*For correspondence:
sp693@cam.ac.uk (SP);
dr360@medschl.cam.ac.uk (DR)
Present address: †Center for
Molecular Biology of Heidelberg
University, Heidelberg, Germany;
‡Greenase Biosynthesis Ltd,
Xiamen, China
Competing interest: See
page 24
Funding: See page 24
Received: 08 June 2017
Accepted: 22 October 2017
Published: 24 October 2017
Reviewing editor: Franz-Ulrich
Hartl, Max Planck Institute for
Biochemistry, Germany
Copyright Preissler et al. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
AMPylation targets the rate-limiting step
of BiP’s ATPase cycle for its functional
inactivation
Steffen Preissler*, Lukas Rohland†, Yahui Yan, Ruming Chen‡, Randy J Read,
David Ron*
Cambridge Institute for Medical Research, University of Cambridge, Cambridge,
United Kingdom
Abstract The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein
folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to
ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of
unfolded ER proteins suggests a post-translational mechanism for adjusting BiP’s activity to
changing levels of ER stress, but the underlying molecular details are unexplored. We present
biochemical and crystallographic studies indicating that irrespective of the identity of the bound
nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound
chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but
appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of
modified BiP to attain high affinity for its substrates. These findings suggest a molecular
mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with
substrates whilst conserving ATP.
DOI: https://doi.org/10.7554/eLife.29428.001
Introduction
Compartment-specific chaperones contribute substantially to folding of newly synthesized polypepti-
des and to protein turnover and thereby facilitate maintenance of proteome integrity (Bukau et al.,
2006). The abundant Hsp70-type chaperone BiP (or Grp78) is a central component of the chaperone
repertoire of the endoplasmic reticulum – the gateway to the secretory pathway of eukaryotic cells.
BiP mRNA and protein levels have long been known to respond to changes in the burden of
unfolded secretory proteins; induction of BiP-encoding mRNA being a hallmark of the unfolded pro-
tein response (UPR) (Chang et al., 1989; Kozutsumi et al., 1988). However, in the secretory path-
way fluctuations in the unfolded protein load occur on time scales that are too short to be
accommodated solely by slow and costly antagonistic regulation of BiP levels by transcription and
protein degradation. Therefore, post-translational mechanisms for rapidly adjusting the level of
active BiP to the burden of unfolded proteins in the ER (ER stress) have long been suspected to exist
(Freiden et al., 1992; Gaut, 1997; Laitusis et al., 1999), but the details have only come into focus
recently.
BiP binding to its clients is in competition with inactivating oligomerization due to self-association
via substrate interactions amongst individual BiP molecules. BiP oligomers likely serve as a repository
from which pre-existing active chaperone can be rapidly recruited when the concentration of
unfolded clients increases (Preissler et al., 2015a). A second mechanism involves the covalent modi-
fication of BiP by reversible AMPylation (Ham et al., 2014; Sanyal et al., 2015). When the burden of
unfolded proteins in the ER declines, the ER-localized enzyme FICD (or HYPE) uses ATP to transfer
adenosine monophosphate (AMP) onto the hydroxyl side chain of a single residue within BiP,
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 1 of 28
RESEARCH ARTICLE
threonine 518 (T518) (Preissler et al., 2015b). With mounting ER stress, an altered functional form
of the same enzyme, FICD, rapidly removes the AMP, restoring the hydroxyl side chain to T518 and
BiP to its ground state (Preissler et al., 2017).
Deregulated FICD activity strongly induces the UPR; an observation consistent with modified BiP
being less able to buffer ER stress (Ham et al., 2014; Preissler et al., 2015b; Preissler et al., 2017).
The inactivating nature of the modification is further supported by observations that in vitro AMPy-
lated BiP binds a model peptide substrate less stably than the unmodified chaperone and by evi-
dence for an ill-defined defect in the responsiveness of AMPylated BiP to the stimulatory effect of
J-domain proteins (Preissler et al., 2015b).
Like other members of its family, BiP consists of an N-terminal nucleotide binding domain (NBD)
and a C-terminal substrate binding domain (SBD), both of which are connected by a conserved
hydrophobic linker. BiP, and Hsp70s in general, interact transiently with extended, usually hydropho-
bic, amino acid sequences exposed on the surface of their client proteins (Blond-Elguindi et al.,
1993; Flynn et al., 1991). These substrate interactions are modulated by nucleotide binding and
hydrolysis at the NBD that are accompanied by substantial conformational changes in both domains
(Mayer, 2013). Substrate binding is facilitated by J-domain containing co-chaperones (J-proteins)
that stimulate ATP hydrolysis by Hsp70s in proximity of the substrate. The concerted action of chap-
erone and J-protein yields a substrate binding machine of ultra-high affinity, characterized by fast ini-
tial association of the Hsp70/BiP with the substrate (high ‘on’ rates) in the ATP-bound state and slow
dissociation (low ‘off’ rates) upon J domain-driven ATP hydrolysis (De Los Rios and Barducci, 2014;
Misselwitz et al., 1998).
Here we have combined biochemical and structural approaches to dissect how AMPylation influ-
ences the J protein-driven ATPase cycle of BiP to cause its functional inactivation. Our findings
reveal an unanticipated role for the structural modification of a residue on the far reaches of BiP’s
SBD - the AMPylated T518 - in affecting an allosteric transition that is essential for Hsp70s to achieve
ultra-affinity for binding their substrates.
Results
AMPylation biases BiP towards a conformation normally attained by
the ATP-bound chaperone
Nucleotide binding and hydrolysis markedly affect interactions between the NBD and SBD of
Hsp70s/BiP. In both the ADP-bound and nucleotide-free (or apo) state the two domains are
undocked, substrates are bound tightly (with low ‘off’ rates), and the hydrophobic interdomain linker
is exposed to the solvent (Bertelsen et al., 2009). In the ATP-bound state the two domains are
docked against each other, substrates exchange rapidly (with high ‘on’ and ‘off’ rates), and the inter-
domain linker is tucked against the NBD and relatively shielded from the solvent (Kityk et al., 2012;
Kumar et al., 2011; Qi et al., 2013; Yang et al., 2015). These mechanisms initially characterized in
the bacterial DnaK likely extend to all members of the family, including BiP.
The bacterial protease SubA has remarkable specificity for BiP’s interdomain linker, cleaving it
between L416 and L417 (Paton et al., 2006). It is thus a useful tool to probe the linker’s disposition
vis-a`-vis the aforementioned allosteric transitions. However, the tendency of ADP-bound (or apo) BiP
to oligomerize confounds interpretation of the effects of nucleotide on SubA-mediated linker cleav-
age, because engagement of the interdomain linker of one BiP molecule in the SBD of another pro-
tects the linker from cleavage (Preissler et al., 2015a). The net result of these two competing
processes manifests in complete resistance of a substantial fraction of ADP-bound wildtype BiP
(BiPWT) to cleavage by SubA (Preissler et al., 2015a’, and reaction 1 in Figure 1 and Figure 1—fig-
ure supplement 1). Interestingly, AMPylation rendered the otherwise refractory fraction of ADP-
bound BiP susceptible to cleavage (compare reactions 1 and 5 in Figure 1, and Figure 1—figure
supplements 1, 2 and 3).
The competing effect of linker protection by inter-molecular engagement in the SBD of ADP-
bound BiP oligomers can be nearly eliminated by a mutation in the SBD, V461F, that enfeebles sub-
strate binding without affecting the allosteric transitions caused by nucleotide binding and hydrolysis
(Preissler et al., 2015a). When the same experiment was performed with an ADP-bound substrate
binding-deficient BiPV461F mutant, the linker was rapidly cleaved nearly to completion. Cleavage of
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 2 of 28
Research article Biochemistry Biophysics and Structural Biology
ADP (1)
NBD
SBD
5 15 30 45- 5 15 30 45
ATP (2)
-90 90
FL
ADP (5)
NBD
SBD
5 15 30 45- 5 15 30 45
ATP (6)
-90 90
FL
ADP (3)
NBD
SBD
5 15 30 45- 5 15 30 45
ATP (4)
-90 90
FL
ADP (7)
NBD
SBD
5 15 30 45- 5 15 30 45
ATP (8)
-90 90
FL
BiPWT BiPV461F
CBB CBB
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
75
50
37
25
20
100
150
250
SubA (min):
U
n
m
o
d
if
ie
d
75
50
37
25
20
100
150
250
SubA (min):
A
M
P
y
la
te
d
(kDa)
(kDa)
75
50
37
25
20
100
150
250
75
50
37
25
20
100
150
250
(kDa)
(kDa)
SubA SubA
SubASubA
unmodified + ADP (3)
unmodified + ATP (4)
AMPylated + ADP (7)
AMPylated + ATP (8)
- 15 30 45 60 75 90
0.0
0.2
0.4
0.6
0.8
1.0
N
o
rm
a
li
z
e
d
 f
u
ll
-l
e
n
g
th
 (
F
L
)
SubA treatment (min)
- 15 30 45 60 75 90
0.0
0.2
0.4
0.6
0.8
1.0
N
o
rm
a
li
z
e
d
 f
u
ll
-l
e
n
g
th
 (
F
L
)
SubA treatment (min)
unmodified + ADP (1)
unmodified + ATP (2)
AMPylated + ADP (5)
AMPylated + ATP (6)
Figure 1. AMPylation biases BiP towards an ATP bound-like conformation. Coomassie-stained (CBB) SDS-PAGE gels of purified unmodified or
AMPylated wildtype BiP (BiPWT) and oligomerization-deficient V461F mutant (BiPV461F) proteins digested with the linker-specific SubA protease for the
indicated times in presence of ADP or ATP. The intact proteins (FL) and isolated nucleotide binding domain (NBD) and substrate binding domain (SBD)
are indicated. The time-dependent change of the intact BiP (FL) signals were quantified, normalized to the initial value (set to 1), and plotted in the
graphs below. The numbering (1–8, in parentheses) refers to the explanatory cartoons of these limited proteolysis reactions that analyze the nucleotide-
dependent conformational states of unmodified and AMPylated BiP (Figure 1—figure supplement 1). Shown are representative experimental
observations reproduced three times (Figure 1—figure supplement 2).
DOI: https://doi.org/10.7554/eLife.29428.002
The following figure supplements are available for figure 1:
Figure supplement 1. Cartoons depicting the conformational states of BiP in the reactions presented in Figure 1.
DOI: https://doi.org/10.7554/eLife.29428.003
Figure supplement 2. AMPylation alters the sensitivity of BiP to cleavage by SubA.
DOI: https://doi.org/10.7554/eLife.29428.004
Figure supplement 3. Analysis of unmodified and AMPylated BiP by mass spectrometry.
DOI: https://doi.org/10.7554/eLife.29428.005
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 3 of 28
Research article Biochemistry Biophysics and Structural Biology
BiPV461F was slower in the presence of ATP, consistent with a domain-docked conformation that par-
tially protects the linker from cleavage (compare reactions 3 and 4 in Figure 1 and Figure 1—figure
supplements 1 and 2). AMPylation, which exposed the interdomain linker of ADP-bound BiPWT to
cleavage by SubA, had a stabilizing effect on the interdomain linker of the ADP-bound mutant
BiPV461F (compare reactions 3 and 7 in Figure 1 and Figure 1—figure supplements 1 and 2), result-
ing in very similar cleavage kinetics of AMPylated BiPWT and AMPylated BiPV461F in both nucleotide
states (compare the lower panels of Figure 1).
These observations are consistent with the notion that AMPylation biases BiP towards a domain-
docked conformation with higher substrate exchange kinetics (the substrate being the interdomain
linker in these reactions, set up in the absence of other clients). Thus, in the case of ADP-bound
BiPWT, AMPylation likely impaired substrate interaction-dependent oligomerization and thereby
increased BiP’s cleavability by SubA, whereas the protective effect of AMPylation on the interdomain
linker due to enhanced domain docking dominated in the context of the V461F mutation. The
enhanced protection of the linker of ATP-bound AMPylated BiPWT and AMPylated BiPV461F (com-
pared to their non-AMPylated ATP-bound versions) was a conspicuous and reproducible finding
(compare reactions 6 and 8 with 2 and 4 in Figure 1 and Figure 1—figure supplements 1 and
2). These observations suggest that AMPylation not only influences the ADP-bound state to resem-
ble the ATP-bound state but also accentuates features normally found in the ATP-bound chaperone.
