From the Medical Research Council Laboratory of
Molecular Biology and § Centre for Protein Engineering,
Medical Research Council Centre, Hills Road,
Cambridge CB2 2QH, United Kingdom
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ABSTRACT |
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Cytosolic phospholipase A2 (cPLA2) is a
calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic
acid-containing membrane phospholipids to initiate the biosynthesis of
eicosanoids and platelet-activating factor, potent inflammatory
mediators. The calcium-dependent activation of the enzyme
is mediated by an N-terminal C2 domain, which is responsible for
calcium-dependent translocation of the enzyme to membranes
and that enables the intact enzyme to hydrolyze membrane-resident
substrates. The 2.4-Å x-ray crystal structure of this C2 domain was
solved by multiple isomorphous replacement and reveals a -sandwich
with the same topology as the C2 domain from phosphoinositide-specific
phospholipase C
1. Two clusters of exposed hydrophobic residues
surround two adjacent calcium binding sites. This region, along with an
adjoining strip of basic residues, appear to constitute the membrane
binding motif. The structure provides a striking insight into the
relative importance of hydrophobic and electrostatic components of
membrane binding for cPLA2. Although hydrophobic interactions
predominate for cPLA2, for other C2 domains such as in
"conventional" protein kinase C and synaptotagmins, electrostatic
forces prevail.
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INTRODUCTION |
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Mammalian phospholipases A2 (PLA2s)1 are a large superfamily of enzymes with a common function of catalyzing the release of fatty acid from the sn-2 position of membrane phospholipids, but with distinct structural and biochemical characteristics and different roles in signal transduction and general lipid metabolism (reviewed in Refs. 1-3). Cytosolic PLA2 (cPLA2) is an 85-kDa protein with no sequence homology to any other PLA2s (Refs. 4 and 5; reviewed in Refs. 3 and 6-8). This enzyme preferentially hydrolyzes phospholipids containing arachidonate at the sn-2 position, thus providing free arachidonic acid for the biosynthesis of eicosanoides, potent inflammatory lipid mediators. cPLA2 is found in a variety of cells where it can act as a receptor-regulated enzyme that can mediate agonist-induced arachidonic acid release (9, 10). cPLA2 is activated by low levels of calcium (11-14), but calcium is not directly involved in catalysis and is required only for membrane binding (15, 16). The enzyme translocates from cytosol to membranes in the presence of physiologically relevant, submicromolar calcium levels (5, 17, 18). The increase in intracellular calcium triggered by calcium ionophores or agonists such as histamine, thrombin, bradykinin, or IgE/antigen causes the translocation of the enzyme from the cytosol to the nuclear membrane and endoplasmic reticulum (19-22). Interestingly, a number of enzymes involved in eicosanoid metabolism, such as prostaglandin endoperoxide synthase 1 and 2, 5-lipoxygenase, and 5-lipoxygenase-activating protein also localize to the nuclear envelope and endoplasmic reticulum (19, 23). In addition to calcium, the activation of cPLA2 by some agonists requires phosphorylation of the enzyme (7, 8).
Using limited proteolysis and deletion analysis, two functionally distinct domains have been mapped in cPLA2: an N-terminal domain responsible for Ca2+-dependent phospholipid binding known as a CaLB or a C2 domain and a C-terminal, Ca2+-independent catalytic domain capable of hydrolyzing monomeric substrates but unable to associate with membranes (16).
In this paper, we describe the crystal structure and calcium binding characteristics of the N-terminal domain of cPLA2 that is responsible for Ca2+-dependent translocation and membrane binding of the enzyme. The structure accounts for the observed preference of cPLA2 for phospholipids with hydrophobic features such as phosphatidylcholine and explains previous observations suggesting the importance of the hydrophobic forces involved in binding of the cPLA2 C2 domain to phospholipid membranes (15, 24, 25). Implications for the relative contributions of the hydrophobic/electrostatic interactions in the membrane binding of the C2 domains from other well characterized proteins are also presented.
