000g.
after 3 days. For purification, the cells were resuspended in 150 mM
For purification, cells were resuspended in PBS (Gibco) supple-
NaCl, 50 mM HEPES pH 7.4 with 0.1 mg/ml DNase I, 1:100 protease mented with 1 mM PMSF and 0.025 mg/ml DNase I. Cells were lysed
inhibitor cocktail (Sigma), 0.1 mg/ml PMSF and were lysed by dounce by three cycles of sonication (50% amplitude and 50% duty cycle).
homogenization before solubilization by addition of 1% n-dodecyl- Cell debris was pelleted by ultracentrifugation at 35,000 rpm, in a
β-ꢂ-maltopyranosinde (DDM; Antrace), 0.2% cholesteryl hemisuc- type T45 Ti rotor, for 30 min. The supernatant was incubated with
cinate (CHS; Anatrace) and 10% glycerol. After 1 h of solubilization, Ni-NTA resin for 1 h and was subsequently washed with 10 cv. 150 mM
cell debris was pelleted by centrifugation at 30,000 rpm in a type NaCl, 50 mM Tris-HCl pH 7.5 and 10 cv. 300 mM NaCl, 40 mM Tris-
T45 Ti rotor (Beckmann). The supernatant was added to anti-1D4 HCl pH 8.0 and 50 mM imidazole, before elution with 5 cv. 300 mM
antibody-coupled beads and was incubated for 1 h. The beads then were NaCl, 40 mM Tris-HCl pH 8.0 and 400 mM imidazole. Immediately
washed with 2 × 20 column volumes (cv.) of washing buffer (150 mM afterwards, the protein buffer was exchanged into 150 mM NaCl and
NaCl, 40 mM HEPES pH 7.4, 0.03% DDM and 0.006% CHS). Then the 50 mM Tris-HCl pH 7.5.
protein was eluted by incubation of washing buffer, supplemented
with 0.5 mg/ml 1D4 peptide for 1 h.
Enzymatic biotinylation of MSP1D1
MSP1D1 fused to a C-terminal Avitag was biotinylated in biotinylation
buffer (75 mM NaCl, 50 mM bicine pH 8.3, 10 mM Mg acetate, 10 mM
Overexpression and purification of ALG6, ALG9 and ALG12
Synthetic genes (GenScript) of Flag-EYFP-PreScission-full length ALG6 ATP and 0.25 mM biotin) with 2 µM BirA protein, overnight at 4ꢁ°C. 3C
UniProtKB: Q12001), ALG9 (UniProtKB: P53868) or ALG12 (UniProtKB: protease (2.5 µM) was added for 6 h at 4ꢁ°C. 3C-protease and BirA were
(
P53730) from S. cerevisiae were optimized for expression in Homo removed by reverse binding to Ni-NTA. Subsequently, the biotinylated
sapiens (GeneArt, Thermo Fisher). Proteins were expressed, and cells MSP1D1 protein was desalted into 150 mM NaCl, 50 mM HEPES pH 7.4
were lysed, solubilized and pelleted as described above for ALG3. The and subsequently used for nanodisc reconstitution as described above.
supernatant was added to anti-Flag antibody-coupled resin and was
incubated for 1 h. The beads then were washed with 2 × 10 cv. washing Synthetic antibody generation
buffer (150 mM NaCl, 40 mM HEPES pH 7.4, 0.03% DDM and 0.006% For phage display experiments, enzymatically biotinylated MSP1D1
CHS). Then the protein was eluted by incubation of washing buffer, sup- was used for the nanodisc reconstitution and no reverse binding or
plemented with 0.2 mg/ml Flag peptide for 1 h. For functional assays, cleavage after nanodisc reconstitution was performed. Aggregates
ALG6 was eluted with washing buffer supplemented with 3C-protease. were removed by SEC after the nanodisc reconstitution.
For the substrate-bound structure, the immobilized ALG6 was solu-
To validate the immobilization efficiency of the final sample, pull-
bilized, immobilized to Flag resin and washed as described above. down assays of the target proteins immobilized on streptavidin Mag-
Subsequently the immobilized protein was washed with 10 cv. washing neSphere paramagnetic particles (Promega) were performed, followed
13,38
buffer supplemented with 0.1% digitonin (Huberlab) and with 2 × 10 cv. by biopanning using Fab Library E ; DNA was kindly provided by S.
digitonin washing buffer (150 mM NaCl, 40 mM HEPES pH 7.4 and Koide (University of Rochester Medical Center, Rochester, NY, USA)
0.1% digitonin). The sample was eluted with 3C-protease and was sub- and the phage library was prepared by S. Mukherjee (University of
sequently purified by size-exclusion chromatography (SEC) in the Chicago, Chicago, IL, USA). Five subsequent rounds of selection were
same buffer.
performed in 25 mM HEPES pH 7.4 and 150 mM NaCl (selection buffer)
39,40
supplemented with 1% bovine serum albumin (BSA)
.
ALG6-mutant generation
The phage display selection was performed according to pub-
39,41
ALG6 mutants were generated by site-directed mutagenesis PCR and lished protocols . In brief, in the first round, 400 nM Flag-EYFP-
were expressed and purified by immobilization to Flag-resin and elu- Prescission-ALG6 in nanodiscs was immobilized onto paramagnetic
tion with peptide as describe above for wild-type ALG6. The proteins particles for biopanning, followed by three washes and resuspension
were further purified by SEC.
of the particles directly in log-phase Escherichia coli XL-1 blue cells
for infection. Phages were amplified in 2×YT media supplemented
with ampicillin (100 µg/ml) and M13-KO7 helper phage (10 p.f.u./ml).
9
Nanodisc reconstitution of ALG6
A mixture of yeast polar lipids (Yeast Extract Polar, Avanti Polar Lipids) From the second to the fifth round, sorting was performed using a
and CHS (80:20, w/w) was diluted to a concentration of 10 mM in buffer Kingfisher magnetic beads handler (Thermo Fisher) with decreas-
(
150 mM NaCl and 40 mM HEPES pH 7.4). Lipids were solubilized by ing concentration of target protein: 200 nM for the second round,
addition of 10 mM DDM and sonicated for 20 min. Purified EYFP-Pre- 100 nM for the third round, 60 nM for the fourth round and 30 nM
Scission-ALG6 in DDM:CHS-supplemented buffer, purified MSP1D1 for the last round. Phage elution was done by disrupting immobilized
37
protein and solubilized lipids were mixed in a ratio of 1:6:390. After nanodiscs with 1% Fos-choline 12 in selection buffer. In addition,
incubation for 10 min at 4ꢁ°C, the mixture was incubated for 20 min for rounds 2–5, each phage pool was negatively selected against
at room temperature. Detergent was removed by addition of 0.8 mg streptavidin-coated particles and then used as an input for the next
activated Biobeads (Bio-Rad) per ml of reconstitution mixture and round. Throughout the entire selection process, soluble competitors
subsequent incubation at 4ꢁ°C overnight. Biobeads were removed and were included in excess: 2 µM non-biotinytaled empty nanodiscs
the reconstitution mixture was 10× diluted in washing buffer (150 mM (same reconstitution procedure as for ALG6, without the addition

