Báo cáo khoa học: Structural framework of the GABARAP–calreticulin interface – implications for substrate binding to endoplasmic reticulum chaperones potx

Structural framework of the GABARAP–calreticulin
interface – implications for substrate binding to
endoplasmic reticulum chaperones
Yvonne Thielmann
1
, Oliver H. Weiergra
¨
ber
1
, Jeannine Mohrlu
¨
der
1,2
and Dieter Willbold
1,2
1 Institut fu
¨
r Neurowissenschaften und Biophysik, Molekulare Biophysik, Forschungszentrum Ju
¨
lich, Germany
2 Institut fu
¨
r Physikalische Biologie und BMFZ, Heinrich-Heine-Universita
¨
tDu
¨
sseldorf, Germany
The neurotransmitter 4-aminobutyrate (GABA) medi-
ates synaptic inhibition in the brain and the spinal
cord [1]. GABA receptors can be categorized into type A
(GABA

A
) receptors, which are ligand-gated chloride
channels, and type B (GABA
B
) receptors, which are
G-protein-coupled and modulate the activity of potas-
sium and calcium channels [2]. GABA
A
receptors are
relevant drug targets for benzodiazepines, barbiturates
Keywords
4-aminobutyrate type A receptor-associated
protein (GABARAP); calreticulin;
protein–protein interaction; structure model;
X-ray crystallography
Correspondence
O. H. Weiergra
¨
ber, Institut fu
¨
r
Neurowissenschaften und Biophysik,
Molekulare Biophysik, Forschungszentrum
Ju
¨
lich, 52425 Ju
¨
lich, Germany
Fax: +49 2461 612020
Tel: +49 2461 612028

E-mail:
D. Willbold, Institut fu
¨
r Physikalische
Biologie und BMFZ, Heinrich-Heine-
Universita
¨
t, 40225 Du
¨
sseldorf, Germany
Fax: +49 2461 612023
Tel: +49 2461 612100
E-mail:
Database
The atomic coordinates and structure
factor amplitudes (code 3DOW) have been
deposited in the Protein Data Bank
()
(Received 14 October 2008, revised
2 December 2008, accepted 12 December
2008)
doi:10.1111/j.1742-4658.2008.06857.x
The 4-aminobutyrate type A receptor-associated protein (GABARAP) is a
versatile adaptor protein that plays an important role in intracellular vesi-
cle trafficking, particularly in neuronal cells. We have investigated the
structural determinants underlying the interaction of GABARAP with cal-
reticulin using spectroscopic and crystallographic techniques. Specifically,
we present the crystal structure of GABARAP in complex with its major
binding epitope on the chaperone. Molecular modeling of a complex con-
taining full-length calreticulin suggests a novel mode of substrate interac-

tion, which may have functional implications for the calreticulin ⁄ calnexin
family in general.
Abbreviations
CRT(178–188), CH
3
CO-SLEDDWDFLPP-NH
2
; ER, endoplasmic reticulum; GABA, 4-aminobutyrate; GABARAP, 4-aminobutyrate type A
receptor-associated protein; GATE-16, Golgi-associated ATPase enhancer of 16 kDa; HSQC, heteronuclear single quantum coherence;
P-domain, proline-rich domain; SPR, surface plasmon resonance; Ubl, ubiquitin-like protein.
1140 FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS
and general anesthetics [3]. GABA
A
receptor-associ-
ated protein (GABARAP) was initially found in a
two-hybrid screen to interact with the cytoplasmic loop
connecting transmembrane helices 3 and 4 of the
GABA
A
receptor c2-subunit. This interaction was con-
firmed by colocalization experiments in cultured corti-
cal neurons and by coimmunoprecipitation of
GABARAP with GABA
A
receptor subunits from
brain extracts [4].
GABARAP belongs to a protein family that is evo-
lutionarily highly conserved, from yeast to mammals.
Atg8 from Saccharomyces cerevisiae has been identified
as an essential regulator of the autophagic machinery,

which serves to nonselectively sequester cytoplasmic
material for vacuolar degradation [5]. Mammalian
orthologs of this family include glandular epithelial
cell protein 1, Golgi-associated ATPase enhancer of
16 kDa (GATE-16), light chain 3 of microtubule-asso-
ciated protein 1, and GABARAP [3].
All these proteins belong to the superfamily of
ubiquitin-like proteins (Ubls). They share the charac-
teristic b-grasp fold, as first demonstrated by the
crystal structure of GATE-16 [6], and are subject to a
modification process that is similar to the ubiquitin-
type conjugation machinery. After proteolytic
cleavage, leading to exposure of a C-terminal glycine
residue, these Ubls are coupled to an E1 enzyme via
a thioester bond, further transferred from the E1
enzyme to an E2 enzyme, and finally conjugated to
phosphatidylserine or phosphatidylethanolamine. Con-
sequently, at the end of the conjugation process,
GABARAP and related proteins are attached to
cellular membranes instead of proteins, as in the case
of ubiquitin [7,8].
Available crystal structures [9–11] as well as NMR
structures of GABARAP [12] show the expected simi-
larity to other Ubls. In GABARAP, the Ubl core
domain comprising the b-grasp fold is extended by an
N-terminal segment containing two additional a-heli-
ces. We have recently determined the first three-dimen-
sional structure of GABARAP complexed with a
ligand [13]. This structure highlights the interactions of
apolar residues of a synthetic peptide with GABA-