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EXPERIMENTAL PROCEDURES |
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Cloning and Expression--
DNA fragments encoding residues
1-137, 1-141, or 17-141 of human cPLA2 were amplified by polymerase
chain reaction using a plasmid containing the gene for the intact
enzyme that was generously provided by Zeneca Pharmaceuticals,
Macclesfield, UK. The BglII site was incorporated at the 5
end of the coding sequence and an EcoRI site followed by a
stop codon was incorporated at the 3
end to enable cloning into
BamHI and EcoRI-cut vector mini-pRSETA. This
vector is a version of pRSETA (Invitrogen) modified so to encode a
17-residue N-terminal tail (MRGSHHHHHHGLVPRGS) containing a
His6 tag for affinity purification and a thrombin cleavage
site rather than the original enterokinase cleavage site for the tag removal.2 The expressed
proteins after thrombin cleavage retain at their N terminus two
additional amino acids (Gly-Ser) encoded by the vector. In addition to
this deviation from the native sequence, constructs 1-141 and 17-141
have two substitutions at their C terminus, C139A and C141S, that were
introduced to eliminate possible complications that could be caused by
cysteine residues during the refolding procedure (see below). For the
purpose of heavy atom derivatives, a construct was made with a
substitution of only one of these cysteines (C141S) while preserving
the other cysteine (Cys-139). The proteins were expressed in
Escherichia coli BLR(DE3) (Novagen). Cells were grown at
37 °C, induced at an A600 ~ 0.5 with 0.5 mM isopropyl-1-thio-
-D-galactopyranoside at
25 °C for 12 h, and harvested by centrifugation.
Purification and Refolding-- The insoluble fraction containing the fusion protein was isolated from 0.5 liters of cells by sonication and centrifugation. The pellets were solubilized in 8 M urea, Tris-HCl, pH 7.2, for 2 h at room temperature and centrifuged to remove the insoluble debris, and the denatured fusion protein was bound to 10 ml of Ni2+-nitrilotriacetic acid-agarose (Qiagen) equilibrated in the same buffer. After washing the column with several column volumes of 8 M urea-Tris buffer, the protein was eluted with 300 mM imidazole in the 8 M urea-Tris buffer, concentrated with Centriprep 10 to 1 ml, and renatured by slow dilution into a rapidly stirred solution of 1.5 M urea, 50 mM Tris-HCl, pH 7.2. The solution was left at room temperature for 1 h and then dialyzed against 50 mM Tris-HCl, pH 8.0. The protein was concentrated using a Centriprep 10 and passed through a gel filtration Superdex 75 16/60 column (Pharmacia), equilibrated in 50 mM Tris-HCl, pH 8.0. The peak of the monomeric, refolded protein was cleaved with ~50 units of thrombin/milligram of protein at 4 °C to remove the tag. The sample was further purified by chromatography on a MonoQ 10/10 column equilibrated in 50 mM Tris-HCl, pH 8.0, and eluted with a gradient of sodium chloride. Protein was concentrated to ~18 mg/ml and stored at 4 °C.
Crystallization -- The crystals were grown at 21 °C in hanging drops by the vapor diffusion method. Protein (5-10 mg/ml) was first incubated with 5 mM calcium chloride for at least 30 min and then mixed with an equal volume of a precipitant containing 1 M sodium acetate, 0.1 M Hepes, pH 7.5, and 50 mM CdSO4. Crystals appeared after 1 week and slowly grew over 3 weeks to about 0.05 × 0.05 × 0.5 mm. Crystals had P3121 symmetry with a = 79.41 and c = 70.67.