Structure of AMPylated BiP
The limited proteolysis experiments suggested that further insight into the consequences of AMPyla-
tion on BiP’s conformation might be provided by a structure of the AMPylated chaperone in the apo
or ADP-bound state, in which AMPylation exerts its most prominent effect. Most crystal structures of
intact Hsp70 chaperones have been obtained in the ATP state, but a previous success in crystallizing
an ADP-Pi-bound bacterial Hsp70 (Geobacillus kaustophilus DnaK) in the domain-undocked conforma-
tion has been reported (Chang et al., 2008). To facilitate crystallization we deleted the N-terminal
nine unstructured residues from mature Chinese hamster BiP as well as a large part of the flexible heli-
cal lid (SBDa) at the C-terminus (Figure 2A), a deletion that favored crystallization of the ADP-bound
G. kaustophilus DnaK. Truncation of the lid enhances the substrate binding and release kinetics of
DnaK and BiP but preserves nucleotide-dependent allosteric regulation (Buczynski et al., 2001;
Chambers et al., 2012;Misselwitz et al., 1998; Pellecchia et al., 2000). Additionally, we introduced
a T229A mutation that strongly inhibits the ATPase activity of BiP (Gaut and Hendershot, 1993;
Wei et al., 1995) and favors FICD-mediated AMPylation over ATP hydrolysis (Preissler et al., 2015b).
The bacterially expressed and affinity-purified protein was AMPylated by exposure to active FICDE234G
in presence of ATP, followed by addition of EDTA to remove the bound nucleotide and size-exclusion
chromatography to separate BiP from FICDE234G. Stoichiometric AMPylation of BiP was confirmed by
intact protein mass spectrometry (see Materials and methods section).
Crystals of AMPylated BiP diffracted well and a structural model of 1.9 A˚ resolution was derived
(PDB 5O4P; Table 1). In the crystals BiP adopted a domain-docked conformation. Accordingly, the
interdomain linker was well structured and bound to the NBD by parallel b-augmentation
(Figure 2B). The main-chain conformation of AMPylated BiP was nearly identical to that of ATP-
bound full-length human BiP (PDB 5E84) (Yang et al., 2015) (Ca alignment RMS = 0.551 A˚;
Figure 2C). However, the nucleotide-binding pocket in the NBD of AMPylated BiP was occupied by
several water molecules and a sulfate group was found at the position where the g-phosphate of
ATP would otherwise be located (Figure 2D and Figure 2—figure supplement 1A).
Although the crystalized AMPylated BiP was clearly ATP-free, it adopted a conformation that is
characteristic of the ATP-bound state. This surprising feature extends to the fine details, as the NBD
of nucleotide-free AMPylated BiP assumed a conformation that resembled the ATP-bound NBD of
intact BiP (Yang et al., 2015) (Figure 2C and D) and deviated considerably in structure from the iso-
lated NBD, whether crystallized in the apo state or with ADP or ATP (Macias et al., 2011;
Wisniewska et al., 2010) (Figure 2—figure supplement 1B). This feature is likely imparted on the
NBD allosterically, as a consequence of AMPylation, because the T229A mutation did not abolish
the nucleotide-dependent allosteric regulation of BiP nor bias the protein towards an ATP-bound
conformation (Figure 2—figure supplement 2A and B).
As expected of BiP/Hsp70 in the domain-docked conformation, the truncated lid of the SBD
(SBDa) was in the open position and the substrate binding groove was substantially wider than that
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 4 of 28
Research article Biochemistry Biophysics and Structural Biology
90o
NBD
Linker
SBDα
SBDβ
Linker
NBD
SBDα
SBDβ
Linker
NBD
L7,8
L7,8
A
B
D
F
NBD SBDβ SBDα
T229A T518-AMP
28 549
1 19 654529
409-419
Linker
ShaBiP
haBiP28-549
huBiP•ATP
L1,2L4,5
L3,4
L5,6
L7,8
SBDα
SBDβhuBiP•ATP
*
T518
T518
T518
E
C
huBiP
T518
SBDα
SBDα
SBDβ
SBDα
huBiP•ATP
NBD
ATP
SO4
2-
Figure 2. Crystal structure of in vitro AMPylated apo BiP. (A) Schematic representation of Chinese hamster BiP (haBiP) and the derivative used for
crystallization (haBiP28-549). The following features are indicated: signal sequence (grey; S), nucleotide binding domain (blue; NBD), interdomain linker
Figure 2 continued on next page
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 5 of 28
Research article Biochemistry Biophysics and Structural Biology
of the isolated SBD of human BiP (PDB 5E85, which reflects the state of the SBD of the domain-
undocked ADP-bound chaperone, Bertelsen et al., 2009; Yang et al., 2015) (Figure 2E). Differen-
ces between the SBD of AMPylated apo BiP and ATP-bound BiP were also noted in the distal loops
of the SBDb subdomain (Figure 2E and F). In particular the conformation of loop L7,8, harboring the
AMPylation site residue T518, differed substantially between both structures. Although no clear den-
sity for the entire AMP moiety was observed, several indications point to the presence of the modifi-
cation. First, the side chain of T518 is oriented outwards and protrudes into the solvent (Figure 2F).
The missing density may thus be explained by flexibility of the hydrophilic AMP exposed on the pro-
tein surface. Second, additional electron density extends from the T518 side chain hydroxyl group,
consistent with the phosphodiester linkage to adenosine (Figure 2—figure supplement 1C). Third,
whereas the surrounding loops are engaged with the neighboring molecule at the crystal packing
interface, loop L7,8 does not form such contacts, providing space in the crystal to accommodate
AMP and affording substantial flexibility to the region, as reflected in the high B-factors of L7,8 of
AMPylated BiP (Figure 2—figure supplement 1D).
The aforementioned features were observed in four additional crystal forms of AMPylated BiP
derived from independent protein preparations and from molecules arranged in a different space
group (Table 1 and Figure 2—figure supplement 3A) and supported by intact protein mass spec-
trometry, pointing to the presence of the modification in the crystallized protein (Figure 2—figure
supplement 4A and B). In all structures BiP adopted a very similar domain-docked conformation in
absence of density for ATP in the NBD and all reveal additional density protruding from the T518
side chain (Figure 2—figure supplement 3A–C). Furthermore, their L7,8 loop regions appeared
more disordered than surrounding parts of the SBDb (indicated by higher B-factors, Figure 2—fig-
ure supplement 3D). These findings indicate that the bulky modification favored solvent exposure
of loop L7,8 and prevented T518 from forming intramolecular contacts observed in structures of
unmodified BiP or other Hsp70s.
Notably, a structure of AMPylated BiP from a crystallization reaction supplemented with ADP
showed that BiP adopted the domain-docked, loop L7,8-exopsed conformation even when ADP was
bound (Table 1 and Figure 2—figure supplement 5A–E). Thus, despite lack of density correspond-
ing to the bulk of the modification, the structural data are in agreement with the results of the lim-
ited proteolysis experiments (Figure 1) and strongly support the conclusion that AMPylation favors a
Figure 2 continued
(magenta; Linker), substrate binding subdomain b (orange; SBDb), substrate binding subdomain a (green; SBDa), and the AMPylation site (T518-AMP).
(B) Ribbon representation of the structure of crystallized haBiP28-549 (PDB 5O4P) in two orientations with coloring as in ‘A’. (C) Comparison of haBiP28-549
(PDB 5O4P) with the structure of unmodified ATP-bound human BiP (huBiP, PDB 5E84; grey). Loop L7,8 comprising the AMPylation site (T518) is
indicated, otherwise coloring as in ‘A’. (D) Same as in ‘C’ showing only the NBDs. The inset is a close-up view of the nucleotide binding cleft. The ATP
(green) bound by the unmodified huBiP and a sulfate bound by AMPylated haBiP (blue) are indicated in stick diagram. Note that the sulfate group fully
occupies the position of the terminal g-phosphate of the bound ATP. (E) Comparison of the SBD of AMPylated haBiP28-549 (PDB 5O4P; coloring as in
‘A’) with the isolated SBD of unmodified huBiP (PDB 5E85, grey). A C-terminal substrate peptide (yellow) occupies the peptide binding groove of
unmodified huBiP. The AMPylation site (T518) is indicated (red). (F) As in ‘C’ comparing only the substrate binding domains. The asterisk (*) marks the
peptide binding groove. The inset shows a close-up view of L7,8. Note the outward orientation of the T518 side chain in the AMPylated haBiP
28-549 (red)
compared to its inward orientation in unmodified huBiP (yellow).
DOI: https://doi.org/10.7554/eLife.29428.006
The following figure supplements are available for figure 2:
Figure supplement 1. Features of the AMPylated apo BiP structure point to the absence of nucleotide in the NBD and AMPylation of T518.
DOI: https://doi.org/10.7554/eLife.29428.007
Figure supplement 2. The T229A mutation does not alter BiP’s nucleotide-dependent allostery.
DOI: https://doi.org/10.7554/eLife.29428.008
Figure supplement 3. Structures of AMPylated BiP in a different crystal form and in the presence of diverse ligands exhibit consistent modification-
dependent features.
DOI: https://doi.org/10.7554/eLife.29428.009
Figure supplement 4. Mass spectrometry analysis of BiP used for crystallization.
DOI: https://doi.org/10.7554/eLife.29428.010
Figure supplement 5. Crystal structure of ADP-bound AMPylated BiP.
DOI: https://doi.org/10.7554/eLife.29428.011
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 6 of 28
Research article Biochemistry Biophysics and Structural Biology
state of BiP that, in absence of ATP, is very similar to the conformation of ATP-bound unmodified
BiP.
AMPylation impairs BiP oligomerization
The data presented above reveal that AMPylation biases BiP’s conformational equilibrium towards
an ATP bound-like, domain-docked state that is predicted to weaken substrate binding. BiP mole-
cules bind each other through typical chaperone-substrate interactions. In cells, these interactions
assemble inactive BiP oligomers to buffer short-term fluctuations in the ER unfolded protein load,
whereas in vitro BiP oligomerization is a convenient means to probe its affinity for substrates
(Preissler et al., 2015a). Purified AMPylated BiP migrates mostly as a monomer on native-PAGE
gels and AMPylation increases the monomeric fraction of endogenous BiP from mammalian cells,
suggesting that the modification might interfere with the ability of BiP to oligomerize
(Preissler et al., 2015b). To explore this in further detail, we monitored the oligomeric state of BiP
by size-exclusion chromatography.
Thereby, monomers of purified BiP were separated from earlier eluting oligomers, which are sta-
bilized by absence of nucleotide or by presence of ADP - with dimers dominating at the concentra-
tion tested. In contrast, AMPylated BiP eluted mainly as a monomer (Figure 3A and B) consistent
Table 1. Data collection and refinement statistics
AMPylated haBiP28-549 Apo Apo Apo ADP ADP
Data collection
Synchrotron stations (DLS) I02 I24 I24 I24 I24
Space group P21 P212121 P212121 P212121 P212121
Cell dimensions
a,b,c; (A˚) 68.33, 118.65, 83.15 64.65, 69.07, 122.23 69.088, 75.408, 98.308 69.089, 75.310, 98.350 69.05, 75.61, 97.50
a, b, g ; (0) 90, 97, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90
Resolution, (A˚) 67.76–1.86 (1.91–1.86)* 61.12–2.0 (2.05–2.00) 75.41–1.67 (1.7–1.67) 98.35–1.71 (1.74–1.71) 97.5–1.59 (1.61–1.59)
Rmerge 0.089 (0.83) 0.158 (1.31) 0.086 (0.794) 0.096 (1.002) 0.131 (0.906)
Rmeas 0.122 (1.119) 0.185 (1.532) 0.096 (0.871) 0.108 (1.104) 0.145 (1.022)
<I/s (I)> 9.6 (1.4) 8.5 (1.5) 12 (2.1) 13 (2.2) 8.8 (2.2)
CC1/2 0.995 (0.533) 0.995 (0.522) 0.997 (0.888) 0.998 (0.820) 0.990 (0.836)
Completeness, % 99.4 (99.1) 99.9 (100) 98.3 (100) 99.9 (100) 100 (99.9)
Redundancy 3.4 (3.5) 7.1 (7.2) 6.6 (6.7) 6.6 (6.7) 6.5 (6.4)
Refinement
Rwork 0.195 0.240 0.210 0.210 0.200
Rfree 0.212 0.280 0.250 0.250 0.220
No. of reflections 104104 35827 56476 53637 66447
No. of atoms 8750 3994 4281 4245 4156
Average B-factors 24.9 35.7 23.14 23.3 30.09
RMS deviations
Bond lengths (A˚) 0.007 0.007 0.007 0.009 0.008
Bond angles, (0) 1.261 1.151 1.184 1.485 1.332
Ramachandran favoured region, % 99 97.49 98.05 98.44 99.03
Ramachandran outliers, % 0.19 0 0 0 0
MolProbity score (percentile†) 0.67 (100%) 0.83 (100%) 0.67 (100%) 0.78 (100%) 0.74 (100%)
PDB code 5O4P 6EOB 6EOC 6EOE 6EOF
* Values in parentheses are for the highest-resolution shell.