RAP’s hydrophobic pockets. These pockets were
probed previously with indole derivatives [14] and have
also been described for GATE-16 [6].
Despite this structural knowledge for GABARAP,
data for complexes with its native interaction partners
as well as conjugating enzymes are still needed to
understand its biological function on a molecular
level. We have previously identified calreticulin and
the heavy chain of clathrin as potential binding part-
ners [15,16]. In the case of calreticulin, immunofluo-
rescence staining of neuronal cells revealed significant
colocalization with GABARAP in punctuate
structures, probably corresponding to a vesicular
compartment [15].
Calreticulin is a multifunctional lectin-like 46 kDa
Ca
2+
-binding chaperone predominantly located in the
endoplasmic reticulum (ER). It is found in a wide
range of species and is involved in intracellular Ca
2+
homeostasis as well as ER Ca
2+
storage capacity [17].
Within secretory pathways, it functions as an impor-
tant chaperone involved in quality control [18]. Studies
on calreticulin knockout mice indicate that the protein
is essential for early cardiac development [19].
Recently, cell surface calreticulin has attracted particu-
lar attention because of its role as a phagocytic signal

on apoptotic cells, implicating the protein in processes
such as autoimmunity and cancer [20]. Moreover, it
was found to be retrotranslocated from the ER lumen
into the cytosol [21], and has been ascribed specific
functions in protein transport and gene expression (see
Discussion for details). The N-terminal and C-terminal
segments of calreticulin are predicted to fold into a
composite globular domain, whereas the intervening
sequence forms an arm-like structure often referred to
as the proline-rich domain (P-domain) [17].
In this study, we investigated the interaction of
GABARAP with different calreticulin fragments,
including the complete P-domain as well as an
undecamer peptide {CH
3
CO-SLEDDWDFLPP-NH
2
[CRT(178–188)]} comprising the principal GABARAP-
binding motif [15]. In particular, we determined the
three-dimensional structure of the latter peptide associ-
ated with the GABARAP molecule. The binding mode
of this native ligand turned out to differ significantly
from the artificial peptide investigated previously.
Moreover, our data provide evidence for additional
contacts mediated by the calreticulin P-domain. On
the basis of these observations, we present a detailed
molecular model of the native complex. Beyond the
specifics of this particular interaction, our model offers
conceptual insights into the function of the calnexin ⁄
calreticulin family in general.

Results
Binding constants determined by surface
plasmon resonance (SPR) spectroscopy
Using SPR, the binding of an analyte in solution to an
immobilized partner can be measured directly [22].
Therefore, we investigated the interaction of GABA-
RAP with the calreticulin P-domain (amino acids 177–
288) and related peptides with this technique (Fig. 1).
Y. Thielmann et al. Complex structure of GABARAP and calreticulin
FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS 1141
Evaluation of steady-state binding signals yielded
dissociation constants of 930 ± 120 nm for the
GABARAP–P-domain interaction (Fig. 1A,B) and
11.5 ± 1.1 lm for G ABARAP binding to CR T(178–188 )
(Fig. 1C,D). Comparison of the dissociation constants
of both complexes suggests that binding of additional
residues not included in the undecamer peptide may
account for the higher affinity of the P-domain. In
fact, full-length calreticulin binds with an even lower
dissociation constant of 64 nm and an estimated mean
lifetime of 20 min [15]. Therefore, the globular domain
is likely to contribute to the association with GABA-
RAP as well. We also investigated a variant of
CRT(178–188) in which the tryptophan residue was
replaced by alanine [W183A-CRT(178–188)]. Binding
of this peptide to GABARAP was not saturable up to
a ligand concentration of 1 mm (data not shown).
Obviously, the mutation shifted the dissociation
constant from 11.5 lm into the millimolar range. We
conclude that the tryptophan side chain plays a key

role in the affinity of the CRT(178–188)–GABARAP
complex.
Characterization of complexes by NMR
spectroscopy
High-resolution liquid-state NMR spectroscopy is a
powerful technique for in vitro studies of the structure
and dynamics of soluble biological macromolecules.
NMR also allows the identification and characteriza-
tion of molecular interactions of soluble complexes
[23].
1
H
15
N heteronuclear single quantum coherence
(HSQC) experiments performed with GABARAP and
the W183A-CRT(178–188) peptide showed only small
changes of chemical shifts for distinct amino acids
(Fig. 2B). In contrast, incubation with the native
CRT(178–188) ligand induced large chemical shift
changes throughout the GABARAP spectrum and the
disappearance of certain peaks (Fig. 2A). Again, the
mutation of Trp183 to alanine in CRT(178–188) had a
tremendous effect on the binding properties of the
molecule. Similar to the results with CRT(178–188),
HSQC titration experiments with GABARAP and the
entire P-domain (Fig. 2C) showed large chemical shift
changes. In addition, we observed disappearance of
AC
BD
Fig. 1. SPR measurements of calreticulin fragments binding to immobilized GABARAP. (A) Calreticulin P-domain and (C) CRT(178–188) were