Diffraction Data Collection--
For data collection, crystals
were flash-frozen in nylon loops using a nitrogen gas stream at 100 K. For the freezing, crystals were briefly transferred to a cryoprotectant
solution consisting of 66 mM Hepes, pH 7.5, 660 mM sodium acetate, 33 mM CdSO4, and 25% (v/v) glycerol. Table I summarizes
the data sets collected. Each data set was collected from a single
crystal and processed using the program MOSFLM (26). The protein with a
double cysteine substitution (C139A,C141S) was used for all the data
sets except for the K2PtCl4 (I) derivative data
set for which a protein with a single cysteine substitution (C141S) was
used. For the heavy atom derivatives, crystals were soaked for 20 h in the reservoir solution containing 1 mM
K2PtCl4 (data set K2PtCl4 (I)) for
5 h in 5 mM K2PtCl4 (data set
K2PtCl4 (II)) or for 4 h in 10 mM LaCl3. Crystals were also grown in the presence of 10 mM L--glycerophosphocholine or
L-
-glycerophosphoserine, but no density corresponding to
these compounds was observed.
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Structure Solution--
Most crystallographic calculations were
carried out using the Collaborative Computing Project Number 4 (CCP4)
program suite (27). Molecular replacement using either PLC-1-C2
(2ISD) or SytIA-C2 (1RSY) located a single molecule in the asymmetric
unit; however, several loops were impossible to trace, and extensive efforts to build and refine a model using the molecular replacement phases proved unsuccessful. Consequently, several heavy atom derivative data sets were collected. One platinum site was located by a Patterson map search using the program SHELX (28). Other sites for other derivatives were located by difference Fourier analysis. Using the
platinum and lanthanum derivative-phased multiple isomorphous replacement map, two strong peaks were located at the same position for
all data sets. These sites were interpreted as cadmium sites. The
program SHARP (29) was used to refine all heavy atom parameters using
both anomalous and isomorphous differences. The MIRAS map showed only
one molecule in the asymmetric unit. Following solvent flattening with
the program SOLOMON (30) using a solvent content of 76%, the electron
density was easily interpretable, and a model was built using the
program O (31) and refined with the program REFMAC (32) using all data
between 15 Å and 2.4 Å (9,339 reflections). The final conventional
R-factor was 22.8% with a free R-factor of 27.2% (based on 1,003 random reflections in thin shells of resolution) for a model consisting
of 1,001 protein atoms, 151 waters, two calcium ions, and two cadmiums.
The average B-factor was 28 Å2. The r.m.s. deviations from
ideal geometry were 0.007 Å and 1.8° for bond lengths and bond
angles. According to analysis by PROCHECK (33), 86% of residues are in
the most favorable regions of the Ramachandran plot, and none are in
disallowed areas. The overall average G-factor from PROCHECK was
0.08. In the final SIGMAA-weighted 2 m|F0|-D|FC| electron density map (34),
there is no break in the main chain density contoured at 1.2
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RESULTS AND DISCUSSION |
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The Domain Boundaries for the cPLA2 C2 Domain--
To pursue
structural studies of the isolated cPLA2 C2 domain, we made several
constructs with slightly different boundaries for the expression in
E. coli. Two topologies for C2 domains have been described:
one present in the first C2 domain of synaptotagmin I (SytI-C2A) (36)
(S variant or topology I) and another present in the
phosphoinositide-specific phospholipase C (PLC-C2) (37) (P variant
or topology II) (35, 38). The two topologies can be best described as
circular permutations of each other. PLC-
1 starts with a strand
corresponding to the
2 strand of (SytI-C2A) and ends with a strand
corresponding to the
1 strand of SytI-C2A. Choosing wrong boundaries
for the isolated domain could greatly destabilize the protein by
truncating either the N-terminal or the C-terminal strand.
The Overall Fold--
The overall fold of the cPLA2 C2 domain is
illustrated in Fig. 1A. As
with the C2 domain from PLC-1 (PLC
-C2) and the C2A domain from
Syt I (SytI-C2A), it consists of an anti-parallel
-sandwich with two
4-stranded sheets. Sheet I of the
-sandwich consisting of strands 3, 2, 5, and 6 and has a concave surface to it, whereas sheet II has a
convex appearance. The topology of the domain is identical to that of
PLC
-C2 and is a circular permutation of the SytI-C2A topology (Fig.
