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INTRODUCTION |
Cytosolic phospholipase A2
(cPLA2)1 is an
85-kDa protein that hydrolyzes phospholipids containing arachidonate at
the sn-2 position (Refs. 1 and 2; reviewed in Refs. 3-6).
This enzyme has no sequence homology to any other phospholipase
A2 and is capable of functioning in receptor-regulated,
agonist-induced arachidonic acid release (7, 8). Recent work with mice
lacking a gene for cPLA2 has demonstrated that
cPLA2 has a critical, nonredundant role in the production
of eicosanoids and platelet-activating factor in the process of
inflammation, anaphylaxis, and reproduction (9, 10). The activity of
cPLA2 is regulated by calcium; however, the role of calcium
is to promote membrane binding rather than participating in catalysis
directly. An increase in intracellular calcium triggered by calcium
ionophores or agonists such as histamine or IgE/antigen causes the
translocation of cPLA2 from the cytosol to the nuclear
membrane and endoplasmic reticulum (11-14), where it co-localizes with
other enzymes involved in eicosanoid metabolism, such as
prostaglandin-endoperoxide synthase-1 and -2, 5-lipoxygenase, and
5-lipoxygenase-activating protein (11, 15). The activity of
cPLA2 is also regulated via phosphorylation of the enzyme, and for at least some agonists, phosphorylation of cPLA2 is
a requisite step in its activation (4, 5). Detailed studies of the role
of calcium and phosphorylation in arachidonic acid release have shown
that a sustained increase in intracellular calcium is sufficient to
induce arachidonic acid release, whereas either sustained
phosphorylation of cPLA2 or a transient increase in calcium
alone is not sufficient, but they can synergize in this process
(16).
Recent work suggests that cPLA2 has three functionally
distinct domains: an N-terminal C2 domain necessary for
Ca2+-dependent phospholipid binding, a
C-terminal Ca2+-independent catalytic region capable of
hydrolyzing monomeric substrates but unable to associate with membranes
(17), and a putative pleckstrin homology domain within this region that may be responsible for the ability of the enzyme to make specific 1:1
interactions with phosphatidylinositol 4,5-bisphosphate (18). The
structure of the N-terminal C2 domain of cPLA2 has been
determined using both crystallographic (19) and NMR (20) techniques. The domain consists of an anti-parallel
-sandwich composed of two
four-stranded sheets. The overall fold of the cPLA2 C2
domain is similar to the C2 domains from other proteins: synaptotagmin I (SytI-C2A) (21), phospholipase C
1 (22), protein kinase C
(23),
and protein kinase C
(24). The topology of the domain is identical
to that of phospholipase C
1 and the recently reported calcium-independent C2 domain from protein kinase C
(23), whereas it
is a circular permutation of the SytI-C2A and protein kinase C
topology. Consistent with the equilibrium binding and stopped-flow kinetic experiments that demonstrated cooperative binding of two calcium ions to the cPLA2 C2 domain (25), the crystal
structure of cPLA2-C2 has shown two adjacent calcium ions
bound at one end of the domain via residues at the bases of three loops
known as calcium-binding regions (CBRs) 1-3 (19).
NMR studies with dodecylphosphocholine micelles have identified several
discrete regions in cPLA2-C2 that show large chemical shift
perturbations in 15N heteronuclear single quantum
correlation spectra upon micelle binding (20). These regions, which
include residues within the three CBRs, residues at the ends of strands
immediately adjacent to the CBRs, and residues of strands 2 and 3, could thus directly interact with phospholipids or have their
conformation changed by phospholipid binding.
In this paper, we have employed a set of green fluorescent protein
(GFP) fusions of cPLA2 deletion variants to show that the C2 domain is both necessary and sufficient for translocation of the
enzyme to internal membranes in response to calcium. We have also
demonstrated that a set of hydrophobic residues present in the
calcium-binding loops at one end of the domain is necessary for
Ca2+-dependent membrane translocation in
vivo and phospholipid binding in vitro. Membrane
binding is accompanied by penetration of two loops, CBR1 and CBR3, into
the hydrophobic core of the phospholipid bilayer.