injected into the flow cell at a range of concentrations (10 n
M to 5 lM, and 100 nM to 100 lM, respectively). Sensorgrams are shown in dark
gray, with black bars indicating the average response at equilibrium for every concentration. In (B) and (D), the respective average responses
(d) are fitted to a 1 : 1 binding model (black curves).
Complex structure of GABARAP and calreticulin Y. Thielmann et al.
1142 FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS
resonances caused by broadening of the line width of
the chemical shift. Line broadening was reduced by
heating the sample from 25 to 35 °C (data not shown),
which probably relates to an increased tumbling rate
at the higher temperature. According to the known
assignment of native GABARAP resonances, the
major binding site for all calreticulin fragments is
located in the hydrophobic pockets hp1 (Ile21, Tyr25,
Ile32, Lys48, and Leu50) and hp2 (Lys46, Tyr49,
Phe60, and Leu63).
Structure of the GABARAP–CRT(178–188)
complex
The three-dimensional structure of the GABARAP–
CRT(178–188) complex was investigated by X-ray
crystallography. Using poly(ethylene glycol) MME 550
as precipitating agent, we obtained crystals belonging
to space group I23, containing one copy of the com-
plex in the asymmetric unit. Initial phases were deter-
mined by molecular replacement with the crystal
structure of GABARAP [9] as a search model, and the
structure was refined to 2.3 A
˚
. Several segments in the
GABARAP structure display elevated temperature fac-

tors and weaker electron density, which indicates
enhanced conformational freedom. This applies to the
N-terminus as well as the a3–b3 and b3–a4 loops of
GABARAP. The N-terminal four residues of the pep-
tide ligand (Ser178 to Asp181) could not be built,
because the electron density was very sparse in the
respective region. A remarkable lattice contact is estab-
lished by a Zn
2+
tethering three symmetry-equivalent
copies of GABARAP; these molecules contribute resi-
dues His69 (no. 1), His99 and Glu101 (no. 2) and
Glu112 (no. 3) to ion coordination. Figure 3 shows a
sketch of the overall structure of the complex (for a
close-up view including GABARAP side chains, see
Fig. S1). The GABARAP molecule (shown as a ribbon
model) displays a b-grasp fold (light blue), which is
A
B
C
Fig. 2.
1
H
15
N-HSQC spectra of GABARAP and calreticulin con-
structs. (A) Superimposed HSQC spectra of [
15
N]GABARAP alone
(red contour lines) and in the presence of a stoichiometric equiva-
lent of CRT(178–188) (black). Large chemical shift changes appear

throughout the spectrum. (B) Superimposed HSQC spectra of
[
15
N]GABARAP alone (red contour lines) and in the presence of a
four-fold stoichiometric excess of W183A-CRT(178–188) (blue).
Minor chemical shifts of distinct amino acids occur. (C) Superim-
posed HSQC spectra of [
15
N]GABARAP alone (red contour lines)
and in the presence of 0.5 (blue) and 1 (green) stoichiometric equiv-
alents of the calreticulin P-domain. During titration, large chemical
shift differences appear throughout the spectrum. In addition, line
broadening is observed.
Y. Thielmann et al. Complex structure of GABARAP and calreticulin
FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS 1143
characteristic for the superfamily of Ubls. This com-
pact domain consists of a four-stranded mixed b-sheet
(strands labeled b1 through b4) and two a-helices (a3
and a4) packed against its concave surface. A specific
feature of the GABARAP family is an extension by
two N-terminal helices (a1 and a2) on the convex face
of the b-sheet. CRT(178–188) assumes an extended
conformation and makes close contact with the
GABARAP molecule, burying 490 A
˚
2
of solvent-acces-
sible surface. The central part of the ligand (Asp184 to
Leu186; gray in Fig. 3) forms main chain hydrogen
bonds with strand b2 of GABARAP (Lys48 and

Leu50), and can thus be thought of as an intermolecu-
lar extension of the central b-sheet. In contrast, the
terminal peptide segments (dark blue) are engaged in
side chain hydrogen bonds to Lys48, Glu17 and Arg28
of GABARAP. Overall, the interaction between
CRT(178–188) and GABARAP appears to be domi-
nated by hydrophobic contacts established by Trp183,
Phe185 and Leu186 of the peptide. The individual side
chains involved are listed in Table 1. The calreticulin
peptide is anchored by the indole moiety of Trp183,
which contacts residues from helix a2, strands b1 and
b2 and the a4–b4 loop (hp1, see below). The side chain
of Phe185 reaches out across strand b2, interacting
with apolar side groups from the a2–b1 loop. Finally,
the C-terminal part of CRT(178–188) is held in posi-
tion by hydrophobic contacts of Leu186 with
strand b2, helix a3 and the b2–a3 loop (hp2). As
expected, the GABARAP residues involved in ligand
binding agree well with those displaying medium to
slow exchange rates in our NMR experiments
(included in Table 1). Notably, the hydrophobic pock-
ets engaged in complex formation of GABARAP and
CRT(178–188) are also crucial for the GABARAP–K1
peptide complex (see below for details).
Conformational changes upon complex formation
The substantial structural knowledge available for
GABARAP [9–13] enables us to delineate the require-
ments and consequences of complex formation.
Figure 4 shows an alignment of nonliganded GABA-
RAP [12] (light gray) with the GABARAP–K1 peptide