of ALG6 and using non-biotinylated MSP1D1) and 2 µM purified EYFP
protein. Phage pools from the fourth and fifth round were used for EM data collection
binder screening.
Data were recorded on a Titan Krios electron microscope (Thermo Fisher
Scientific) operated at 300 kV, equipped with a Gatan BioQuantum 1967
filter with a slit width of 20 eV and a Gatan K3 camera. Movies were col-
Single-point phage ELISA was performed to screen and validate lected semi-automatically using SerialEM at a nominal magnification
Synthetic antibody screening
44
39
the binding affinities of selected binders . Briefly, E. coli XL-1 blue of 105,000 and a pixel size of 0.42 Å per pixel in super-resolution mode.
were transfected with selected phage pools and plated on LB/agar The defocus range was −0.6 to −2.8 µm. Each movie contained 40 images
2
plates with ampicillin. Colonies were used to inoculate 2×YT media per stack with a dose per frame of 2.3 electrons/Å .
9
with 100 µg/ml ampicillin and 10 p.f.u./ml M13-KO7 helper phage.
Overnight amplified cultures were pelleted by centrifugation and EM data processing, model building and refinement
tenfold diluted supernatants were used for ELISA. For this purpose, For the apo structure of ALG6 in nanodiscs, 6,801 movies were cor-
45
9
6-well plates coated with 2 µg/ml NeutrAvidin (Thermo Fisher) rected for beam-induced motion using MotionCor2 . The micro-
were blocked with selection buffer with 1% BSA, and Flag-EYFP- graphs were visually inspected and 5,143 were used for further
4
6
Prescission-ALG6 protein reconstituted in biotinylated nanodiscs processing in RELION 3.0 . Contrast transfer function (CTF) was
47
(
50 nM) was immobilized in each well. Subsequent ELISA assays were estimated using gCTF . Particles (n = 3,437,475) were auto-picked
performed in selection buffer with 2% BSA. Diluted phage particles and extracted with threefold binning to a pixel size of 2.52 Å/pixel.
were assayed against the target protein by using an HRP-conjugated After two subsequent rounds of 2D classification, 445,702 particles
anti-M13 monoclonal antibody (GE Healthcare). Detection was done were used to generate an initial model. The particles were subjected
with TMB substrate (Thermo Fisher) quenched with 1.0 M HCl after to two subsequent rounds of 3D classification and the remaining
3
min and subsequent absorbance measurement at 450 nm. Bioti- 171,764 particles were subjected to 3D refinement. Subsequently,
nylated empty nanodiscs were used as a control and only target- the particles were re-extracted at 0.84 Å/pixel and subjected to 3D
specific binders were selected. refinement yielding a map at 3.2 Å resolution. Subsequent particle
Selected binders were sequenced at the University of Chicago Compre- polishing and 3D refinement lead to a map at 3.1 Å resolution. Appli-
hensive Cancer Center DNA Sequencing facility and cloned into a pRH2.2 cation of a tight mask that masked out most of the flexible constant
vector (kind gift of S. Sidhu (University of Toronto, Toronto, ON, Canada)) domain yielded a map at 3.0 Å resolution.
using the In-Fusion Cloning kit (Takara). The antigen-binding fragments
For the substrate-bound structure in digitonin, 6,187 movies were
45
were expressed in E. coli BL21 gold cells and purified by Protein A chroma- corrected for beam-induced motion using MotionCor2 . The micro-
42
tography and ion-exchange chromatography as described previously . graphs were visually inspected and 4,000 were used for further pro-
46
47
Subsequently, multi-point protein ELISA was performed to estimate cessing in RELION 3.0 . CTF was estimated using gCTF . Particles
the binding affinity. ELISA plate preparation and target immobilization (n = 995,878) were auto-picked and extracted with threefold binning
were done similar to single-point ELISA, followed by purified Fabs in to a pixel size of 2.52 Å/pixel. After two subsequent rounds of 2D clas-