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EXPERIMENTAL PROCEDURES |
GFP-cPLA2 Fusion Constructs--
Fig. 1 illustrates
the various constructs that were employed in our studies. For in
vivo translocation experiments, a series of human
cPLA2 constructs fused to the C terminus of GFP were made:
full-length cPLA2 (amino acids 1-749), a variant removing a short flexible N-terminal peptide (amino acids 17-749), a C2 domain-deleted variant (amino acids 148-749), or a C2 domain-only variant (amino acids 17-141). In the context of the full-length cPLA2 construct, three single or multiple site-specific
mutants were designed to examine the effect of mutations of a
calcium-binding residue (D43N) and residues proposed to be important
for phospholipid binding (M38N/L39A and Y96S/V97S/M98Q). Except for the
GFP-cPLA2-(17-141) fusion, all other GFP-cPLA2
fusions had a His6 tag directly fused to the C terminus of
the construct. GFP-cPLA2 fusions were cloned in a vector
(26) with a cytomegalovirus immediate-early gene promoter and the GFP
protein (preceded by the sequence MVTSPVEK) containing several
substitutions previously described to enhance fluorescence (S65T) and
to increase solubility and thermostability of GFP (V163A/S175G/I167T)
(27). All the cPLA2 constructs except for
cPLA2-(17-141) were cloned as
BamHI/XbaI fragments into the GFP-containing
vector cut with the same enzymes (the internal BamHI site in
the cPLA2 gene was mutated), and the resulting constructs had three residues (LGS) between the GFP and cPLA2
sequences. The cPLA2-(17-141) construct was cloned as a
BglII/EcoRI fragment into the vector, and the
final construct had LGSMEQKLISEEDLRS between the GFP and
cPLA2 sequences.
Escherichia coli Expression and Purification of Wild-type and
Mutant cPLA2 C2 Domains--
A plasmid expressing the C2
domain of cPLA2 (residues 17-141 of the intact enzyme) was
described previously (19). For in vitro binding studies,
site-specific mutants of this domain were prepared by polymerase chain
reaction-directed mutagenesis and expressed in mini-pRSET, a version of
pRSET (Invitrogen) modified to encode a 17-residue N-terminal tag
(MRGSHHHHHHGLVPRGS) containing a His6 tag for affinity
purification and a thrombin cleavage site. For the Trp mutants used for
fluorescence quenching studies, the wild-type Trp residue at position
71 was mutated to Phe, and a single Trp residue was introduced at
various positions in the C2 domain. The wild-type cPLA2 C2
domain was insoluble and expressed in inclusion bodies. The proteins
were refolded in vitro using the protocol essentially as
described previously (19). The inclusion body pellet from 0.5 liter of
cell culture was dissolved in 40 ml of 8 M urea and 50 mM Tris-HCl, pH 7.2 (denaturation buffer), by stirring for
2 h at room temperature and centrifuged for 30 min at 17,000 rpm
in a Sorvall SS34 rotor to remove the insoluble debris. Denatured
protein was bound in batch to 1.5 ml of
Ni2+-nitrilotriacetic acid-agarose (QIAGEN Inc.), and the
resin was washed four times with 50 ml (each wash) of denaturation
buffer. Protein was eluted with 10 ml of 300 mM imidazole
in denaturation buffer. The protein was concentrated to 10-15 mg/ml
using a Centriprep 10 at room temperature, and 1 ml of the purified
protein was renatured by adding 50-µl aliquots into a rapidly stirred
solution of 50 mM Tris-HCl, pH 7.2, and 1.5 M
urea at room temperature. After stirring for 1 h, the solution was
filtered through a 0.2-µm filter (Millex-GV, Millipore Corp.) and run
on a 1.6-ml Poros 20HQ column equilibrated in 50 mM Tris,
pH 8.0. The renatured protein was eluted with a gradient of 0-1
M NaCl (the folded protein eluted with ~150
mM NaCl). The C2 domain mutant M38N/L39A and all the Trp
mutants were insoluble and were refolded in the same way as the wild
type, whereas two of the site-specific mutants (D43N and
Y96S/V97S/M98Q) were soluble and required no refolding. The proteins
were purified on Ni2+-agarose as described above, but
without urea, and by ion-exchange chromatography on a Poros 20HQ column.
Translocation Studies by Fluorescence Analysis--
PtK2 cells
(potooro kidney cells) were microinjected with plasmid DNA (50 µg/ml,
except for GFP-cPLA2-(17-141), which was injected at 10 µg/ml). Microinjected cells were incubated overnight to allow
adequate expression of the plasmids, and culture medium was replaced
with Hanks' balanced salt solution without phenol red containing 1.25 mM CaCl2 (catalog No. 14025, Life Technologies, Inc.). The GFP fluorescence of the live cells was recorded using an MRC
1024 confocal imaging system both before and after addition of
4-bromo-A23187 calcium ionophore (Alexis) at a concentration of 20 µM for 3-5 min.