complex [13] (shades of red) and the complex investi-
gated in this study (shades of blue). (For this compari-
son, the solution structure of nonliganded GABARAP
(1KOT) was preferred over available X-ray structures
(1GNU, 1KJT), because the latter contain a lattice
contact that partially mimics the effect of ligand bind-
ing. In contrast, crystal packing interactions in the K1
and CRT(178–188) complexes do not involve the
hydrophobic surface of GABARAP, suggesting that
this part of the structure should be relatively unaf-
fected.) The overview at the top gives an impression of
the overall variation among the three structures. The
most significant backbone displacements occur in
Fig. 3. Overview of the GABARAP–CRT(178–188) complex. GABA-
RAP is depicted as a ribbon model with the b-grasp domain and
the N-terminal extension colored in light blue and light gray, respec-
tively. The ligand backbone is shown in dark blue (terminal seg-
ments) and gray (b-strand). The apolar side groups docking to
GABARAP are drawn in stick mode (gold).
Table 1. Overview of hydrophobic interactions between GABARAP
and CRT(178–188), as revealed by the crystal structure, and extent
of chemical shift changes of GABARAP resonances in the corre-
sponding
1
H
15
N-HSQC experiment. +, minor effect; ++, large
chemical shift change; +++, absence of peak from spectrum; NA,
not applicable, since prolines do not appear in
1

H
15
N-HSQC spectra;
NE, not evaluated due to spectral overlap.
CRT(178–188)
GABARAP
Contacts in
crystal structure
Effects in HSQC
experiment
Trp183 Ile21 ++
Pro30 NP
Ile32 ++
Lys48 +++
Leu50 +++
Phe104 +++
Phe185 Arg28 +
Tyr25 +++
Leu186 Tyr49 +++
Val51 NE
Phe60 ++
Leu63 ++
Ile64 NE
Arg67 ++
Complex structure of GABARAP and calreticulin Y. Thielmann et al.
1144 FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS
helix a3 and the adjacent a3–b3 loop. A more detailed
view of the alignment is given in the bottom panels of
Fig. 4 for the two hydrophobic pockets. Binding of
Trp183 (dark blue) in hp1 induces a slight shift of

helix a2 (blue), similar to the effect of Trp11 in the K1
peptide (dark red). Notably, the two complexes differ
in both the position and conformation of these trypto-
phan side chains. Specifically, Trp183 extends deeper
into the pocket, and this is reflected by the side chain
configuration of Lys48 and Phe104, which are altered
most notably as compared to the GABARAP–K1
complex. Binding of Phe185 does not have obvious
consequences for the conformation of either hp1 or
hp2. In contrast, Leu186 (dark blue) leads to a large
displacement of helix a3 (blue). Binding of this leucine
alone exhibits almost the same effect as was reported
for Trp6 and Leu9 in the GABARAP–K1 complex
(shades of red), resulting in hp2 assuming an open
conformation. This spatial rearrangement appears to
be chiefly mediated by the displacement of Leu63. On
the other hand, the side chain conformation of Arg67
remains similar to that of the nonliganded protein,
thus not exposing additional hydrophobic surface,
which is needed in the GABARAP–K1 complex for
insertion of Trp6 (dark red) into hp2.
Model of the GABARAP–calreticulin interaction
Unfortunately, attempts to cocrystallize GABARAP
with the entire calreticulin molecule or the P-domain
have been unsuccessful. In order to gain more insight
into the three-dimensional arrangement of the native
complex, we have built a homology model incorporat-
ing available data on the soluble portion of calnexin
[24] and the calreticulin P-domain [25], in addition to
the GABARAP–CRT(178–188) complex investigated

in this study (Fig. 5). The GABARAP-binding epitope
on calreticulin is located at the N-terminal junction
between the globular domain and the arm domain.
Intriguingly, the corresponding residues could not be
resolved in the X-ray structure of calnexin serving as
the major template, suggesting significant conforma-
tional freedom in this region. In agreement with this
notion, our model indicates that, at least when com-
plexed with GABARAP (blue), this portion of calreti-
culin forms a protrusion emerging from the base of the
hp1
hp2
Fig. 4. Comparison of GABARAP structures.
Top: overview of nonliganded and liganded
GABARAP structures in ribbon and coil rep-
resentation: light and dark blue, GABARAP
and CRT(178–188) (this study); light and
dark red, GABARAP and K1 peptide [13];
light gray, nonliganded GABARAP [12].
Bottom: detailed view of hydrophobic
pockets hp1 and hp2. The structures are
depicted as above, with selected side
chains taking part in the interaction
appearing as stick models. For visual clarity,
additional GABARAP side chains involved in
the interaction (Glu17, Tyr25, Arg28, Pro30,
Ile32, Tyr49, Leu50, Val51, Phe60 and Ile64)
have been omitted.
Y. Thielmann et al. Complex structure of GABARAP and calreticulin
FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS 1145