threefold dilutions (starting from 3 µM) assayed against target protein sification, 287,977 particles were selected and were used to calculate
by HRP-conjugated mouse anti-human IgG F(ab’)2 monoclonal anti- an initial model. After one round of 3D classification, 115,590 parti-
body ( Jackson). Detection was done as described above. Data points cles were selected for one round of 3D refinement and subsequent
were plotted assuming sigmoidal dose response and EC50 values were re-extraction at 0.84 Å/pixel. Subsequent 3D refinement yielded a
calculated for affinity estimation.
final map at 3.9 Å.
Model building was performed in Coot . Most of the Fab, except for
the binding interface, was built based on a pre-existing model at high res-
48
Fab-binding characterization using SEC
For SEC binding analysis, 3C-protease-cleaved ALG6 in DDM:CHS-sup- olution (Protein Data Bank (PDB) ID 5UCB). The structures were refined
4
9
50
plemented buffer was incubated with fourfold molar excess of Fab for in PHENIX and ligand files were generated in PHENIX eLBOW . The cor-
3
51
0 min on ice and subsequently analysed by SEC. Peak fractions were relation between model and map was validated using PHENIX mtriage .
subsequently analysed by SDS–PAGE.
Thermostability experiments were performed as described previ- Figure preparation and data analysis
43
52
ously , with 3C-protease-cleaved ALG6 in DDM:CHS-supplemented Local resolution estimations were calculated in ResMap . Figures were
53
54
55
buffer pre-incubated with threefold molar excess of Fab for 30 min prepared in PyMol and UCSF Chimera and UCSF ChimeraX . Graphs
on ice and by measuring A280 instead of fluorescence during SEC.
were generated in GraphPad Prism 8. Protein sequence alignments were
performed using CLUSTAL W , Clustal Omega and EMBOSS Needle .
56
57
58
EM sample preparation
Figure 3d was generated using the following structures: S. cerevisiae
For the apo structure, ALG6 in MSP1D1 lipid nanodiscs was incubated ALG6 (this study), Campylobacter lari PglB (PDB 3RCE), Cupriavidus
with 6AG9-Fab in 1:2 molar ratio for 1 h at 4ꢁ°C and subsequently purified metallidurans ArnT (PDB 5F15) and S. cerevisiae PMT1 (PDB 6P25).
by SEC. Pooled peak fractions were concentrated to 0.33 mg/ml. Quan-
tifoil holey carbon grids, Cu, R 1.2/1.3, 300 mesh, were glow discharged Chemical synthesis of substrate analogues
for 45 s, 25 mA using a PELCO easiGLOW glow discharger. Sample (4 µl) β-Mannosyl (3) and β-glucosyl (4) triesters were synthesized starting
was applied to the cryo-EM grids and blotted for 1–2 s before plunge from 2,3,4,6-tetracetylated glycosides with an optimized procedure
59
freezing in a liquid ethane–propane mixture with a Vitrobot Mark IV based on a previously described method . Diphenyl-protected glyco-
Thermo Fisher Scientific) operated at 4ꢁ°C and 100% humidity. syl phosphates were readily hydrogenated and coupled to farnesyl-
For the substrate-bound structure, ALG6 in digitonin-supple- citronellol in presence of trichloroacetonitrile. Acetyl deprotection
mented buffer at 7.2 mg/ml was incubated with 1.5-fold excess of gave β-glycosyl phosphoisoprenoids Dol25-P-Man (5) and Dol25-P-
AG9-Fab for 1 h at 4ꢁ°C. Subsequently, the sample was purified Glc (6). To generate potentially clickable analogues of 6, an azide
(
6
6
0
by SEC and peak fractions were pooled and were concentrated to was introduced in the 6 position following known procedure ,
.3 mg/ml. The sample was supplemented with a final concentration and in the 4 position introducing a short linker via O-alkylation of
6
61
of 150 µM Dol25-P-Glc and incubated for 1.5 h before grid preparation. orthogonal-protected glucopyranoside with bromoacetonitrile fol-
Grids were prepared the same way as for the nanodisc sample, but lowed by reduction of the nitrile with BH3•SMe2 (ref. ) and acylation
with a 3.5-s blotting time. of the primary amine. β-Phosphorylation of the azide-bearing lactols
62