† 100th percentile is the best among structures of comparable resolution.
DOI: https://doi.org/10.7554/eLife.29428.012
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 7 of 28
Research article Biochemistry Biophysics and Structural Biology
A670 158 44 17 (kDa)
BiP
BiP-AMP
6 8 10 12 14
A
2
3
0
 n
m
 (
a
.u
.)
0
100
200
300
400
500
6 8 10 12 14
A
2
3
0
 n
m
 (
a
.u
.)
0
600
200
400
800
BiP
BiP + ADP
BiP + ATP
670 158 44 17 (kDa)
6 8 10 12 14
A
2
3
0
 n
m
 (
a
.u
.)
0
600
200
400
800
BiP-AMP
BiP-AMP + ADP
BiP-AMP + ATP
670 158 44 17 (kDa)
A
2
3
0
 n
m
 (
a
.u
.)
0
600
200
400
800
6 8 10 12 14
ADP
ATP
670 158 44 17 (kDa)
A
2
3
0
 n
m
 (
a
.u
.)
0
600
200
400
800
670 158 44 17 (kDa)
6 8 10 12 14
5 µM J + ATP
5 µM J* + ATP
10 µM J + ATP
10 µM J* + ATP
A
2
3
0
 n
m
 (
a
.u
.)
0
100
200
300
400
500
600
6 8 10 12 14
670 158 44 17 (kDa)
BiP + ATP
BiP + ATP + 1x J
BiP + ATP + 2x J
BiP + ATP + 4x J
BiP
BiP + ATP + 8x J
A
2
3
0
 n
m
 (
a
.u
.)
0
600
200
400
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670 158 44 17 (kDa)
6 8 10 12 14
BiP
BiP + ATP + J
BiP + ATP + J*
670 158 44 17 (kDa)
A
2
3
0
 n
m
 (
a
.u
.)
0
600
200
400
800
6 8 10 12 14
Time (min)
BiP-AMP
BiP-AMP + ATP + J
BiP-AMP + ATP + J*
6 8 10 12 14
A
2
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0
 n
m
 (
a
.u
.)
0
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670 158 44 17 (kDa)
BiP
BiP + ATP + J
BiP + ATP + J*
6 8 10 12 14
Time (min)
A
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 n
m
 (
a
.u
.)
0
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670 158 44 17 (kDa)
BiP-AMP
BiP-AMP + ATP + J
BiP-AMP + ATP + J*
5
 µ
M
 J
1
0
 µ
M
 J
B
C
D
M
II
IIIHMW
oligomers
ATP
M
II
III
HMW
oligomers
ATP/ADP
ATP
ATP
ATP
ATP
ATP ATP
M
II
III
HMW
oligomers
M
II
III
ATP/ADP
M
II
III
HMW
oligomers
M
II
III
M
II
III
HMW
oligomers
M
II
III
J/J*
Figure 3. AMPylation inhibits J protein-mediated BiP oligomerization. (A) Peptide bond absorbance traces (A230 nm) of wildtype unmodified (BiP) and
AMPylated (BiP-AMP) BiP proteins (both at 50 mM) resolved by size-exclusion chromatography in absence of added nucleotide. The elution peak
Figure 3 continued on next page
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 8 of 28
Research article Biochemistry Biophysics and Structural Biology
with previous evidence of impaired substrate interactions (Preissler et al., 2015b). Despite its bias
towards the conformation normally imposed by ATP binding, AMPylated BiP retained further
responsiveness to ATP, as the remaining oligomers of both unmodified and modified BiP disas-
sembled into monomers upon exposure to ATP (Figure 3B).
Basal ATP hydrolysis rates of Hsp70s are low. Under physiological conditions this process requires
stimulation by co-chaperones, which share a structurally conserved J-domain that interacts with the
chaperone to promote ATP hydrolysis and thereby stable substrate binding (Liberek et al., 1995).
In the absence of other substrates, addition of a J-domain promotes oligomerization of Hsp70, as
J-stimulated ATP hydrolysis (and the relatively slow intrinsic exchange of ADP for ATP) dynamically
enforces a regime of enhanced substrate interactions between individual chaperone molecules
(King et al., 1995; King et al., 1999). This feature was therefore exploited to investigate the func-
tional consequences of BiP AMPylation on J-mediated substrate binding.
In the presence of ATP, increasing concentrations of the J-domain of ERdj6 purified as a glutathi-
one S transferase (GST) fusion protein (GST-JWT, Petrova et al., 2008) progressively shifted the
equilibrium from monomers towards BiP oligomers (Figure 3C). J-mediated oligomers were much
larger than those observed in the absence of nucleotide or in presence of ADP (compare Figure 3C
with Figure 3A and B). BiP oligomerization was dependent on the functionality of the J-domain, as
no oligomers were established by a mutant J-domain in which the histidine residue of its conserved
HPD motif - essential for stimulation of the ATPase activity of Hsp70s (Wall et al., 1994) - was
exchanged to glutamine (GST-JQPD; Figure 3D). Importantly, J domain-mediated BiP oligomeriza-
tion was nearly absent when AMPylated BiP was tested in the assay (right panels in Figure 3D).
Thus, AMPylation interfered with BiP’s cooperation with a J-domain co-chaperone to form a stable
substrate interaction.
AMPylation of BiP affects its J protein-mediated interactions with
substrates
BiP oligomerization represents a particular type of substrate interactions; to study J domain-medi-
ated substrate interactions by an alternative approach we took advantage of the well-documented
ability of Hsp70s (Mayer et al., 1999; Suh et al., 1998) and BiP in particular to recognize J-proteins
as substrates in vitro (Misselwitz et al., 1999). Accordingly, in the presence of ATP, BiPWT co-puri-
fied efficiently with GST-JWT coupled to glutathione sepharose beads in a co-purification assay
(Petrova et al., 2008’, and lane 8 in Figure 4A). This interaction was observed neither in presence
of ADP nor when mutant GST-JQPD was immobilized, nor when substrate binding-deficient BiPV461F
was used (Figure 4A). The assay therefore reported on a J protein-dependent substrate interaction
of BiP. Importantly, AMPylated BiP did not bind stably to beads carrying GST-JWT in presence of
ATP, pointing to defective J protein-mediated high-affinity substrate interactions imposed by the
modification (lane 10 in Figure 4A).
Consistent with its bias towards the ATP-like state, AMPylated BiP retains the ability to bind sub-
strates, but with accelerated release kinetics (Preissler et al., 2015b’, Figure 8E therein). To circum-
vent the limitation of post-equilibrium methods (such as co-purification or size-exclusion
chromatography) to report on transient substrate interactions, a bio-layer interferometry (BLI) assay
was established to monitor J protein-mediated BiP engagement of substrates in real-time. A biotiny-
lated GST-J fusion protein and a model BiP substrate peptide (P15, Misselwitz et al., 1998) were
co-immobilized on the surface of streptavidin-coated BLI sensors (Figure 4B). The sensors were then
Figure 3 continued
corresponding to BiP monomers (M) and earlier eluting peaks (II and III) representing BiP dimers and trimers, respectively, as well as high-molecular
weight (HMW) oligomers are indicated. Note the enhanced monomer signal and paucity of oligomers in the sample of AMPylated BiP. (B) As in ‘A’ but
in presence of ADP or ATP (both at 1.5 mM). Note the nearly complete disassembly of BiP oligomers in the ATP-containing sample. (C) Where
indicated the same experiment as in ‘A’ was performed in presence of ATP (1.5 mM) and different concentrations of the wildtype (J) or inactive QPD
mutant (J*) J-domain of ERdj6 (1  represents 1.25 mM of J-domain). Note the J-domain concentration-dependent increase in peaks II and III and the
formation of faster eluting species (before 8 min) representing large BiP oligomers. (D) Comparison of the elution profiles of unmodified (left) and
AMPylated BiP (right) treated as in ‘C’. Note the near absence of early-eluting oligomeric species in the AMPylated BiP sample. A representative
experiment of three independent repeats is shown.
DOI: https://doi.org/10.7554/eLife.29428.013
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 9 of 28
Research article Biochemistry Biophysics and Structural Biology
BA
B
BLI sensor
ATP
BiP
B
GST
J
B BBB
1 2 3 4A
D
P
C
Input
BiP
GST-J/JQPD
CBB
1 2 3 4 5 6 7 8 9 10 11 12
75 kDa
50 kDa
37 kDa
20 kDa
100 kDa
150 kDa
250 kDa
25 kDa
- - - - D T D T D T D T D T
-- + -
-+ - -
-- - -
-- - -
-- - -
++ + +
+- - +
-+ + -
-+ + -
-- - -
++ + +
-- - -
- -
- -
+ +
- -
13 14
GSH pull-down
ADP/ATP:
GST-JQPD:
GST-J:
BiP-AMP:
BiP:
BiPV461F-AMP:
BiPV461F:
+- - -
-- - +
-- - -
-- - -
-- - -
+- - +
+ +
- -
P15
-40 -30 -20 -10 0 10 20 30 40 50 60
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Time (min)
B
in
d
in
g
 (
n
m
)
Association DissociationP15 peptide B
a
s
e
li
n
e
W
a
s
h
G
S
T
-J
W
T
/J
Q
P
D
I II III IV V VI VII
1
2
5
6
7
3
8
4
1:
2:
3:
4:
5:
6:
7:
8:
Loading (II & IV)
GST-JWT
GST-JWT
GST-JQPD
GST-JWT
GST-JWT
GST-JQPD
GST-JWT
GST-JWT
→ P15 
→ P15 
→ P15 
→ P15 
→ P15 
→ P15 
→ P15 
→ P15 
Association (VI)
→ BiPWT
→ BiPT518E
→ BiPT518E
→ BiPWT
→ BiPWT
→ BiPWT-AMP
→ BiPWT-AMP
→ BiPWT-AMP
+ ATP 
+ ATP
+ ATP
+ ATP 
+ ATP 
+ ATP 
+ ATP 
+ ATP 
Dissociation (VII)
→ ATP 
→ ATP 
→ ADP 
→ ADP 
→ ATP 
→ ATP 
→ ADP 
→ ATP 
SA
Figure 4. AMPylation inhibits J protein-mediated substrate binding. (A) Coomassie-stained SDS-PAGE gels of proteins eluted from a
glutathione (GSH) sepharose matrix. Unmodified or AMPylated wildtype or V461F mutant BiP proteins were incubated in presence of ADP or ATP with
wildtype or QPD mutant J-domain coupled via a GST-fusion protein to GSH sepharose beads and subsequently washed extensively. The input and the
bound proteins (GSH pull-down) eluted by denaturation in SDS are indicated. (B) Cartoon depicting possible interactions between soluble BiP (the
Figure 4 continued on next page
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 10 of 28
Research article Biochemistry Biophysics and Structural Biology
introduced into solutions containing BiP to detect its association, followed by transfer to BiP-free sol-
utions to record dissociation. In presence of ATP unmodified BiPWT was rapidly recruited to sensors
carrying GST-JWT and P15 (traces 1 and 2 in Figure 4C). This interaction required a functional
J-domain (trace 3 in Figure 4C) and was dependent on BiP’s ability both to bind substrate and to
hydrolyze ATP (Figure 4—figure supplement 1).
