GABAA receptors are relevant drug targets for benzodiazepines, barbiturates Keywords 4-aminobutyrate type A receptor-associated protein GABARAP; calreticulin; protein–protein interaction
Trang 1interface – implications for substrate binding to
endoplasmic reticulum chaperones
Yvonne Thielmann1, Oliver H Weiergra¨ber1, Jeannine Mohrlu¨der1,2and Dieter Willbold1,2
1 Institut fu¨r Neurowissenschaften und Biophysik, Molekulare Biophysik, Forschungszentrum Ju¨lich, Germany
2 Institut fu¨r Physikalische Biologie und BMFZ, Heinrich-Heine-Universita¨t Du¨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
(GABAA) receptors, which are ligand-gated chloride
channels, and type B (GABAB) receptors, which are G-protein-coupled and modulate the activity of potas-sium and calcium channels [2] GABAA 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: o.h.weiergraeber@fz-juelich.de
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: d.willbold@fz-juelich.de
Database
The atomic coordinates and structure
factor amplitudes (code 3DOW) have been
deposited in the Protein Data Bank
(http://www.pdb.org)
(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), CH3CO-SLEDDWDFLPP-NH2; 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.
Trang 2and general anesthetics [3] GABAA
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
GABAAreceptor c2-subunit This interaction was
con-firmed by colocalization experiments in cultured
corti-cal neurons and by coimmunoprecipitation of
GABARAP with GABAA 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
Ca2+-binding chaperone predominantly located in the endoplasmic reticulum (ER) It is found in a wide range of species and is involved in intracellular Ca2+ homeostasis as well as ER Ca2+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 {CH3CO-SLEDDWDFLPP-NH2 [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)
Trang 3Evaluation 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 GABARAP binding to CRT(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 interaccharacteriza-tions of soluble complexes [23] 1H15N 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
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 l M, and 100 n M to 100 l M , 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).
Trang 4resonances caused by broadening of the line width of the chemical shift Line broadening was reduced by heating the sample from 25 to 35C (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 Zn2+ 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 [15N]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.
Trang 5characteristic 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˚2of
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 GABAGABA-RAP–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
Trang 6helix 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.
Trang 7arm 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
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.
Trang 8members 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 b2 of
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
a3b1 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, a3b1 inte-grins colocalize with GABAA 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 GABAAreceptors 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 Ca2+ -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
Trang 9binding pocket for Glc1Man9GlcNAc2 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 Ca2+ 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 21.5C Biosensor data were prepared by double referenc-ing [38] The biaevaluation software package was used for data analysis
Trang 10NMR 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 [15N]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 [15N]GABARAP and
690 lm W183A-CRT(178–188) were used These spectra,
together with a third one of 200 lm [15N]GABARAP
with-out ligand, were recorded at 10C The buffer conditions for
experiments with GABARAP and calreticulin P-domain
were 25 mm sodium phosphate (pH 7.0), 100 mm NaCl,
3 mm EDTA and 7% (v⁄ v) deuterium oxide The initial
concentration of [15N]GABARAP was 680 lm, and final
concentrations were 365 lm [15N]GABARAP and 400 lm
P-domain; spectra were recorded at 25C 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 ZnSO4
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˚3ÆDa)1and 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 (http://molprobity.biochem.duke.edu), the model exhibits good geometry with one residue (His69) in the dis-allowed region This histidine is involved in coordination of
a Zn2+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
Cell dimensions
Refinement
No atoms
rmsd