2). Recently, this topology was predicted
for the cPLA2 C2 domain based on the sequence analysis of 65 C2 domains
and the known structure for the two topological variants (35). Although
naturally occurring circular permutations have been noted previously
for other proteins (39), the C2 domains have strikingly conserved
three-dimensional structures despite the topological variations. A
structurally conserved core of 105 residues of the C2 domain of cPLA2
can be superimposed on those of PLC-
1-C2 and SytI-C2A with 1.3 and
1.4 Å r.m.s. deviation, respectively. As shown in Fig. 2, the residues
that superimpose well among the three C2 domains are two of the
connecting loops (CBR2 and CBR3) and all of the
-strands except the
edge strand 7 of sheet II. Two loops involved in calcium binding, CBR2
and CBR3, have main-chain conformations that are essentially identical to the conformations observed for PLC
-C2 and for SytIA-C2. The third
loop involved in calcium binding, CBR1, is longer than in either
PLC-
1 or SytI-C2A and has a turn of
-helix. Among the three C2
domain structures, CBR1 has the most variable loop conformation. From
sequence analysis of C2 domains, it is clear that this region has the
greatest variability in both length and character (35, 40). The curved
shape of the domain is caused by four
-bulges that represent a
conserved feature of the cPLA2, SytI-C2A, and PLC
-C2 domains.
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Calcium Binding to cPLA2 C2 Domain--
The C2 domain of cPLA2 was
co-crystallized with calcium, and the structure reveals that it binds
two Ca2+ ions at one end of the domain between three loops,
previously named CBRs: CBR1, CBR2, and CBR3 (Fig. 1B, Fig.
2) (38). As with PLC-C2, the two calcium sites are adjacent and
about 4 Å apart. Calcium site I has a coordination that is
approximately a pentagonal bipyramid with one of the equatorial
positions missing where the two sites adjoin each other. The
coordination at site II is best described as an octahedron with one of
the equatorial vertices represented by a bidentate interaction with
Asp-93. Calcium site I has six ligands with an average distance to the
calcium of 2.5 Å and consists of one side-chain oxygen from each of
Asp-40, Asp-43, Asn-65, the carbonyl oxygen of Thr-41, and two water
molecules (Fig. 1, Fig. 3). The seven
ligands for site II have an average distance to the calcium of 2.5 Å and consist of one side-chain oxygen from each of Asp-40, Asp-43, and
Asn-95, both side-chain oxygens of Asp-93, the carbonyl oxygen of
Ala-94, and one water molecule. The exposed apices of each calcium site
are occupied by waters (Fig. 1C). These waters would
presumably be displaced so that the calcium ions could form a direct
interaction with the phosphate moiety of a lipid headgroup. Two of the
residues ligating the Ca2+ ions, Asp-40 and Asp-43, form
one interaction with each Ca2+ ion. This arrangement of
Ca2+ binding sites would be expected to lead to cooperative
Ca2+ binding because the binding of one ion would position
the ligands of the second ion as well as neutralize the negative charge
in the binding site, allowing the acidic side chains for the second site to more closely approach each other.
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cPLA2 Has a Preference for Head Groups with Hydrophobic
Features--
Both CBR1 and CBR3 of cPLA2 have a prominent cluster of
hydrophobic residues (Fig. 1C, Fig.
4). In CBR1, these hydrophobic residues
(Phe-35, Met-38, and Leu-39) are arrayed on the exposed face of the
-helical segment, and in CBR3, the hydrophobic residues (Tyr-96,
Val-97, and Met-98) are also exposed and include the apex of the loop.
The exposed hydrophobic area in these two loops is 780 Å2.
This hydrophobic patch may have a significant contribution to the
membrane binding of this domain. Indeed, both intact cPLA2 and the
cPLA2 C2 domain show calcium-dependent, preferential
binding to phospholipids with hydrophobic features of the headgroup
such as phosphatidylcholine in preference to phosphatidylserine,
phosphatidylinositol (PI) or phosphatidic acid (13, 16, 35). cPLA2 is
also known to bind very tightly to lipid vesicles consisting of
synthetic phospholipid phosphatidylmethanol,
1,2-dimyristoyl-sn-glycero-3-phosphomethanol and
1,2-dioleoyl-sn-glycero-3-phosphomethanol (47, 48). Although there are no data on the binding of the isolated cPLA2 C2 domain to
these phospholipids, the calcium-dependence of their binding to intact
cPLA2 suggests that the site of interaction is in the C2 domain.