Dissociation of the bound BiPWT was faster in presence of ATP compared with ADP (Figure 4C,
compare dissociation phase of traces 1 and 2). This finding indicated that the apparent plateau in
binding (observed in the preceding association phase, with both BiP and ATP present in solution)
was enforced by cycles of nucleotide exchange-driven BiP dissociation and J protein-driven ATP
hydrolysis-dependent rebinding, as formalized in the ultra-affinity model for J-mediated Hsp70-sub-
strate interactions (De Los Rios and Barducci, 2014). In contrast, AMPylated BiPWT showed a
severely reduced binding plateau to GST-JWT-coupled sensors in presence of ATP and its dissocia-
tion kinetics were faster in presence of ADP compared to unmodified BiPWT (traces 4 and 5 in
Figure 4C).
Mutant BiPT518E, which mimics aspects of AMPylation (Preissler et al., 2015b), behaved similarly
to AMPylated BiPWT in this assay (compare traces 7 and 8 to 4 and 5 in Figure 4C). ATP further
accelerated dissociation of the bound AMPylated BiPWT and BiPT518E, paralleling unmodified BiPWT.
These BLI experiments and the oligomerization assays both reveal a consistent defect in the ability
of modified BiP to achieve J protein-mediated ultra-affinity substrate binding, whilst showcasing the
residual responsiveness of the modified protein to ATP.
AMPylation of BiP does not alter its non-substrate interactions with the
J-domain
The defect in J protein-mediated substrate interaction imposed by AMPylation may have arisen from
impairment in the initial interaction between AMPylated BiP and the J-domain or from a defect in
subsequent stimulated ATP hydrolysis (the functional output of the initial engagement), or a combi-
nation of both. To distinguish amongst these possibilities we needed to deconvolute the two com-
ponents that contribute to the binding of BiP to immobilized J-protein: (i) the initial engagement of
the J-domain with ATP-bound BiP (involving non-substrate, protein-protein interactions) and (ii) typi-
cal substrate interactions catalyzed by J protein-mediated ATP hydrolysis; the latter is expected to
dominate in binding assays (Mayer et al., 1999; Suh et al., 1998).
In an effort to measure the non-substrate interaction of BiP with the J-domain the T229A muta-
tion (that inhibits ATP hydrolysis without affecting nucleotide binding-coupled allostery, Figure 2—
figure supplement 2) and the V461F mutation (that inhibits substrate binding without affecting allo-
stery, Figure 1 and Figure 2—figure supplement 2) were combined to generate BiPT229A-V461F. BLI
sensors coated with immobilized GST-JWT (in absence of a substrate peptide) were exposed to dif-
ferent concentrations of unmodified or AMPylated BiPT229A-V461F in presence of ATP. Plotting the
Figure 4 continued
analyte) and biotinylated GST-J co-immobilized with a BiP substrate peptide (P15) on a bio-layer interferometry (BLI) sensor (the ligand). Streptavidin
(SA)-coated BLI sensors (grey), the biotin moiety (B) covalently attached to GST-J (yellow), biotinylated P15 substrate peptide (red), and ATP-bound BiP
in solution are shown. Arrows indicate J domain-mediated BiP interactions with GST-J in trans (1), P15 (2), GST-J in cis (3) and J domain-mediated BiP
oligomerization (4). (C) Plot of the BLI signal as a function of time in experiments as cartooned in ‘B’ and tabulated below. The individual steps of the
experiment (I-VII) are indicated: After an initial equilibration step (I) biotinylated wildtype or QPD mutant GST-J corresponding to an interference signal
difference of ~0.4 nm was immobilized (II). Following a short wash step (III) the binding sites on the sensors were saturated with biotinylated P15
peptide (IV), followed by an extended wash step to establish a stable baseline signal (V). The sensors were then introduced into solutions containing
unmodified (BiP) or AMPylated (BiP-AMP) wildtype BiP or BiPT518E mutant proteins to detect their association with the sensors in presence of ATP (VI).
Dissociation of BiP from the sensor was measured in protein-free solutions containing either ADP or ATP (VII). A representative experiment is shown
and the same result was observed in at least three independent repeats. Note that in the presence of ADP the dissociation of AMPylated BiP
(32.9  104 ± 19.0104 s1; trace 5) and BiPT518E (16.5  104 ± 7.3104 s1, trace 8) was faster than unmodified BiP (7.9  104 ± 5.3104 s1;
trace 2). The dissociation rate constants represent mean values with standard deviations.
DOI: https://doi.org/10.7554/eLife.29428.014
The following figure supplement is available for figure 4:
Figure supplement 1. J domain-mediated substrate interactions depend on BiP’s ATPase activity and ability to bind substrates.
DOI: https://doi.org/10.7554/eLife.29428.015
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 11 of 28
Research article Biochemistry Biophysics and Structural Biology
steady state binding amplitudes against the BiP concentrations revealed very similar binding con-
stants (Kd » 14 mM; Figure 5). The similar shape of the curves also indicated that AMPylation did
not significantly alter the association or dissociation kinetics.
Several control experiments confirmed that substrate interactions had indeed a negligible contri-
bution to the observed binding of BiPT229A-V461F to GST-JWT: First, the dissociation rate of the
B
iP
T
2
2
9
A
-V
4
6
1
F
-A
M
P
B
iP
T
2
2
9
A
-V
4
6
1
F
Ass. (ATP) Diss. (ATP)
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Time (min)
B
in
d
in
g
 (
n
m
)
0 5 10 15 20
0.0
0.2
0.4
0.6
0.8
[µM]
Steady state binding
Ass. (ATP) Diss. (ATP)
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Time (min)
B
in
d
in
g
 (
n
m
)
0 5 10 15 20
0.0
0.2
0.4
0.6
0.8
[µM]
Steady state binding
21 µM
14 µM
7 µM
3.5 µM
1 µM
BiP
21 µM
14 µM
7 µM
3.5 µM
1 µM
BiP
14.05
3.13
0.96
KD
SD
R2
14.29
4.66
0.92
KD
SD
R2
B
GST
J
Sensor
ATP
BiP
B
GST
J
Sensor
ATP
AMP
BiP
Figure 5. AMPylation does not alter non-substrate interactions between BiP and a model J-domain. Bio-layer
interferometry (BLI) experiment based on the experiment outlined in Figure 4C to measure the functionally
relevant non-substrate interactions between an ATPase and substrate binding BiP mutant protein (BiPT229A-V461F)
and a model J-domain fused to GST (GST-J). The cartoons on the left depict the anticipated interactions between
BiP in solution and the J-domain on the sensor surface during the association step and plots of BLI binding signals
against time are shown on the right. After an initial equilibration step the sensors were saturated with biotinylated
GST-J followed by another wash step to achieve a stable baseline signal (not shown). The sensors were then
exposed to solutions containing different concentrations of unmodified or AMPylated BiPT229A-V461Fproteins to
measure their association with the sensors. Dissociation was detected in protein-free solutions containing ATP.
Representative plots of recorded binding signals of the association and dissociation steps are shown. The insets
present plots of the plateau binding amplitudes during the association step against BiP concentrations of three
independent experiments as well as the obtained dissociation constant (KD) values with the corresponding
standard deviations and the R2 values of the fits.
DOI: https://doi.org/10.7554/eLife.29428.016
The following figure supplements are available for figure 5:
Figure supplement 1. The non-substrate interaction between BiPT229A-V461F and the BLI sensor requires a
functional J-domain.
DOI: https://doi.org/10.7554/eLife.29428.017
Figure supplement 2. The non-substrate interaction between BiPT229A-V461F and the J-domain is sensitive to
elevated salt concentrations.
DOI: https://doi.org/10.7554/eLife.29428.018
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 12 of 28
Research article Biochemistry Biophysics and Structural Biology
complex was very similar in presence of ADP and ATP (Figure 5—figure supplement 1; a stark con-
trast to the nucleotide-dependent dissociation kinetics of BiPWT from BLI sensors loaded with GST-
JWT and P15, Figure 4C). Second, consistent with the predicted ionic character of the interaction
between the HPD motif of the J-domain and BiP/Hsp70, binding was sensitive to increasing salt con-
centrations, whereas substrate interactions between BiPWT and the sensor remained nearly unaf-
fected (Figure 5—figure supplement 2A and B). Thus, AMPylation did not alter BiP’s HPD motif-
dependent, non-substrate interaction with the J-domain.
AMPylation inhibits the J protein-stimulated ATPase activity of BiP, the
rate-limiting step of its nucleotide-driven cycle
The observations described in the previous section suggested that defective J protein-stimulated
ATP consumption by AMPylated BiP, observed in a relatively crude, cumulative assay
(Preissler et al., 2015b’, Figure 8C therein), arose from interference with the ATP hydrolysis step
that follows normal engagement of the J-domain. To investigate further the direct effect of AMPyla-
tion on BiP’s J protein-stimulated ATPase activity, the rates of an individual round of ATP hydrolysis
were measured in a ‘single-turnover’ format. In presence of non-functional GST-JQPD, unmodified
BiPWT showed a very low basal ATP hydrolysis rate (Figure 6A), as expected (Kassenbrock and
Kelly, 1989; Mayer et al., 2003; Wei and Hendershot, 1995). Basal ATP turnover of AMPylated
BiPWT was only slightly (but not significantly) decreased. In contrast, exposure to a wildtype
J-domain increased the ATPase rate of unmodified BiPWT more than 4-fold but had almost no effect
on the rate of ATP hydrolysis by AMPylated BiPWT (Figure 6A). By uncoupling ATP hydrolysis from
nucleotide exchange this assay points directly to an AMPylation-induced defect in the ability of ATP-
bound BiP to respond to the stimulatory effect of J-protein on ATPase activity. This defect ade-
quately explains much of the previously observed slower J protein-stimulated ATP hydrolysis by
AMPylated BiP.
Nonetheless, the effect of AMPylation was not limited to the ability of BiP to respond to J-pro-
tein. Release of ADP (and subsequent binding of ATP) re-establishes the low-affinity (high ‘off’ rate)
state and is therefore crucial to BiP’s ability to complete its substrate binding cycle. To determine if
AMPylation affects the release of ADP (which in the presence of excess ATP is the rate-limiting step
for nucleotide exchange, Mayer et al., 2003; Theyssen et al., 1996) we compared the dissociation
rate of a fluorescent ADP derivative (MABA-ADP) from unmodified and AMPylated BiP in a stopped-
flow apparatus connected to a fluorometer. MABA-ADP dissociated from unmodified BiP with a rate
constant of 9.7  102 ± 1.8103 s1 (within the range reported previously, Mayer et al., 2003).
AMPylation imposed a modest but reproducible defect on the release of MABA-ADP
(7.2  102 ± 7.1103 s1; Figure 6B). This was likely a direct effect of the modification, rather
than a consequence of the inability of AMPylated BiPWT to form oligomers in the ADP-bound state,
because unmodified oligomerization-deficient BiPV461F had the same MABA-ADP release rate as
unmodified BiPWT (9.4  102 ± 3.1103; Figure 6B).
ER calcium concentrations are high and it has been shown that BiP binds ADP with higher affinity
at physiological calcium concentrations (Lamb et al., 2006). Moreover, nucleotide exchange factors,
such as Grp170, enhance ADP release from BiP in the ER (Behnke et al., 2015; Weitzmann et al.,
2006). Accordingly, addition of calcium to the reaction caused a general (~5 fold) decrease in the
MABA-ADP release rate (Figure 6C). Slightly slower MABA-ADP release imposed by BiP AMPylation
was observed under these more physiological conditions too, whereas AMPylation had no significant
effect on Grp170-stimulated ADP release (Figure 6C and Figure 6—figure supplement 1). Further-
more, under the conditions studied here, the J domain-stimulated ATPase rate of unmodified BiP
remained slower than even the rate of spontaneous ADP release. While both steps are impaired by
AMPylation, the former is more severely affected. This identifies inhibition of the rate-limiting step
of BiP’s ATPase cycle as the main mechanism of its functional inactivation by AMPylation.