Calcium Binding Assays--
The ability of the recombinant
cPLA2 C2 domains to bind calcium was tested qualitatively
by calcium-dependent band shift on nondenaturing
polyacrylamide gels. Recombinant C2 domains (0.5 mg/ml) were mixed in a
20-µl reaction with 2 mM EDTA in the presence or absence
of 2.5 mM CaCl2 for 5 min at room temperature,
and 4-µl aliquots were analyzed by electrophoresis on native 20%
polyacrylamide Phast gels (Amersham Pharmacia Biotech). The positions
of the bands were visualized by staining with Coomassie Brilliant Blue R-250.
The Ca2+ binding affinity of recombinant C2 domains (wild
type and M38N/L39A and Y96S/V97S/M98Q mutants) was measured by
isothermal titration calorimetry with an Omega calorimeter (MicroCal
Inc.) using the same conditions as described previously for the
cPLA2 C2 domain (20). All proteins used for titrations were
dialyzed against 20 mM Tris, pH 7. Aliquots (4 µl each)
of calcium solution (2.4 mM) were injected at 4.6-min
intervals into the protein solution (40 µM) in a 1.34-ml
sample cell at 27 °C. Control experiments were carried out in the
absence of protein to determine the heats of dilutions, which were
subtracted from the apparent heat of binding prior to data analysis.
Data were analyzed using the ORIGIN software package provided with the calorimeter.
Protein Binding to Large Multilamellar Vesicles--
Binding of
recombinant C2 domains to large multilamellar vesicles prepared with
phosphatidylcholine from brain (Avanti Polar Lipids) was carried out as
described previously (28). The reaction mixtures (400 µl) contained 5 µg of C2 domains (final concentration of 0.77 µM), 100 µg of large multilamellar vesicles (final concentration of 330 µM), and 2 mM EDTA with or without 2.2 mM CaCl2. After incubation for 5 min at room
temperature, the reaction mixtures were centrifuged for 15 min at
4 °C, and the pellets were analyzed by SDS-polyacrylamide gel electrophoresis.
Fluorescence Measurements--
Fluorescence measurements were
carried out on a Hitachi F-4500 fluorescence spectrometer. The
intrinsic fluorescence of Trp-71 that is present in the wild-type C2
domain and D43N, M38N/L39A, and Y96S/V97S/M98Q mutants was measured
using an excitation wavelength of 284 nm, and emission scans were taken
from 300 to 400 nm.
Binding of C2 domains to large unilamellar vesicles was quantified by
measuring fluorescence resonance energy transfer (FRET), using Trp-71
as the donor and phosphatidylcholine vesicles containing dansyl-PE (5%
mol/mol; Molecular Probes, Inc.) as the acceptor, at 20 °C.
Phosphatidylcholine (PC from brain; Avanti Polar Lipids) and dansyl-PE
in chloroform were mixed, dried under a stream of nitrogen and then in
vacuo for at least 1 h, and resuspended in buffer F (150 mM NaCl and 20 mM Tris, pH 7.5). Large
unilamellar vesicles were prepared by extrusion through a polycarbonate
filter (100-nm pore size). Binding reactions contained 0.5 µM C2 domain, 1 mM EDTA plus 2 mM
CaCl2 (or EDTA alone), and various amounts of phospholipids
in 2 ml of buffer F. Samples were excited at 284 nm (5-nm slit width),
and emission was monitored at 530 nm (10-nm slit width); a 450-nm
long-pass filter was placed on the emission side. Relative FRET was
calculated as (F+Ca
F
Ca)/
Fmax, where
F+Ca represents the emission of the vesicles and
the C2 domain in the presence of saturating levels of calcium,
F
Ca represents the emission of the vesicles
and the protein in the absence of calcium, and
Fmax is the maximal energy transfer obtained from the binding curve. The data were analyzed by plotting relative FRET against the total lipid concentration and fitting the data to the
binding equation y = n(x/(Kd + x)),
where y represents relative FRET, x is the total
lipid concentration, n is a normalization constant, and
Kd is the apparent equilibrium dissociation constant.