Article
63
was performed using diallylphosphoryl chloride to avoid hydro-
Data availability
genation steps. The desired configuration was confirmed by ROESY
(
Supplementary Methods). Diallyl phosphates were deprotected, Atomic coordinates of the apo-ALG6 and Dol25-P-Glc-bound ALG6
converted to the phosphoric acid and coupled with farnesylcitron- models were deposited in the RCSB PDB under accession number 6SNI
1
1
ellol , as described above. More detailed procedures are described for apo-ALG6 and 6SNH for Dol25-P-Glc-bound ALG6. The 3D cryo-EM
in Supplementary Methods.
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.
Synthesis and purification of Dol25-PP-GlcNAc Man9
2
10
Dol25-PP-GlcNAc Man was produced as previously described . Dol25-
2
9
3
7. Denisov, I. G., Grinkova, Y. V., Lazarides, A. A. & Sligar, S. G. Directed self-assembly of
monodisperse phospholipid bilayer nanodiscs with controlled size. J. Am. Chem. Soc.
26, 3477–3487 (2004).
PP-GlcNAc Man (20 µM) and Dol25-P-Man (120 µM) were mixed in
2
5
1
50 mM NaCl, 50 mM HEPES pH 7.4, 0.03% DDM and 0.006% CHS. Puri-
1
fied ALG3 (1.5 µM) was added and the mixture was incubated overnight
at 4ꢁ°C. The reaction was inactivated by heating for 10 min at 95ꢁ°C. After
cooling down to 4ꢁ°C, the reaction was supplemented with 10 mM MgCl2,
and 1.5 µM ALG9 and 1.5 µM ALG12 were added and incubated overnight
at 4ꢁ°C. For purification of Dol25-PP-GlcNAc Man , the reaction was
38. Fellouse, F. A., Wiesmann, C. & Sidhu, S. S. Synthetic antibodies from a four-amino-acid
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2
9
proteins using antibody phage display with nanodiscs. Structure 24, 300–309 (2016).
41. Hornsby, M. et al. A high through-put platform for recombinant antibodies to folded
lyophilized followed by a first extraction with CHCl :MeOH (2:1, v/v)
3
to remove contaminants of smaller LLO species. The remaining pel-
let was then subjected to extraction with CHCl :MeOH:H O (10:10:3,
proteins. Mol. Cell. Proteomics 14, 2833–2847 (2015).
2. Borowska, M. T., Dominik, P. K., Anghel, S. A., Kossiakoff, A. A. & Keenan, R. J. A YidC-like
4
3
2
protein in the archaeal plasma membrane. Structure 23, 1715–1724 (2015).
v/v/v). Subsequently, the extracted substrate was dried under N and
2
43. Hattori, M., Hibbs, R. E. & Gouaux, E. A ꢀluorescence-detection size-exclusion
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was resuspended in 150 mM NaCl and 40 mM HEPES pH 7.4. The yield
10
was quantified as described previously .
4
Note that a similar synthetic route for in vitro synthesis of an LLO
6
4
analogue was previously reported . However, the lipid carrier in that
study was a phytanol moiety instead of dolichol.
improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
6. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure
4
determination. J. Struct. Biol. 180, 519–530 (2012).
In vitro glycosyl transfer assays
Glycosyl transfer reactions for ALG1, ALG2 and ALG11 were per-
formed as previously described . Glycosyl transfer reactions for
ALG3, ALG9 and ALG12 were performed in reaction buffer (150 mM
NaCl, 50 mM HEPES pH 7.4, 0.03% DDM and 0.006% CHS) supple-
mented with 10–20 µM acceptor substrate (starting from purified
47. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12
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(
4
8. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta
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structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
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2
5
addition and heat inactivation of ALG enzymes) and 50–120 µM donor
substrate (Dol25-P-Man). Reactions for ALG9 and ALG12 were sup-
plemented with 10 mM MgCl . Protein concentrations were ranging
2
5
5
from 0.5 to 2 µM. Reactions were carried out overnight at 4ꢁ°C. Reac-