Discussion
AMPylation is a reversible modification of BiP that correlates inversely with the burden of unfolded
proteins in the ER. In the resting ER of secretory cells up to one of every two molecules of the chap-
erone may be found in the modified state (Chambers et al., 2012’, Figure 1 therein). Our efforts to
understand the consequences of this physiologically-entrained, high-stoichiometry modification of
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 13 of 28
Research article Biochemistry Biophysics and Structural Biology
B
iP
V
46
1F
A
0.000
0.004
0.008
0.012
 A
T
P
 h
y
d
ro
ly
s
is
 r
a
te
(s
-1
)
BiP:
BiP-AMP:
- -
- -
+ +
+ +
GST-JWT: + +- -
GST-JQPD: + +- -
B
B
iP
B
iP
-A
M
P
0.00
0.02
0.04
0.06
0.08
0.10
0.12
D
is
s
o
c
ia
ti
o
n
 r
a
te
 o
f
M
A
B
A
-A
D
P
 (
s
-1
)
C
0.010
0.006
0.002
****
B
iP
B
iP
 +
 G
rp
17
0
B
iP
-A
M
P
B
iP
-A
M
P
 +
 G
rp
17
0
0.00
0.02
0.04
0.06
0.08
0.10
*
D
is
s
o
c
ia
ti
o
n
 r
a
te
 o
f
M
A
B
A
-A
D
P
 (
s
-1
)
n.s.
****
n.s.
****
n.s.
****
no calcium 2 mM calcium
0 0
.5
1 2 3 5 1
0
1
5
2
0
ADP
ATP
ADP
ATP
Time (min):
G
S
T
-J
Q
P
D
G
S
T
-J
B
iP
B
iP
-A
M
P
Autoradiograph
ADP
ATP
ADP
B
iP
B
iP
-A
M
P
ATP
Figure 6. Effect of AMPylation on J domain-stimulated ATPase activity of BiP and ADP release from BiP. (A) Shown is an autoradiograph of 32P-labeled
ATP and ADP separated by thin-layer chromatography, the products of a single-turnover ATPase assay to analyze the effect of AMPylation on ATP
hydrolysis by BiP. Pre-formed complexes between purified unmodified or AMPylated wildtype BiP protein and a-32P-ATP were incubated without or
with wildtype GST-J or the QPD mutant for the indicated times prior analysis by thin-layer chromatography. A representative experiment is shown on
the left and the signals from five repeats of the experiment were quantified and the calculated ATP hydrolysis rates are presented on the graph. Error
bars represent standard deviations. ****p<0.0001, n.s. p>0.05. (B) Measurement of nucleotide release from BiP in absence of calcium. Unmodified or
AMPylated wildtype or V461F mutant BiP proteins were incubated with the fluorescent ADP derivative MABA-ADP and the dissociation of the formed
complexes was measured upon dilution with a solution containing excess of ATP to prevent re-binding of MABA-ADP. The dissociation rates of at least
three independent repeats are shown. Error bars represent standard deviations. ****p<0.0001, n.s. p>0.05. (C) A similar experiment as in ‘B’ was
performed in presence of 2 mM calcium in the solution and without or with Grp170. The dissociation rates of at least five independent repeats are
shown. Error bars represent standard deviations. *p=0.0281, ****p<0.0001, n.s. p>0.05.
DOI: https://doi.org/10.7554/eLife.29428.019
The following source data and figure supplement are available for figure 6:
Source data 1. Source data and calculated rates for the single-turnover ATPase assays shown in Figure 6A.
DOI: https://doi.org/10.7554/eLife.29428.021
Figure 6 continued on next page
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 14 of 28
Research article Biochemistry Biophysics and Structural Biology
an important ER chaperone have yielded two key findings: Regardless of the identity of the bound
nucleotide, AMPylation favors the domain-docked conformation of BiP; a conformation that specifies
unstable substrate binding and is normally associated with ATP-bound Hsp70s. In this domain-
docked conformation AMPylated BiP is recognized by J-proteins, however, the modification of T518
imposes an intrinsic blockage on BiP’s ability to respond to the co-chaperone by accelerated ATP
hydrolysis.
AMPylation weakens BiP-substrate interactions
ADP-bound BiP adopts a heterogeneous ensemble of conformations with a bias towards the
domain-undocked state, whereas ATP binding strongly favors domain docking (Marcinowski et al.,
2011). The findings presented here suggest that even when bound to ADP the equilibrium of AMPy-
lated BiP is shifted towards a conformation in which the SBD and NBD are docked onto each other,
thus resembling the ATP-bound state of unmodified BiP. This is evident both in solution-based pro-
tease-sensitivity experiments that track the disposition of the interdomain linker of BiP (a proxy for
its conformational state, Zhuravleva et al., 2012) and in the crystal structure of AMPylated BiP,
which is found in a domain-docked conformation despite the absence of ATP in the nucleotide bind-
ing domain (Figure 7A).
The substrate-binding domain of AMPylated apo BiP is found in a conformation typical of ATP-
bound Hsp70/BiP (Kityk et al., 2012; Yang et al., 2015) and its widened substrate binding groove
likely underlies the destabilizing effect of AMPylation on substrate binding. This structural feature is
concordant with the enfeebling effect of AMPylation on BiP oligomerization in vitro (noted here),
with previous observations that AMPylation correlates with an increased pool of monomeric cellular
BiP and with higher substrate dissociation rates from ADP-bound AMPylated BiP observed in vitro
(Preissler et al., 2015b’, Figure 8E therein).
Loop L7,8, comprising T518, and the connected b-sheet in the SBDb subdomain undergo major
rearrangements during the transition between the nucleotide-dependent conformations
(Yang et al., 2015; Zhuravleva and Gierasch, 2015). In the ADP-bound state the T518 side chain is
oriented inwards and contributes to contacts amongst other loop residues. These contacts are lost
upon ATP binding (Yang et al., 2015), confining AMPylation to the ATP-bound conformation of BiP
(Preissler et al., 2015b’, Figure 7 therein). In the structures presented here the modified T518 side
chain was flipped outward and pointed into the solvent. This suggests that the bulky and hydrophilic
AMP moiety sterically prevents loop L7,8 from reverting to its ADP-bound position. As a conse-
quence, the connected downstream b-sheet8 likely retains its conformation even in absence of ATP,
biasing the SBD towards a conformation associated with high substrate ‘off’ rates (Zhuravleva and
Gierasch, 2015) and stabilizing the contacts at the interface between the SBD and NBD, including
the linker region. The net effect is to interfere with the allosteric transition to the domain-undocked
conformation, normally observed in the ADP-bound state.
The benefit of inactivating BiP in a conformation that precludes stable engagement of substrates
may arise in circumstances of an excess of BiP over its client proteins; for example, in the declining
phase following a physiological burst of secreted protein synthesis. By interfering with the ability of
excess BiP to engage substrates, AMPylation works against the tendency of the over-chaperoned ER
to degrade clients that would otherwise be secreted (Dorner et al., 1992). Furthermore, over-
expression of BiP that is locked in a conformation of slow substrate release is detrimental to ER func-
tion (Hendershot et al., 1995), suggesting how the emergence of a mechanism for inactivating
excess BiP in the alternative conformation, specified by AMPylation, may have been selected.
Figure 6 continued
Source data 2. Source data and calculated rates for the MABA-ADP release measurements shown in Figure 6B.
DOI: https://doi.org/10.7554/eLife.29428.022
Source data 3. Source data and calculated rates for the MABA-ADP release measurements shown in Figure 6C.
DOI: https://doi.org/10.7554/eLife.29428.023
Figure supplement 1. Grp170 stimulates MABA-ADP release from BiP.
DOI: https://doi.org/10.7554/eLife.29428.020
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Research article Biochemistry Biophysics and Structural Biology
JJ
BiP-AMP
BiP
NBD
SBD
ATP
Substrate
Pi ADP
Linker
J-domain
AMP
A
B
Substrate
ADP
LinkerNBD
SBD
BiP
BiP-AMP
ADP
AMP
AMP
Figure 7. Hypothesized mechanisms by which AMPylation inactivates BiP. (A) ADP-bound unmodified BiP is strongly biased towards a domain-
undocked conformation with low substrate ‘off’ rates. AMPylation (BiP-AMP) biases the ADP-bound chaperone towards a more domain-docked state
with higher substrate ‘off’ rates. As a consequence, AMPylation enfeebles BiP-client interactions. (B) The contacts formed between the docked
nucleotide binding domain (NBD) and substrate binding domain (SBD) of ATP-bound BiP are proposed to inhibit its basal ATPase activity (red arrow).
J-domain interaction with BiP likely weakens these inhibitory interdomain contacts favoring ATP hydrolysis (Kityk et al., 2015) and triggering complete
domain undocking, exposure of the interdomain linker (green), and stable substrate binding (red). By an allosteric mechanism, AMPylation on threonine
518 further strengthens the domain-docked conformation of ATP-bound BiP, which also strengthens the ATPase-inhibitory interdomain contacts (bold
red arrow). This elevates the threshold for J domain-mediated stimulation of ATPase activity.
DOI: https://doi.org/10.7554/eLife.29428.024
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Research article Biochemistry Biophysics and Structural Biology
AMPylation interferes with BiP’s responsiveness to J-proteins
The consequences of AMPylation to BiP’s functional interactions with ER-localized J-proteins may be
even more important than the effects of AMPylation on substrate binding in the ADP-bound state.
In cells the interaction of Hsp70 proteins with their clients is governed by J-domain co-chaperones
that instruct a non-equilibrium ATP hydrolysis-driven conformational cycle (Kampinga and Craig,
2010). The initial interactions between the ATP-bound chaperones and their substrates occur with
high association rates followed by nucleotide hydrolysis that then strongly decreases the dissociation
of the chaperone-substrate complex. Under physiological conditions (of excess ATP) J-proteins
recruit ATP-bound Hsp70s to their substrates and thus couple substrate binding to acceleration of
ATP hydrolysis, which increases the effective binding affinity beyond the equilibrium values observed
in either nucleotide-bound state, thus establishing a regime of ultra-affinity (De Los Rios and Bar-
ducci, 2014).
AMPylation strongly interferes with the ability of J-proteins to impart ultra-affinity on BiP. This is
reflected both by the dramatic defect in J protein-driven BiP oligomerization and by the defect in J
protein-mediated BiP association with a model substrate in BLI experiments. Both the higher dissoci-
ation rate of substrates from ADP-bound AMPylated BiP and the defect in J protein-stimulated ATP
hydrolysis appear to contribute to the inability of AMPylated BiP to attain ultra-affinity for its sub-
strates. Thus, by targeting the rate-limiting step of its ATPase cycle AMPylation favors the retention
of BiP in an inert, mostly ATP-bound conformation, whilst impeding wasteful ATP hydrolysis.
The significance of the modest, but reproducible defect observed in nucleotide exchange from
AMPylated BiP, remains to be determined. The rate of ATP hydrolysis we observed was slower than
ADP dissociation, suggesting that AMPylated BiP spends most of its time in the ATP-bound state (a
feature that is likely accentuated in cells by the presence of nucleotide exchange factors). Nonethe-
less, a slowing of nucleotide exchange would serve to further limit ATP consumption by AMPylated
BiP, whilst retaining even the small ADP-bound AMPylated fraction of BiP in a relatively inert state.
Insights into AMPylation-mediated interference with J-protein
stimulated ATP hydrolysis
AMPylation does not affect the ability of BiP to engage in protein-protein interactions with the
J-domain - unmodified and AMPylated BiP both bind the J-domain with similar affinities - rather
AMPylation blocks the subsequent stimulation of ATP hydrolysis. Can this be explained by the struc-
tural effect of the modification?
It has been proposed that the interdomain contacts formed between the SBD and NBD in the
ATP-bound conformation of the E. coli Hsp70 DnaK communicate to the active site within the NBD
to inhibit ATP hydrolysis (Kityk et al., 2015). Docking of the SBD against the NBD likely exerts a
similar inhibitory effect in BiP, as the ATPase activity of the isolated NBD is 2-fold greater than that
of intact BiP (Hendershot et al., 1995) and BiP’s ATPase activity is increased 2-fold by mutations
that interfere with domain docking (Awad et al., 2008). Furthermore, the SBD-NBD contacts identi-
fied by Kityk et al. (Kityk et al., 2015) as having a role in enforcing the inhibited state, are largely
conserved in the crystal structure of AMPylated BiP.