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A Model for the cPLA2 C2 Domain Interaction with
Membranes--
The types of residues displayed on the surface of the
cPLA2 C2 domain suggest an interaction with membranes such that the hydrophobic CBR3 inserts into the membrane, CBR1 interacts with the
hydrophobic portions of the lipid head group (e.g. the
methylene and methyl groups of a choline moiety), and a patch of basic
residues arrayed along strand 3 makes weaker electrostatic
interactions with the negatively charged lipid head groups (Fig. 4 and
5). In the presence of bound calcium, the
surface of cPLA2 shown in Fig. 4 would become positively charged and
would presumably be the surface directed toward the membrane. In
PLC-
1, the analogous surface is exposed in the structure of the
intact enzyme, whereas much of the rest of the surface of the domain is
involved in interaction with the EF-hand domain and the linker sequence
between the catalytic domain and the C2 domain (37, 42, 53, 54).
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Hydrophobic-Electrostatic Switch of the cPLA2 C2 Domain-- The cPLA2 C2 domain interaction with membranes may be analogous to the hydrophobic-electrostatic switch that modulates reversible membrane binding of several myristoylated proteins such as the myristoylated alanine-rich protein kinase C substrate (MARCKS) or Src (66, 67). In those examples, both the hydrophobic interaction of the myristoyl group with the membrane interior and the electrostatic, surface interaction of a cluster of basic residues with polar head groups of acidic phospholipids are necessary to anchor the proteins to the membrane. In the case of cPLA2, the electrostatic switch is Ca2+, which changes the electrostatic properties of the C2 domain surface, i.e. neutralizes a cluster of negative charges in the CBR region and enables membrane binding, whereas in the case of MARCKS peptide, the switch is the phosphorylation of the peptide that introduces negative charges and prevents membrane binding. It is unknown whether calcium directly interacts with the phosphate group of phospholipids or whether it plays only an indirect, electrostatic role by neutralizing the negative charge of the acidic cluster. Our extensive efforts to crystallographically resolve this issue did not succeed. Although we were able to grow the crystals of the C2 domain of cPLA2 in the presence of calcium and a millimolar range of glycerophosphocholine and glycerophosphoserine, there was no structural evidence for binding of these compounds to the C2 domain.
The C2 domain of cPLA2 enables reversible, calcium-regulated binding of the intact enzyme to membranes. The control of this localization provides a powerful regulation of the enzyme activity. Once bound to membranes, cPLA2 could undergo processive catalysis (47, 68), a catalytic mode that has been observed in vitro for many enzymes involved in lipid signaling and metabolism in which multiple rounds of hydrolysis occur before the enzyme releases from the interface (69, 70). ![]() |
ACKNOWLEDGEMENTS |
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We thank Zeneca Pharmaceuticals for the clone of intact cPLA2, the staff of beamlines EMBL BW7B, Hamburg and Daresbury synchrotron radiation source stations 7.2 and 9.6, UK, Ed Walker for help in synchrotron data collection, Eric de La Fortelle for advice on SHARP, and Gérard Bricogne for help with BUSTER. We are grateful for the support by the MRC/DTI/ZENECA LINK Program (to R. L. W.). We thank the European Union for the support of synchrotron visits to EMBL Hamburg through the Human Capital and Mobility Programme access to Large Installation Project.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1RLW) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ To whom correspondence should be addressed: Tel.: 44-1223-402171; Fax: 44-1223-412178; E-mail: rlw{at}mrc-lmb.cam.ac.uk.
1 The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; PLC, phospholipase C; CBR, calcium binding region; PI, phosphatidylinositol; PKC, protein kinase C; MARCKS, myristoylated alanine-rich protein kinase C substrate.
2 M. Proctor and M. Bycroft, unpublished data.
3 S. Fong and M. Bycroft, unpublished data.
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REFERENCES |
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