arm domain, largely devoid of tertiary interactions
with neighboring segments (Fig. 5A,C). We therefore
propose that the residues forming the N-terminal junc-
tion between the two domains of calreticulin (as well
as calnexin) constitute a versatile interaction site that
may be adapted to accommodate a variety of ligands.
The potential implications for the structure and func-
tion of these chaperones are discussed below.
Figure 5B,D includes surface representations of
GABARAP (light gray), with colored patches denoting
residues with major shifts in our HSQC titrations with
CRT(178–188) (blue) and the calreticulin P-domain
(red). Localization of these amino acids is consistent
with the spatial arrangement of GABARAP and
calreticulin in our model.
Discussion
Members of the GABARAP family of Ubls have been
implicated in several aspects of membrane vesicle traf-
ficking in eukaryotic cells. An important step towards
understanding these functions at a molecular level was
the discovery of a peculiar conjugation mechanism
resulting in covalent linkage of these proteins to mem-
brane lipids [7,8]. On the other hand, knowledge of the
protein–protein interactions implicated in the various
biological roles of GABARAP and its relatives is only
beginning to emerge. In a search for novel cellular
targets of GABARAP, we have recently identified
calreticulin and the heavy chain of clathrin as potential
binding partners [15,16]. In both cases, binding activity
could be narrowed down to short contiguous peptide

sequences comprising 11 and 13 amino acids, respec-
tively, centered on a hydrophobic motif (WxFL).
In the current study, we present the first three-
dimensional structure of GABARAP complexed with a
fragment of such a proposed physiological ligand. Our
data largely confirm previous assignments of hydro-
phobic patches on the surface of GABARAP, which
we have shown to interact with indole derivatives as
well as a high-affinity artificial ligand (K1) [13].
Although a detailed analysis of the GABARAP–
CRT(178–188) interface reveals significant differences
with respect to the K1 peptide, both complexes are
critically dependent on the presence of at least one
tryptophan side chain in the ligand. In its central part,
the calreticulin peptide assumes an extended conforma-
tion, aligning parallel to strand b2 of GABARAP.
Protein–protein interactions via formation of inter-
molecular b-sheets have been observed for several
AB
CD
Fig. 5. Model of the GABARAP–calreticulin interaction, shown in two orientations. (A, C) Overview of the GABARAP–calreticulin complex;
GABARAP is shown in light blue and the CRT(178–188) segment in dark blue, with the apolar side chains drawn as stick models (gold); the
globular domain and P-domain of calreticulin are depicted in light and dark red, respectively. The calreticulin P-domain bends around the
bound GABARAP molecule. (B, D) Detailed view of the GABARAP surface (light gray) in complex with calreticulin, oriented as in (A) and (C),
respectively. Blue surface patches indicate the GABARAP residues that are most strongly affected in HSQC spectra in the presence of
CRT(178–188); additional candidates found with the calreticulin P-domain are marked in red.
Complex structure of GABARAP and calreticulin Y. Thielmann et al.
1146 FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS
members of the ubiquitin superfamily [26,27]. Intrigu-
ingly, one of the GABARAP crystal structures has

revealed self-association by a similar mechanism, with
the N-terminal six amino acids binding to strand b2of
a neighboring molecule [11].
What is the biological significance of the GABA-
RAP–calreticulin complex? Although its high affinity
and estimated lifetime are clearly indicative of a
relevant interaction, definition of the precise biochem-
ical context in which it naturally occurs has remained
a challenge. As long as direct experimental evidence
for a biological function of this complex is missing,
even fortuitous binding cannot be completely
excluded. This seems rather unlikely, however, given
that the two proteins not only interact with apprecia-
ble affinity in vitro, but also colocalize in vivo. Inter-
estingly, conventional knowledge indicates that the
subcellular locations of these two proteins should be
mutually exclusive: GABARAP has largely been
found associated with intracellular membranes [28],
and the lack of sorting signals together with the
C-terminal conjugation mechanism suggests that it is
linked to phospholipids on the cytosolic leaflet of
such membranes. Calreticulin, on the other hand, is
well known as a soluble chaperone of the ER lumen
[17]. However, the protein is in fact not restricted to
the ER, but does exert important functions in other
cellular compartments, such as the cytosol [29], the
nucleus [30] and the plasma membrane [20]. Impor-
tantly, calreticulin found at these locations appears
to be derived from the ER pool; export into the
cytosol is accomplished by a retrotranslocation