J-proteins, which bind at the interface between the NBD and SBD, are proposed to weaken the
interaction between the two, relieving the repression and thereby favoring ATP hydrolysis
(Awad et al., 2008). Our findings suggest that BiP AMPylation further biases even the ATP-bound
BiP towards the domain-docked conformation (compare reactions 4 and 8 in Figure 1). This likely
arises from the presence of a bulky modification on T518, which prevents major rearrangements in
the SBDb by enforcing a specific conformation on loop L7,8, thus stabilizing contacts between the
SBD and NBD, frustrating J-protein action (Figure 7B). The importance of long-range allosteric com-
munication between the SBD and NBD of BiP and the impact of AMPylation and other subtle
changes in the SBD on this allostery receive independent support from NMR studies of the Zhurav-
leva lab (Wieteska et al., 2017). Though situated on the opposite end of the SBD, AMPylation allo-
sterically targets the principal mechanism by which J-proteins enhance ATP hydrolysis by the NBD of
Hsp70s.
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Research article Biochemistry Biophysics and Structural Biology
Materials and methods
Plasmids
The plasmids used in this study were described previously or generated by standard molecular clon-
ing techniques and are listed in Supplementary file 2.
Protein purification
Wildtype and mutant Chinese hamster BiP proteins carrying an N-terminal hexahistidine (His6)-tag
were purified as described previously (Preissler et al., 2015b) with modifications. Proteins were
expressed in M15 E. coli cells (Qiagen, Hilden, Germany) and the bacterial cultures were grown at
37˚C to an optical density (OD600nm) of 0.8 in LB medium containing 50 mg/ml kanamycin and 100 mg/
ml ampicillin. Protein expression was induced with 1 mM isopropylthio b-D-1-galactopyranoside
(IPTG) and cells were further incubated at 37˚C for 6 hr before they were harvested by centrifugation.
The cells were lysed with a high-pressure homogenizer (EmulsiFlex-C3; Avestin, Mannheim, Germany)
in buffer A [50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM MgCl2, 0.2% (v/v) Triton X-100, 10% (v/v)
glycerol, 20 mM imidazole] containing protease inhibitors [2 mM phenylmethylsulphonyl fluoride
(PMSF), 4 mg/ml pepstatin, 4 mg/ml leupeptin, 8 mg/ml aprotinin] and 0.1 mg/ml DNaseI. The lysates
were centrifuged for 30 min at 25,000 g and incubated with 1 ml Ni-NTA agarose (Qiagen) per 1 l of
expression culture for 2 hr rotating at 4˚C. The matrix was then transferred to a gravity-flow column
and washed with buffer B [50 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.2% (v/v) Triton X-100, 10% (v/v)
glycerol, 30 mM imidazole] followed by buffer C [50 mM HEPES-KOH pH 7.4, 300 mM NaCl, 5% (v/v)
glycerol, 10 mM imidazole, 5 mM b-mercaptoethanol] and further wash steps in buffer C supple-
mented sequentially with (i) 1% (v/v) Triton X-100, (ii) 1 M NaCl, (iii) 3 mMMg2+-ATP, or (iv) 0.5 M Tris-
HCl pH 7.5. After a further wash step in buffer C containing 35 mM imidazole the retained BiP proteins
were eluted with buffer D [50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 5% (v/v) glycerol, 5 mM b-mer-
captoethanol, 250 mM imidazole] and dialyzed against HKM buffer (50 mM HEPES-KOH pH 7.4, 150
mM KCl, 10 mM MgCl2). The proteins were concentrated using centrifugal filters (Amicon Ultra, 30
kDa MWCO; Merck Millipore, Darmstadt, Germany), snap-frozen in liquid nitrogen, and stored at
80˚C.
To generate nucleotide-free (apo) BiP preparations the purified proteins were further dialyzed
twice for 12 hr at 4˚C against buffer E (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA), twice for
12 hr against buffer E containing 2 mM EDTA, once for 6 hr against buffer E without EDTA, and twice
for 12 hr against HK buffer (50 mM HEPES-KOH pH 7.4, 150 mM KCl). The effectiveness of this treat-
ment to remove bound nucleotide was confirmed by ion pair HPLC as described previously
(Preissler et al., 2017). Nucleotide-free BiP proteins were used in the experiment described in Fig-
ure 2—figure supplement 2.
The E. coli Hsp70 protein DnaK (Figure 6—figure supplement 1B) was expressed as a fusion pro-
tein with an N-terminal His6-Smt3 from a pET24-based plasmid (UK 2243) in E. coli C3013 BL21 T7
Express lysY/Iq cells (New England BioLabs, Ipswich, MA). The cells were grown in LB medium con-
taining 50 mg/ml kanamycin to OD600nm 0.6 at 37˚C and expression was induced with 0.4 mM IPTG
at 30˚C for 4 hr. The cells were lysed and Ni affinity chromatography was performed as described
above (see BiP purification), except that 1 mM b-mercaptoethanol was added to all buffers. For
cleavage of His6-Smt3, Ulp1 enzyme was added to the eluted protein in a 1:1000 mass ratio
together with 1 mM ATP and dialyzed for 16 hr at 4˚C against HKM buffer supplemented with 1 mM
b-mercaptoethanol. After addition of 2 mM ATP the dialyzed solution was immediately passed
through a Superdex 200 10/300 GL gel filtration column (GE Healthcare, Chicago, IL) connected in
series with a 1 ml HisTrap HP column (GE Healthcare) in HKM buffer containing 1 mM b-mercaptoe-
thanol. The DnaK containing elution fractions were pooled, concentrated, and flash frozen in
aliquots.
Human Grp170 protein (UK 1264) carrying an N-terminal His6-tag was expressed in E. coli BL21
(DE3) cells. The cells were grown at 37˚C in LB medium containing 50 mg/ml kanamycin to OD600nm
0.6. The cells were then shifted to 20˚C for 30 min and expression was induced with 1 mM IPTG for 4
hr. The cells were lysed and the protein was affinity purified as described above (see BiP purification)
with the exception that detergent was omitted and 5 mM ATP was present in all solutions. The final
wash steps of the affinity matrix were performed with buffer C containing 5 mM ATP sequentially
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 18 of 28
Research article Biochemistry Biophysics and Structural Biology
supplemented with (i) 0.5 M NaCl, (ii) 0.25 M Tris-HCl pH 7.5, or (iii) 35 mM imidazole. The bound pro-
tein was eluted with buffer D containing 5 mMATP and further purified by size-exclusion chromatogra-
phy using a Superdex 200 10/300 GL column (GE Healthcare) in HKM buffer containing 0.5 mM ATP.
The protein eluted in two main peaks (the earlier one likely representing inactive oligomeric assem-
blies) and Grp170 containing fractions of the later eluting peak were pooled and immediately frozen in
aliquots and stored at 80˚C. SDS-PAGE analysis and Coomassie staining revealed that the final prep-
aration still contained additional faster migrating protein species (Figure 6—figure supplement 1A).
These likely comprise proteolytic fragments of full-length Grp170 as in-gel tryptic digest and mass
spectrometry analysis of the corresponding gel bands identified mainly Grp170-derived peptides (Fig-
ure 6—figure supplement 1A and Supplementary file 1). Accordingly, most species efficiently
bound to Ni-NTA agarose under denaturing conditions (Figure 6—figure supplement 1A). Although
Grp170 is glycosylated in mammalian cells (Lin et al., 1993) bacterially expressed Grp170 stimulated
MADA-ADP release from BiP in a concentration-dependent manner, but not from the bacterial Hsp70
DnaK, indicating its functionality and specificity in this assay (Figure 6—figure supplement 1B).
Expression and purification of N-terminally GST-tagged AMPylation-active GST-FICDE234G mutant
protein was performed as described earlier (Preissler et al., 2015b). The protein was produced in E.
coli C3013 BL21 T7 Express lysY/Iq cells at 37˚C in LB medium containing 100 mg/ml ampicillin.
Expression was induced at OD600nm 0.8 with 0.5 mM IPTG and the cultures were shifted to 20˚C for
16 hr. The cells were harvested and lysed as described above in lysis buffer [50 mM Tris-HCl pH 7.5,
500 mM NaCl, 1 mM MgCl2, 2 mM dithiothreitol (DTT), 0.2% (v/v) Triton X-100, 10% (v/v) glycerol]
containing protease inhibitors and DNaseI. The lysate was centrifuged for 30 min at 25,000 g and
incubated with 0.7 ml Glutathione Sepharose 4B (GE Healthcare) per 1 l of expression culture. After
incubation for 2 hr at 4˚C the beads were washed extensively with wash buffer F [50 mM Tris-HCl pH
7.5, 500 mM NaCl, 1 mM DTT, 0.2% (v/v) Triton X-100, 10% (v/v) glycerol] containing protease inhib-
itors, wash buffer G [50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1% (v/v)
Triton X-100, 10% (v/v) glycerol] containing protease inhibitors, and wash buffer G sequentially sup-
plemented with (i) 1% (v/v) Triton X-100, (ii) 1 M NaCl, (iii) 3 mM ATP, or (iv) 0.5 M Tris-HCl pH 7.5.
Retained protein was eluted with elution buffer H [50 mM HEPES-KOH pH 7.4, 100 mM KCl, 4 mM
MgCl2, 1 mM CaCl2, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, 40 mM reduced glutathione], snap-
frozen in liquid nitrogen, and stored at 80˚C.
The wildtype J-domain (UK 185) of mouse P58/ERdj6 (residues 384–470) and the H422Q mutant
thereof (UK 186) were expressed as GST-fusion proteins (here referred to as GST-JWT and GST-JQPD,
respectively) according to (Petrova et al., 2008) and purified by gluthathione (GSH) affinity chroma-
tography as described above. Proteins were eluted in buffer H containing 1 mM DTT, dialyzed over
night against HKM at 4˚C, and aliquots were frozen in liquid nitrogen and stored at 80˚C. A fraction
of purified GST-JWT or GST-JQPD proteins were adjusted to 160 mM and biotinylated with 1.65 mM
Biotin-maleimide (Sigma, St. Louis, MO, cat. no. B1267) for 1 hr at 24˚C in HKM buffer. The reaction
was stopped on ice and proteins were passed through a Centri.Pure P25 desalting column (emp
BIOTECH, Berlin, Germany) equilibrated with HKM. The protein containing elution fractions were
pooled and aliquots were stored frozen.
AMPylation of purified BiP proteins
AMPylation of purified BiP proteins was performed as previously described (Preissler et al., 2015b)
with minor modifications. Purified BiP proteins were incubated for 6 hr at 30˚C with 0.25 mg bacterially
expressed GST-FICDE234G per 20 mg of BiP protein in presence of 3 mM ATP in buffer I [25 mM
HEPES-KOH pH 7.4, 100 mM KCl, 10 mM MgCl2, 1 mM CaCl2, 0.1% (v/v) Triton X-100] followed by
binding to Ni-NTA agarose beads for 1 hr at 25˚C. The beads were washed with buffer I and eluted in
buffer I containing 350 mM imidazole for 45 min at 25˚C. The eluates were desalted using a Cen-
tri.Pure P25 column equilibrated in HKM buffer. The protein-containing fractions were pooled, frozen
in liquid nitrogen, and stored at 80˚C. Unmodified BiP prepared from mock AMPylation reactions
without enzyme served as a control in the experiments. Intact protein mass spectrometry analysis of a
representative preparation confirmed modification at high stoichiometry (Figure 1—figure supple-
ment 3).
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Research article Biochemistry Biophysics and Structural Biology
Limited proteolysis experiments
For SubA-mediated BiP cleavage experiments purified wildtype or mutant BiP proteins were
adjusted to a concentration of 0.5 mg/ml in HKM buffer containing 3 mM ATP, ADP or no added
nucleotide. The reactions were incubated at 30˚C and samples were taken before and at the indi-
cated time intervals after addition of 20 ng/ml SubA protease. The withdrawn samples were immedi-
ately denatured in SDS-sample buffer and heated for 5 min at 75˚C. The digested samples together
with the undigested controls were analyzed by reducing SDS-PAGE and the proteins on the gels
were visualized by Coomassie staining with InstantBlue solution (expedeon, Over, United Kingdom).
The band intensities were quantified with Image J64 (NIH; RRID: SCR_003070).