tions were stopped by heating at 98ꢁ°C for 10 min. This ensured that,
during the ALG-mediated glycosyl transfer reactions, no other active
ALG enzymes from previous reactions were present and eliminated
fluorescence from EYFP fusion proteins, which otherwise would
affect subsequent tricine gel analysis.
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analysis. Protein Sci. 27, 14–25 (2018).
6. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-speciꢀic
gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).
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5
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7. Sievers, F. & Higgins, D. G. Clustal Omega for making accurate alignments of many
Reactions for ALG6 were carried out in reaction buffer (150 mM NaCl,
protein sequences. Protein Sci. 27, 135–145 (2018).
58. Needleman, S. B. & Wunsch, C. D. A general method applicable to the search for
similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443–453 (1970).
50 mM HEPES pH 7.4, 0.03% DDM and 0.006% CHS) supplemented with
1
0 µM purified Dol25-PP-GlcNAc Man acceptor substrate, 50 µM of the
2
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9. Sabesan, S. & Neira, S. Synthesis of glycosyl phosphates and azides. Carbohydr. Res. 223,
respective donor substrate and 0.2 µM tag-cleaved ALG6. For reactions in
thepresenceofEDTAorFab, theentirereactionmixturewaspreincubated
for 1 h on ice, before adding donor and acceptor substrates. The reactions
were incubated for 6 h at 10ꢁ°C. Subsequently, the reactions were stopped
by heating at 98ꢁ°C for 10 min. Reactions of ALG6 mutants were carried
out as described above, at 10ꢁ°C for 24 h. All ALG reaction products were
analysed by TbSTT3A-mediated oligosaccharyl-transfer and subsequent
1
69–185 (1992).
60. Maunier, V., Boullanger, P., Lafont, D. & Chevalier, Y. Synthesis and surface-active
properties of amphiphilic 6-aminocarbonyl derivatives of d-glucose. Carbohydr. Res.
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reports on the mannosidase conformational coordinate. Angew. Chem. Int. Edn Engl. 53,
1087–1091 (2014).
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2. Malet, C. & Hindsgaul, O. Versatile functionalization of carbohydrate hydroxyl groups
through their O-cyanomethyl ethers. J. Org. Chem. 61, 4649–4654 (1996).
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synthesis of ADP- and polyprenyl-β-mannopyranosides. Org. Lett. 16, 5628–5631 (2014).
4. Li, S. T. et al. Reconstitution of the lipid-linked oligosaccharide pathway for assembly of
10
tricinegelanalysisaspreviouslydescribed . Owingtolowersubstratespeci-
ficity of TbSTT3A for Dol25-PP-GlcNAc Man Glc , oligosaccharyl-transfer
2
9
1
6
reactions for ALG6 reactions were performed for 12 h at 30ꢁ°C. For the oli-
gosaccharyl-transfer reactions for ALG6 reactions that were carried out in
high-mannose N-glycans. Nat. Commun. 10, 1813 (2019).
thepresenceofEDTA, 30µMMnCl wasadded, toavoidinhibitionofSTT3.
2
Acknowledgements This research was supported by the Swiss National Science Foundation
SNF) Sinergia programmes TransGlyco (CRSII3_147632) and GlycoStart (CRSII5_173709) to
(
Statistics and reproducibility
Unless otherwise stated, ALG6 assays and tricine gel-based analyses
were conducted once as shown in the figures.
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 puriꢀication; J. Kowal and I. Manolaridis for help
with EM data collection; and A. Ramirez and A. Alam for helpful discussions.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Author contributions K.P.L. and J.S.B. conceived the project. J.S.B. cloned, screened,
expressed and puriꢀied proteins, reconstituted ALG6 into lipid nanodiscs, performed
biochemical experiments and produced Dol25-PP-GlcNAc
Man using synthetic precursor
2 9