process that is distinct from the pathway taken by
misfolded proteins leading to ubiquitination and
proteasomal degradation [21]. On the basis of these
findings, we shall discuss several cellular processes
that may be envisaged as involving the formation of
a GABARAP–calreticulin complex.
Export of the N-cadherin–b-catenin complex from
the ER has been shown to be dependent on PX-RICS
(a GTPase-activating protein acting on Cdc42) and its
interaction partner GABARAP. In HeLa cells
expressing GABARAP and PX-RICS, knockdown of
either protein with short hairpin RNA prevented
transport of N-cadherin to sites of cell–cell contact.
Exogenous expression of the respective components
restored the subcellular distribution of N-cadherin and
b-catenin [31]. On the other hand, N-cadherin is
downregulated in calreticulin-deficient mouse embry-
onic hearts. This may contribute to the disorganiza-
tion in myocardial architecture that led to death of
the embryos mostly between day 12 and day 14 post
conception [19]. Conversely, if calreticulin is overex-
pressed in fibroblasts, the N-cadherin protein level is
doubled as compared to control cells [32]. Taken
together, both GABARAP and calreticulin are
involved in a process that enriches N-cadherin in the
plasma membrane at cell–cell contacts. According to
these data, it is attractive to speculate that GABA-
RAP may recruit calreticulin to the cytosolic surface
of transport vesicles carrying N-cadherin.
Similar considerations may hold in the case of inte-

grins. Calreticulin has been shown to associate with
a
3
b
1
integrin dimers, and the interaction site has been
mapped to a conserved motif in the intracellular
domain of the integrin a-subunit, thus clearly involving
cytosolic calreticulin [33]. At the same time, a
3
b
1
inte-
grins colocalize with GABA
A
receptors, suggesting a
possible connection to GABARAP [34]. It seems
conceivable that calreticulin may travel to the plasma
membrane on the cytosolic surface of GABARAP-
tagged vesicles loaded with either GABA
A
receptors or
integrins (or both).
For the two scenarios discussed above, the func-
tional role of calreticulin in complex with GABARAP
is still unclear, but may be speculated to involve Ca
2+
-
dependent regulation of subsequent protein–protein
interaction or membrane fusion events.

Recent investigations have established that low
amounts of calreticulin are exposed on the plasma
membrane of most cell types. Intriguingly, surface
expression was found to be significantly enhanced by
cellular stress, acting as a potent ‘eat me’ signal stimu-
lating clearance of apoptotic cells [35]. Moreover,
upregulation of calreticulin exposure in tumor cells by
certain antineoplastic drugs has been shown to
enhance phagocytosis and tumor antigen cross-presen-
tation by dendritic cells [20]. Saturable binding of
exogenous calreticulin to the surfaces of viable as well
as apoptotic cells [35] indicates the presence of specific
receptors. On the basis of our results, we speculate
that GABARAP (or another member of its family)
may constitute such a receptor, tethering calreticulin to
membranes via its phospholipid linkage. However, this
concept requires that GABARAP should be present on
the lumenal leaflet of the ER membrane, which is
topographically equivalent to the outer surface of the
plasmalemma. Such a localization cannot be excluded,
although GABARAP and its relatives do not contain
obvious sorting signals.
Irrespective of the precise physiological role of the
GABARAP–calreticulin complex, its structure sheds
light on a general aspect of calnexin and calreticulin
function. It is widely accepted that these chaperones
provide at least two distinct sites for interaction with
folding intermediates in the ER lumen [17]. A specific
Y. Thielmann et al. Complex structure of GABARAP and calreticulin
FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS 1147

binding pocket for Glc
1
Man
9
GlcNAc
2
oligosaccarides
has been identified on the surface of the globular
domain. In contrast, a general polypeptide interaction
site, which is independent of glycosylation, has been
postulated, but its location has not been established so
far. Such a site can be expected to expose hydrophobic
side chains, conferring the ability to stabilize folding
intermediates and to prevent them from aggregating.
Indeed, the structure of CRT(178–188) in complex
with GABARAP reveals a significant hydrophobic
interface. Its position within the overall structure of
calreticulin, at the socket of the arm-like domain,
makes this segment a particularly favorable candidate
for a substrate recognition site, as it would provide
access to important chaperoning and refolding activi-
ties associated with calreticulin. Specifically, it is
located in the vicinity of the carbohydrate-binding
pocket on the globular domain and of the protein
disulfide isomerase ERp57, which is bound to the
distal part of the arm domain [36]. Although the
precise orientation of the enzyme in this complex is
still unknown, the remarkable flexibility of the arm
domain, as demonstrated by NMR spectroscopy [25],
is likely to enable it to accommodate substrate mole-