Protein crystallization and structure determination
Chinese hamster (Cricetulus griseus) BiP (residues 28–549) with a T229A mutation (Figure 2A) was
expressed as a His6-Smt3 fusion protein (UK 1607) in M15 E. coli cells. The bacterial cultures were
grown at 37˚C to OD600nm 0.8 in LB medium containing 50 mg/ml kanamycin and 100 mg/ml ampicil-
lin and expression was induced with 1 mM IPTG. The cells were further grown at 25˚C for 14 hr, har-
vested and lysed in high-salt buffer (50 mM Tris-HCl pH 7.4, 500 mM NaCl, 1 mM MgCl2, 2 mM
PMSF) containing protease inhibitors and DNaseI as described above. The lysate was cleared by cen-
trifugation for 30 min at 25,000 g, passed through a 0.45 mm syringe filter, and supplemented with
25 mM imidazole. The following purification strategies resulted in protein crystals that were suitable
for X-ray data collection.
Strategy A (PDB 5O4P): The cleared lysate from a 4 l expression culture was passed over a 5 ml
Ni-Sepharose HisTrap HP column (GE Healthcare) at 4˚C. The column was washed sequentially with
30 ml high-salt buffer and 30 ml low-salt buffer (50 mM Tris, pH7.4, 100 mM NaCl, 1 mM MgCl2)
both containing 25 mM imidazole and 1 mM ATP. Bound protein was eluted with low-salt buffer sup-
plemented with 500 mM imidazole (pH 7.4) followed immediately by addition of 10 mM ATP, 10
mM MgCl2 and 1 mM Tris(2-carboxyethyl)phosphine (TCEP). Purified GST-FICD
E234G protein and
Ulp1 protease were added in a 60:1 and 1000:1 (His6-Smt3-haBiP:X) mass ratio, respectively, and
incubated 14 hr at 24˚C to simultaneously allow for AMPylation and cleavage of the His6-Smt3 tag.
The solution was then supplemented with 100 mM EDTA and the protein was further purified by
size-exclusion chromatography using a HiLoad 16/60 Superdex 75 prep grade column (GE Health-
care) equilibrated with GF buffer (5 mM HEPES-KOH pH 7.6, 100 mM NaCl, 0.1 mM EDTA, 0.1 mM
TCEP). A Glutathione Sepharose 4B column (GSTrap 4B; GE Healthcare) was connected in series
with the gel filtration column to retain GST-FICDE234G protein. The eluted protein was concentrated
to >50 mg/ml in presence of 1 mM TCEP and crystallization was performed in 96-well sitting drop
plates by combining 150 nl protein solution with 150 nl reservoir solution and equilibration at 20˚C
against 80 ml reservoir solution. Diffraction quality crystals grew in a solution containing 100 mM
HEPES pH 7.5 and 1.5 M Li2SO4. Crystals were soaked in cryosolution [the precipitant solutions con-
taining 20% (v/v) glycerol] and snap frozen in liquid nitrogen. Diffraction data were collected at the
Diamond Light Source beamline I02 (or I24, Didcot, United Kingdom; see Table 1) and processed
with Mosflm (Battye et al., 2011) and Aimless (Evans, 2011). The structure was solved by molecular
replacement in Phaser (McCoy et al., 2007) by searching 2 copies of the nucleotide binding domain
(PDB 3IUC) and substrate binding domain (PDB 4B9Q). Manual model building was carried out in
COOT (Emsley et al., 2010) and further refinements in refmac5 (Winn et al., 2001) and phenix.
refine (Adams et al., 2010). The final refinement statistics are summarized in Table 1. The structural
graphs were generated with PyMOL software (PyMOL version 1.5.0.4; RRID: SCR_000305).
Strategy B (PDBs 6EOB and 6EOC): Protein was expressed in a 6 l culture and cell lysis was per-
formed as described in strategy A. The lysate was supplemented with 0.1 mM TCEP and incubated
with 6 ml Ni-NTA agarose for 1 hr while slowly rotating at 4˚C. The suspended matrix was then trans-
ferred to a Glass Econo-column (2.5  10 cm; Bio-Rad, Hercules, CA) and washed first with high-salt
buffer containing protease inhibitors and then with wash buffer J (50 mM Tris-HCl pH 8, 500 mMNaCl,
30 mM imidazole). Further wash steps were performed with wash buffer K (50 mM Tris-HCl pH 8, 300
mM NaCl, 10 mM imidazole, 5 mM b-mercaptoethanol) sequentially supplemented with (i) 1 M NaCl,
(ii) 10 mM MgCl2 and 3 mM ATP, (iii) 0.5 M Tris-HCl pH 8 and (iv) 40 mM imidazole. The retained pro-
tein was eluted with elution buffer L (50 mM Tris-HCl pH 7.4, 100 mMNaCl, 250 mM imidazole, 10 mM
MgCl2, 10 mM ATP, 1 mM TCEP). After AMPylation with GST-FICD
E234G and cleavage of the His6-
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Research article Biochemistry Biophysics and Structural Biology
Smt3 tag with Ulp1 (as described in strategy A) the protein solution was incubated with 1 ml Glutathi-
one Sepharose 4B for 2 hr at room temperature (to bind GST-FICDE234G). The matrix was collected by
centrifugation and the supernatant was dialyzed twice for 14 hr against 5 l buffer M (25 mM Tris-HCl
pH 7.4, 50 mMNaCl, 5 mM EDTA) and once for 24 hr against 10 l AEX-LS buffer (25 mM Tris-HCl pH 8,
50 mM NaCl). Afterwards, the protein was subjected to anion exchange chromatography using a 5 ml
HiTrap Q HP column (GE Healthcare) equilibrated in AEX-LS buffer and bound protein was eluted with
AEX-HS buffer (25 mM Tris-HCl pH 8, 1 M NaCl) on a linear gradient (0% to 50% AEX-HS in 20 column
volumes). The purest fractions were pooled and incubated with 1.5 ml Ni-NTA agarose beads in pres-
ence of 25 mM imidazole for 30 min at 4˚C (to bind residual uncleaved protein and free His6-Smt3
tag). To crystallize AMPylated BiP in absence of nucleotide the protein solution was desalted over a
HiLoad 16/60 Superdex 75 prep grade column equilibrated with buffer N (10 mM HEPES-KOH pH 7.4,
100 mM NaCl). The protein solution was then supplemented with 1 mM TCEP and concentrated for
crystallization. Crystals grown in 20% PEG-1000, 0.1 M NaKHPO4 pH 6.2, 0.1 NaCl (PDB 6EOB) and
5% PEG-1000, 0.2 M Li2SO4, 0.1 M Na2HPO4 pH 4.2 (PDB 6EOC) were used for data collection and
the structures were solved as described above.
Strategy C (PDB 6EOE): Protein was expressed, purified, and AMPylated as in strategy B. Protein
obtained after ion exchange and reverse Ni affinity chromatography was incubated with 14 mMMgCl2
and 10 mM ATP for 2 hr on ice before addition of 100 mM EDTA and immediate gel filtration using a
HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) in buffer TN (25 mM Tris-HCl pH 8,
150 mM NaCl). Fractions of the main elution peak (corresponding to monomeric BiP protein) were
pooled, supplemented with 1 mM TCEP, and concentrated for crystallization. The structure of BiP crys-
tallized in 5% PEG-1000, 0.2 M Li2SO4, 0.1 M Na2HPO4 pH 4.2 was solved as described above.
Strategy D (PDB 6EOF): Protein was initially purified according to strategy B except that dialysis
(after AMPylation and removal of the modifying enzyme) was performed against buffer O (25 mM
Tris-HCl pH 7.4, 300 mM NaCl) for 16 hr at 4˚C. The dialyzed solution was passed through a column
containing 2 ml Q Sepharose High Performance matrix (GE Healthcare) by gravity flow. The flow-
trough was concentrated and applied to gel filtration using a Superdex 200 10/300 GL column equil-
ibrated in buffer TN. The purest elution fractions were pooled and dialyzed twice for 24 hr at 4˚C
against 5 l buffer TN containing 5 mM EDTA (to remove nucleotides) and once against the same
buffer without EDTA. The protein solution was then supplemented with 1 mM TCEP and concen-
trated. The crystallization reactions were set up in presence of 10 mM ADP and 14 mM MgCl2. Crys-
tals grown in 9% PEG-1000, 0.2 M Li2SO4, 0.1 M Na2HPO4 pH 4.4 were used for data collection and
the structure was solved as described above.
Analytical size-exclusion chromatography
Analytical size-exclusion chromatography (SEC) was performed as described previously
(Preissler et al., 2015a) with modifications. Purified BiP proteins were adjusted to 50 mM in HKM
buffer and incubated in a final volume of 25 ml at room temperature for 20 min before injection.
Where indicated ADP or ATP (each at 1.5 mM) as well as GST-JWT or GST-JQPD J-domain proteins
(each between 1.25 mM and 10 mM) were added to the reactions. 10 ml of each sample were injected
onto a SEC-3 HPLC column (300 A˚ pore size; Agilent Technologies, Santa Clara, CA) equilibrated
with HKM and the runs were performed at a flow rate of 0.3 ml/min at room temperature. Peptide
bond absorbance at 230 nm (A230 nm) was detected and plotted against the elution time. A gel filtra-
tion standard (Bio-Rad, cat. no. 151–1901) was applied as a size reference and the elution peaks of
Thyroglobulin (670 kDa), g-globulin (158 kDa), Ovalbumin (44 kDa), and Myoglobulin (17 kDa) are
indicated.
Bio-layer interferometry
Bio-layer interferometry (BLI) experiments were performed on the Forte´Bio Octet RED96 System
(Pall Forte´Bio, Menlo Park, CA). To study J domain-dependent substrate interactions (Figure 4C,
Figure 4—figure supplement 1 and Figure 5—figure supplement 2B) unmodified or in vitro
AMPylated BiP proteins were diluted to 15 mM in HKM buffer containing 0.05% (v/v) Triton X-100.
Biotinylated GST-JWT and GST-JQPD were diluted to 20 nM and P15 peptide (ALLLSAPRRGAGKK;
custom synthesized by GenScript, Piscataway, NJ), which was biotinylated on the C-terminal lysine,
was diluted to 50 nM in the same solution. Streptavidin (SA)-coated biosensors (Pall Forte´Bio) were
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Research article Biochemistry Biophysics and Structural Biology
hydrated in assay solution for at least five minutes before the experiment. Where indicated 2 mM
ADP or ATP was added. All solutions were prepared in a final volume of 200 ml on a 96 well micro-
plate (greiner bio-one, Kremsmu¨nster, Austria, cat. no. 655209) and data acquisition was performed
at a shake speed of 600 rpm and at 24˚C with the indicated experimental steps. In brief, after an ini-
tial equilibration step in assay solution biotinylated GST-JWT or GST-JQPD was immobilized until a dif-
ference in the binding signal of 0.4 nm was reached. The sensors were then washed and saturated
with P15 peptide. Following a baseline step the sensors were introduced in BiP protein-containing
solution to measure association followed by dipping into protein-free solution to record BiP dissocia-
tion. The dissociation data from at least three independent repeats were fitted with a one-phase
decay function to determine the dissociation rate constants using the GraphPad Prism 6 software
(GraphPad Software; RRID: SCR_002798).
The experiments to analyze non-substrate interactions between the J-domain and BiP (Figure 5,
Figure 5—figure supplement 1 and Figure 5—figure supplement 2A) were performed likewise
without P15 peptide and the sensors were saturated with biotinylated GST-JWT or GST-JQPD (both at
20 nM) before exposure to unmodified or AMPylated BiPT229A-V461F (UK 1825) at the indicated con-
centrations. During the baseline, association, and dissociation steps 1 mM ATP was present. The dis-
sociation constants were determined by plotting the steady state binding signals against the BiP
concentration and fitting the data with GraphPad Prism 6.