substrates. G.P. synthesized Dol25-P-Glc and derivatives. J.B. synthesized Dol25-P-Man and
Dol25-PP-GlcNAc . 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 reꢀined 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.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
2044-z.
Correspondence and requests for materials should be addressed to K.P.L.
Peer review information Nature thanks Xiaochen Bai and the other, anonymous, reviewer(s) for
their contribution to the peer review of this work.
2
Reprints and permissions information is available at http://www.nature.com/reprints.
Competing interests The authors declare no competing interests.

Article
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-GlcNAc Man and
2 9
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
e, Spatial distribution of particles in the final iteration of 3D refinement.
f, Refined and B-factor-sharpened EM map, coloured by local resolution
5
2
estimation as calculated in ResMap . g, Resolution estimation of the final map
via Fourier shell correlation (FSC), as calculated in RELION 3.0. h, The FSC
4
6
3
.0 . b, Representative cryo-EM micrograph. c, Representative 2D classes.
5
1
d, A representative 2D class with 6AG9-Fab and the nanodisc belt highlighted.
between model and map, as calculated in PHENIX mtriage .

Article
Extended Data Fig. 3 | Molecular interactions at the interface of ALG6 and
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.
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

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
donor substrate Dol25-P-Glc. The red dashed arrows indicate potential binding
sites of the acceptor substrate Dol-PP-GlcNAc
Man . These sites were chosen
2
9
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.
(
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

Article
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
5
8
sequence alignment with EMBOSS needle
.

Article
Extended Data Fig. 7 | Sequence alignment of ALG6 homologues and
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).
orthologues. a, Alignment of amino acid sequences of yeast and human ALG6
5
7
and ALG8 proteins, generated with Clustal Omega (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

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 3.0 . b, A representative cryo-EM micrograph. c, Representative
D 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
5
2
resolution estimation, as calculated in ResMap . g, The FSC between model
4
6
51
and map, as calculated in PHENIX mtriage .
2

Article
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.

a
49
Extended Data Table 1 | Cryo-EM data collection, reꢀinement and validation statistics as deꢀined in PHENIX

Corresponding author(s): Kaspar P. Locher
Last updated by author(s): 20/02/20
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Serial EM 3.6
PyMOL 2.3.3 , PHENIX 1.15-3459, Coot 0.8.9.2 (CCP4), Relion 3.0, MotionCor2, Gctf_v1.06, UCSF Chimera 1.14, UCSF ChimeraX 0.91,
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Atomic coordinates of the apo ALG6 and Dol25-P-Glc bound ALG6 models were deposited in the RCSB Protein Data Bank (PDB) under accession number 6SNI for
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