cules of different size and shape. By virtue of its over-
all concave surface, the chaperone is believed to shield
the folding intermediate from its surrounding, thus
reducing formation of aggregates. The calnexin seg-
ment corresponding to CRT(178–188) differs in
sequence, but displays significant hydrophobic charac-
ter as well. As pointed out previously, the binding
motif of calreticulin considered here is remarkably con-
served between organisms as diverse as slime molds
(exemplified by Dictyostelium discoideum), insects
(Drosophila melanogaster) and vertebrates (Homo
sapiens) [14]. As this similarity even extends to higher
plants (such as Arabidopsis thaliana), it is likely to
reflect a fundamental function of calreticulin acquired
during early eukaroytic evolution. Besides interaction
with substrate proteins, such a function may also
involve the formation of specific complexes with other
chaperones.
In summary, these considerations provide a possible
structural foundation for the well-documented affinity
of calnexin and calreticulin for partially unfolded poly-
peptides. Numerous proteins expressed in the ER have
been shown to preferentially interact with one of these
chaperones. Among other reasons, this may be related
to the differences in the apolar sequence discussed
above.
Current evidence indicates that the calreticulin frac-
tion retrotranslocated into the cytosol exerts distinct
functions that are unrelated to the rather promiscuous
activities within the ER lumen. Indeed, the chemical

milieu in the two compartments is strikingly different,
the most prominent example being the Ca
2+
concen-
tration, which is four orders of magnitude higher in
the ER. Along these lines, it seems conceivable that a
general recognition site for partially folded polypep-
tides, which plays a crucial role in the chaperoning
function of calreticulin, might have been readapted for
specific protein–protein interactions in the cytosolic
environment, such as with GABARAP. The structural
details of such complexes are now beginning to be
unraveled.
Experimental procedures
Expression and purification of proteins
The expression and purification of GABARAP have
been described previously [37]. The calreticulin P-domain
(amino acids 177–288) coding sequence was cloned into
pGEX-6P-2 (GE Healthcare, Munich, Germany) using
BamH1 and Xho1 restriction sites, and expressed in the
Escherichia coli BL21 plysS strain transformed with the
plasmid. After affinity purification using glutathione–Sepha-
rose 4B (GE Healthcare), the fusion protein of glutathione
S-transferase and the P-domain was cleaved with PreScis-
sion protease (GE Healthcare). The final purification step
was size exclusion chromatography using a Superdex 75
matrix (GE Healthcare). The correct molecular mass was
verified by MS.
Peptide synthesis
The two peptides CRT(178–188) and W183A-CRT(178–

188) were custom synthesized and purified to > 95% by
the BMFZ at the University of Du
¨
sseldorf and Jerini
BioTools (Berlin, Germany), respectively.
SPR spectroscopy
SPR studies were carried out on a BiacoreX optical biosen-
sor (GE Healthcare). Following the standard procedure of
the manufacturer for amine coupling, 1.5 lm GABARAP
protein in 10 mm sodium acetate buffer (pH 5.5) was used
to perform coupling to the carboxymethylated dextran
matrix of a CM5 sensor chip surface. A reference surface
was treated identically, but was not exposed to GABARAP
for immobilization. Experiments were performed in 10 mm
Hepes (pH 7.4), 150 mm NaCl, 3 mm EDTA and 0.005%
surfactant P20, using various concentrations of calreticulin
P-domain and peptides at a flow rate of 30 lLÆmin
)1
at
21.5 °C. Biosensor data were prepared by double referenc-
ing [38]. The biaevaluation software package was used for
data analysis.
Complex structure of GABARAP and calreticulin Y. Thielmann et al.
1148 FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS
NMR spectroscopy
All NMR spectra were recorded on a Varian (Darmstadt,
Germany) Unity INOVA spectrometer at a proton frequency
of 600 MHz with a Varian Gen 2 HCN cryogenic probe.
The sample for the CRT(178–188) binding experiment con-
tained 600 lm [

15
N]GABARAP and 600 lm CRT(178–188)
in 25 mm sodium phosphate (pH 7.0), 100 mm KCl, 100 mm
NaCl and 5% (v ⁄ v) deuterium oxide. For the experiment
with W183A-CRT(178–188), 170 lm [
15
N]GABARAP and
690 lm W183A-CRT(178–188) were used. These spectra,
together with a third one of 200 lm [
15
N]GABARAP with-
out ligand, were recorded at 10 °C. The buffer conditions for
experiments with GABARAP and calreticulin P-domain
were 25 mm sodium phosphate (pH 7.0), 100 mm NaCl,
3mm EDTA and 7% (v ⁄ v) deuterium oxide. The initial
concentration of [
15
N]GABARAP was 680 lm, and final
concentrations were 365 l m [
15
N]GABARAP and 400 lm
P-domain; spectra were recorded at 25 °C. Data were
processed with nmrpipe [39] and analyzed with cara [40].
Crystallization
The GABARAP–CRT(178–188) complex was prepared by
combining 700 lm protein and 770 lm peptide in 10 mm
Tris-HCl (pH 7.0). Cocrystallization was achieved using the
hanging-drop vapor diffusion method, with the reservoir
containing 0.1 m Mes (pH 6.5), 27% (v ⁄ v) poly(ethylene
glycol) MME 550 and 10 mm ZnSO