Single-turnover ATPase assay
ATPase assays under single-turnover conditions were performed as described previously
(Mayer et al., 1999) with modifications. Unmodified and AMPylated BiP proteins were adjusted to 30
mM in reaction solution (25 mM HEPES-KOH pH 7.4, 50 mM KCl, 10 mM MgCl2) and incubated in a
final volume of 50 ml with 0.8 mM ATP and 0.6 MBq a-32P-ATP (EasyTide; Perkin Elmer, Waltham, MA)
for 3 min on ice. The formed BiP.ATP complexes were separated from free nucleotide by gel filtration
using illustra Sephadex G-50 NICK columns (GE Healthcare, cat. no. 17-0855-01) that have been pre-
saturated with 1 ml of a 1 mg/ml bovine serum albumin (BSA) solution and equilibrated with ice-cold
reaction solution. Individual elution fractions (two drops each) were collected and the fractions of the
first radioactive peak were pooled and flash frozen in aliquots. The final protein concentration of the
pools was estimated based on the dilution of the sample during separation. For each reaction a fresh
aliquot of the pre-formed complexes was rapidly thawed at room temperature. After 0.5 ml were with-
drawn as a zero time point sample, 6 ml of the remaining BiP.ATP complexes were added to reactions
containing GST-JQPD or GST-JWT proteins (both at 2 mM) in a final volume of 50 ml. The final BiP protein
concentration in the reactions was ~0.8 mM. At the indicated time points 2 ml were withdrawn from the
reactions and spotted onto a thin layer chromatography (TLC) plate (PEI Cellulose F; Merck Millipore,
cat. no. 105579), which was pre-spotted with a mixture of 5 mM ADP and 5 mM ATP. The nucleotides
were then separated by developing the TLC plates with 400 mM LiCl and 10% (v/v) acetic acid as a
mobile phase. The plates were dried, exposed to a storage phosphor screen, and the signals were
detected using a Typhoon Trio imager (GE Healthcare). The signals were quantified using Image J64
and the ADP values were normalized to the total radioactive signal (ADP + ATP) for each time point
and fitted with a single exponential function using the GraphPad Prism 6 software.
ADP release experiments
Non-AMPylated or AMPylated BiP proteins at 1 mM were incubated with 1 mM of the fluorescent
ADP analog MABA-ADP (8-[(4-Amino)butyl]-amino-ADP - MANT; Jena Bioscience,
Jena, Germany, cat. no. NU-893-MNT) (Theyssen et al., 1996) in HKM buffer for 2 hr at 30˚C. After-
wards, the samples were mixed at 25˚C in a 1:1 (v/v) ratio with HKM containing 2 mM ATP using a
stopped-flow reaction analyzer (SX.18MV; Applied Photophysics, Leatherhead, United Kingdom).
The fluorophore was excited at 360 nm and a 420 nm cut-off filter was used to detect emission. The
traces of at least five consecutive injections per sample were averaged and the data were fitted with
a single exponential decay function.
Stimulated MABA-ADP release (Figure 6C and Figure 6—figure supplement 1B and C) was
measured in HKM containing 2 mM CaCl2. For that, 2.5 mM BiP protein was incubated with 2.5 mM
MABA-ADP for 2 hr at 30˚C and then mixed in a 1:1 ratio with the same buffer solution containing 3
mM ATP without or with 1.4 mM Grp170. The decrease in the fluorescent signal was detected at
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Research article Biochemistry Biophysics and Structural Biology
22˚C in a 3 mm quartz cell (Hellma Analytics, Mu¨llheim, Germany) with a fluorescence spectrometer
(LS 55; PerkinElmer) at an excitation wavelength of 360 nm and emission was recorded at 440 nm.
The dissociation rate constants were calculated by fitting a single exponential decay function to the
data using GraphPad Prism 6.
Mass spectrometry
Intact protein mass spectrometry was performed to analyze the AMPylation status of modified BiP
protein used for biochemical experiments (Figure 1—figure supplement 3), to set up crystallization
reactions, and of protein from dissolved crystals (Figure 2—figure supplement 4). For the latter, sev-
eral remaining crystals grown under the condition that yielded structure PDB 6EOE (after having taken
out the one used for X-ray data collection) were washed by dipping them sequentially three times in
water and dissolving in 0.5 M sodium acetate. Liquid chromatography-mass spectrometry (LC–MS)
was performed on a Xevo G2-S Tof mass spectrometer coupled to an ACQUITY UPLC system (Waters,
Elstree, United Kingdom) using an ACQUITY UPLC BEH300 C4 column (1.7 mm, 2.1  50 mm). Sol-
vents A (water with 0.1% formic acid) and B (95% acetonitrile, 4.9% water and 0.1% formic acid) were
used as the mobile phase at a flow rate of 0.2 ml/min. The electrospray source was operated with a
capillary voltage of 2.0 kV and a cone voltage of 40 V. Nitrogen was used as the desolvation gas at a
total flow of 850 l/hr. Total mass spectra were reconstructed from the ion series using the MaxEnt
algorithm preinstalled on MassLynx software (v. 4.1 from Waters; RRID: SCR_014271) according to the
manufacturer’s instructions.
For peptide mass spectrometry (Figure 6—figure supplement 1A and Supplementary file 1)
selected protein bands were excised from an SDS-polyacrylamide gel and subjected to in-gel tryptic
digestion. The resulting peptides were analyzed using a Q Exactive (Thermo
Scientific, Waltham, MA) coupled to an RSLC3000nano UPLC (Thermo Scientific). Files were
searched against a SwissProt database (downloaded 16/06/16, 551,385 entries) using Mascot 2.3
with peptide and protein validation performed in Scaffold Proteome Software 4.3.2 (RRID: SCR_
014345).
Affinity-tag based co-purification
The GSH pull-down experiment (Figure 4A) was performed as described previously (Petrova et al.,
2008). For each reaction 15 ml Glutathione Sepharose 4B beads were incubated with 30 mg GST-JWT or
GST-JQPD protein in HKM buffer containing 2 mM DTT for 30 min at room temperature while slowly
rotating. The beads were collected by centrifugation for 2 min at 100 g and washed twice in buffer P
[20mMHEPES-KOH pH 7.4, 75mMKCl, 10 mMMgCl2, 0.01% (v/v) Tween 20] to remove unbound pro-
tein followed by incubation with 30 mg unmodified or AMPylated BiPWT or BiPV461F proteins in buffer P
supplemented with 3mMADP or ATP (as indicated) for 1 hr at 4˚C. Afterwards, the beads were washed
four times on ice with buffer P containing 1 mMADP or ATP and bound proteins were eluted with 35 ml
2x SDS sample buffer for 5 min at 75˚C. The samples were analyzed by reducing SDS-PAGE and Coo-
massie staining. Samples of the adjusted BiP protein solutions were loaded as an input control.
For the Ni-NTA pull-down experiment (Figure 6—figure supplement 1A) to identify N-terminal
His6-tagged fragments of purified Grp170 (UK 1264) 10 mg protein per sample was incubated for 16
hr at 4˚C with 30 ml Ni-NTA agarose beads in HKM buffer containing 2 mM b-mercaptoethanol and
20 mM imidazole either under native or denaturing [with 6 M guanidine hydrochloride (GdnHCl)]
conditions. The beads were then recovered by centrifugation for 5 min at 500 g and washed with
HKM (native sample) or HKM containing 4 M GdnHCl (denatured sample). After three further wash
steps with HKM the bound proteins were eluted with SDS sample buffer containing 250 mM imidaz-
ole for 5 min at 75˚C. Input samples and eluted proteins were analyzed by reducing SDS-PAGE and
Coomassie staining.
Statistics
Two-tailed unpaired t-tests were performed using GraphPad Prism 6.
Acknowledgements
We thank Cla´udia Rato (Ron lab) for advice and revising the manuscript as well as Robin Antrobus
(CIMR, University of Cambridge, UK) and Dijana Matak-Vinkovic (Department of Chemistry,
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 23 of 28
Research article Biochemistry Biophysics and Structural Biology
University of Cambridge, UK) and their facility teams for mass spectrometry analyses. Further we
thank Chris Johnson (Laboratory of Molecular Biology, Cambridge, UK) for technical support and
access to the stopped-flow setup, Osamu Hori (Kanazawa University, Japan) for his gift of human
Grp170 cDNA, the Huntington lab (CIMR, University of Cambridge, UK) for access to the Octet
machine, as well as Anastasia Zhuravleva and Lukasz Wieteska (University of Leeds, UK) for exchange
of unpublished data. Supported by Wellcome Trust Principal Research Fellowships to DR (Wellcome
200848/Z/16/Z) and RJR (Wellcome 082961/Z/07/Z), a grant from the British Heart Foundation (PG/
12/41/29679) to YY, and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical
Research (Wellcome 100140).
Additional information
Competing interests
David Ron: Reviewing editor, eLife. The other authors declare that no competing interests exist.
Funding
Funder Grant reference number Author
Wellcome Wellcome 200848/Z/16/Z David Ron
Wellcome Wellcome 082961/Z/07/Z Randy J Read
Wellcome Wellcome 100140 David Ron
British Heart Foundation PG/12/41/29679 Yahui Yan
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Steffen Preissler, Conceived and led the project, Designed and conducted most experiments, Crys-
tallized BiP, Analyzed and interpreted the data, Drafted the manuscript; Lukas Rohland, Conducted,
analyzed, and interpreted experiments to study non-substrate BiP-J interactions, Contributed to pro-
tein purification, Revised the manuscript; Yahui Yan, Collected, processed, and analyzed the X-ray
diffraction data, Contributed to interpretation of the structural data and figure preparation; Ruming
Chen, Crystallized BiP and collected X-ray diffraction data, Contributed to X-ray data analysis and
interpretation; Randy J Read, Contributed to analysis and interpretation of the structural data,
Revised the manuscript; David Ron, Oversaw the project conception and design, Constructed plas-
mid DNA, Interpreted the data, Drafted and revised the manuscript
Author ORCIDs
Steffen Preissler https://orcid.org/0000-0001-7936-9836
Lukas Rohland https://orcid.org/0000-0003-1559-5097
Yahui Yan https://orcid.org/0000-0001-6934-9874
Randy J Read https://orcid.org/0000-0001-8273-0047
David Ron https://orcid.org/0000-0002-3014-5636
Decision letter and Author response
Decision letter https://doi.org/10.7554/eLife.29428.038
Author response https://doi.org/10.7554/eLife.29428.039
Additional files
Supplementary files
. Supplementary file 1. Mass spectrometry analysis of the Grp170 protein preparation. Bands of full-
length Grp170 protein and faster migrating species were cut out of a Coomassie-stained SDS-PAGE
Preissler et al. eLife 2017;6:e29428. DOI: https://doi.org/10.7554/eLife.29428 24 of 28
Research article Biochemistry Biophysics and Structural Biology
gel and analyzed by LC-MS/MS after in-gel digest with trypsin. The identified peptides for the bands
indicated in Figure 6—figure supplement 1A are presented.
DOI: https://doi.org/10.7554/eLife.29428.025
. Supplementary file 2. Plasmids used in this study. The table lists the plasmids used in this study
including references (as PMIDs).
DOI: https://doi.org/10.7554/eLife.29428.026
. Transparent reporting form
DOI: https://doi.org/10.7554/eLife.29428.027
Major datasets
The following datasets were generated:
Author(s) Year Dataset title Dataset URL
Database, license,
and accessibility
information
Yahui Yan, Ruming
Chen, David Ron,
Randy J Read
2017 Crystal structure of AMPylated
GRP78
https://www.rcsb.org/
pdb/explore/explore.do?
structureId=5O4P
Publicly available at
the RCSB Protein
Data Bank (Accession
no: 5O4P)
Yahui Yan, Steffen
Preissler, David Ron,
Randy J Read
2017 Crystal structure of AMPylated
GRP78 in apo form (Crystal form 1)
https://www.rcsb.org/
pdb/explore/explore.do?
structureId=6EOB
Publicly available at
the RCSB Protein
Data Bank (Accession
no: 6EOB)
Yahui Yan, Steffen
Preissler, David Ron,
Randy J Read
2017 Crystal structure of AMPylated
GRP78 in apo form (Crystal form 2)
https://www.rcsb.org/
pdb/explore/explore.do?
structureId=6eoc
Publicly available at
the RCSB Protein
Data Bank (Accession
no: 6EOC)
Yahui Yan, Steffen
Preissler, David Ron,
Randy J Read
2017 Crystal structure of AMPylated
GRP78 with nucleotide
https://www.rcsb.org/
pdb/explore/explore.do?
structureId=6EOE
Publicly available at
the RCSB Protein
Data Bank (Accession
no: 6EOE)
Yahui Yan, Steffen
Preissler, Randy J
Read, David Ron
2017 Crystal structure of AMPylated
GRP78 in ADP state
https://www.rcsb.org/
pdb/explore/explore.do?
structureId=6EOF
Publicly available at
the RCSB Protein
Data Bank (Accession
no: 6EOF)
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