4
.
Data collection
The X-ray diffraction dataset was collected at 100 K. Prior
to cryocooling, crystals were soaked once in a reservoir
solution containing 29% (v ⁄ v) poly(ethylene glycol)
MME 550 and 5% (v ⁄ v) glycerol.
A single-wavelength native dataset was recorded at beam-
line ID14-1 of the ESRF (Grenoble, France) tuned to a
wavelength of 0.934 A
˚
on an ADSC-Q4R detector. Data
processing was carried out with the ccp4 [41] software suite
using mosflm and scala.
Structure determination
Cocrystals of GABARAP and CRT(178–188) belonged to
space group I23. The structure was determined by mole-
cular replacement using molrep (ccp4) with a single native
dataset. The search model was created from the crystal
structure of GABARAP (Protein Data Bank code: 1KJT)
[9]. Crystals were found to contain one copy of the complex
per asymmetric unit, corresponding to a Matthews coeffi-
cient of 2.47 A
˚
3
ÆDa
)1
and a solvent content of 50.2%. Fol-
lowing rigid-body refinement using the cns [42] package,
the model was improved by iterative cycles of manual

rebuilding using the program o [43] and refinement with
cns. Later stages of refinement and assignment of water
molecules were carried out with the phenix [44] package. In
order to avoid overfitting in light of the moderate observa-
tions-to-parameters ratio, the relative weight of stereochem-
ical restraints was increased, resulting in a comparatively
low deviation of geometric parameters from library targets.
For statistics on data collection and refinement, see
Table 2.
The final model contains amino acids 1–117 of native
GABARAP with an additional N-terminal glycine–serine
extension and amino acids 182–188 of the calreticulin
ligand. The N-terminal cloning artefact (glycine–serine) is
omitted from residue numbering throughout this article.
Note that all amino acids of the crystallized protein have to
be considered in the Protein Data Bank entry, resulting in
a +2 shift in residue numbers.
According to Ramachandran plots generated with mol-
probity (), the model
exhibits good geometry with one residue (His69) in the dis-
allowed region. This histidine is involved in coordination of
aZn
2+
at the interface of three GABARAP molecules.
Comparative modeling
The molecular model of the human GABARAP–calreticulin
complex was built with the modeller package [45], using
two distinct templates. The template for the calreticulin
moiety was created from the crystal structure of canine
calnexin (Protein Data Bank code 1JHN) [24] by manually

Table 2. Data collection and refinement statistics. Values in paren-
theses are for the highest-resolution shell (2.42–2.30 A
˚
).
Data collection
Space group I23
Cell dimensions
a (A
˚
)(T = 100 K) 97.04
Resolution range (A
˚
) 39.6–2.3
Beamline ESRF ID14-1
Detector ADSC-Q4R
Wavelength (A
˚
) 0.934
R
sym
(%) 5.2 (39.9)
I ⁄ r(I) 24.5 (4.9)
Completeness (%) 99.7 (100.0)
Redundancy 6.8 (6.6)
Refinement
No. reflections 6892
R
work
(%) 23.2
R

free
(%) 27.0
No. atoms
Protein 1036
Ion 1
Solvent 29
rmsd
Bond lengths (A
˚
) 0.003
Bond angles (°) 0.6
Y. Thielmann et al. Complex structure of GABARAP and calreticulin
FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS 1149
replacing the two distal modules of the arm domain by the
terminal portion of the rat calreticulin arm domain solved
by NMR spectroscopy (Protein Data Bank code 1HHN)
[25]. The X-ray structure of human GABARAP complexed
with amino acids 182–188 of human calreticulin (this study)
was introduced as a second template for accurate represen-
tation of the binding interface. Alignment of the calreticulin
sequence to the calnexin-based template was performed
using the structure-sensitive algorithm implemented in
modeller. Ten models of the complex were built in a
single run, employing the automodel environment. Out of
these, the structure with the most favorable values in the
modeller objective function as well as the discrete
optimized protein energy evaluation score was selected for
further analysis.
Molecular graphics
The alignment underlying Fig. 4 was calculated with

lsqman [46], based on Ca coordinates of b-strands 1, 3
and 4 from the GABARAP–CRT(178–188) complex (this
study), the GABARAP–K1 peptide complex ([13], chains A
and C) and nonliganded GABARAP ([12], selecting
model 2 as the best representative of the ensemble). Figures
were generated with molscript [47] and raster3d [48],
using secondary structure assignments as given by the dssp
[49] program. Surface representations were prepared with
grasp [50].
Acknowledgements
The authors wish to thank Olga Dietz for excellent
technical assistance. O. H. Weiergra
¨
ber is grateful to
Georg Bu
¨
ldt for continuous generous support. More-
over, assistance by the ESRF staff at beamline ID14-1
is acknowledged. This study was supported by a
research grant from the Deutsche Forschungsgemeins-
chaft (DFG) to D. Willbold (Wi1472 ⁄ 5).
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Supporting information
The following supplementary material is available:
Fig. S1. Close-up view of the GABARAP–CRT(178–
188) complex.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the
corresponding author for the article.
Complex structure of GABARAP and calreticulin Y. Thielmann et al.
1152 FEBS Journal 276 (2009) 1140–1152 ª 2009 The Authors Journal compilation ª 2009 FEBS