Fluorescence Quenching by Doxyl-labeled
Phosphatidylcholine--
Large unilamellar vesicles were prepared by
extrusion through a polycarbonate filter (100-nm pore size) using an
Avanti Polar Lipids extruder. For cPLA2-C2 binding assays,
the vesicles contained either 100% PC (control) or 90% PC and 10%
doxyl-labeled PC, with a doxyl group at position 7 or 12 of the
sn-2-acyl chain (Avanti Polar Lipids). For SytI-C2A binding
assays, vesicles contained 50% phosphatidylserine and 50% PC
(control) or 50% phosphatidylserine, 40% PC, and 10% doxyl-PC. The
protein sample (final concentration of 5 µM) was diluted
into a 1.6-ml assay containing 250 µM total phospholipid
in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2 mM EDTA in a 3-ml quartz cuvette with a magnetic stirrer.
Fluorescence spectra were taken before and after addition of calcium to
a final concentration of 3 mM. Samples were excited at 288 nm (2.5-nm slit width), and emission spectra were collected from 300 to
450 nm (5-nm slit width) at 20 °C.
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RESULTS |
The C2 Domain of cPLA2 Is Sufficient for
Calcium-dependent Translocation in Vivo--
To determine
if the C2 domain is sufficient for membrane translocation in response
to an increase in intracellular Ca2+ or whether other
features of the intact enzyme are also essential, a series of
GFP-cPLA2 fusions were constructed (Fig.
1), and their translocation was observed
in intact cells using confocal fluorescence microscopy. As shown in
Fig. 2, the GFP fusion of full-length cPLA2 (GFP-cPLA2-(1-749)) translocates from
the cytosol to internal membranes in a pattern that is consistent with
binding to nuclear membranes, the endoplasmic reticulum, and the Golgi.
This is in agreement with previous immunofluorescence studies of
cPLA2 using fixed cells (11-14). Structural studies have
shown that the N-terminal 16 residues of the enzyme are flexible and do
not contribute to the structural integrity of the N-terminal C2 domain
(19, 20). The GFP-cPLA2-(17-749) construct shows
calcium-dependent translocation identical to the
full-length enzyme. Using fixed cells, it was previously shown that an
N-terminal truncation variant of cPLA2 (amino acids
178-749) does not translocate in response to calcium (13). Fig. 2
shows that the deletion variant corresponding to removal of the C2
domain only, the GFP-cPLA2-(148-749) construct, also does
not translocate to internal membranes. However, a GFP fusion with only
the C2 domain, GFP-cPLA2-(17-141), is capable of
Ca2+-dependent membrane translocation in
vivo. These results show that the C2 domain consisting of residues
17-141 of cPLA2 is both necessary and sufficient for
Ca2+-dependent translocation. Although the C2
domain alone enables translocation, membrane interactions by the
remainder of the enzyme including the putative pleckstrin homology
domain may be important for the kinetics of translocation or for the
fine-tuning of the enzyme activity once on the membrane surface (18,
29).

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Fig. 1.
Schematic representation of the
cPLA2 domain structure and constructs used for in
vivo and in vitro studies. A,
shown is a putative domain organization of cPLA2. Only the
N-terminal C2 domain (residues 17-141) has been verified by structural
studies. B, for in vivo translocation studies in
PtK2 cells, full-length, truncated, and/or mutated cPLA2
was fused to the C terminus of GFP. C, shown is a schematic
diagram of C2 domain constructs expressed in E. coli and
used for in vitro binding to phospholipids. PH,
pleckstrin homology.
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Fig. 2.
Calcium ionophore-induced translocation of
GFP-cPLA2 constructs in PtK2 cells. The left
panel for a given construct illustrates fluorescence of a GFP
fusion for a field of transfected cells. The right panel
illustrates the same field 3-5 min after addition of 4-bromo-A23187
calcium ionophore at a concentration of 20 µM.
Translocation was observed only for constructs containing the C2 domain
(residues 17-141). The pattern of fluorescence upon ionophore
treatment suggests translocation to nuclear membranes, the endoplasmic
reticulum, and the Golgi.
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Mutations in Loops CBR1 and CBR3 Selectively Inhibit Phospholipid
Binding by the C2 Domain in Vitro and by the Full-length Enzyme in
Vivo--
Several mutants were examined in the context of the isolated
C2 domain to assess the contributions of various residues to membrane
binding. The experimentally determined structure of the cPLA2 C2 domain served as a guide to select residues that
would be likely candidates for participation in membrane interactions. The double mutant M38N/L39A and the triple mutant Y96S/V97S/M98Q were
examined in order to ascertain the importance of the exposed hydrophobic residues in CBR1 and CBR3, respectively, for membrane binding. In vitro binding studies were carried out for the
isolated C2 domains, whereas the effect of the mutants on in
vivo translocation was assayed in the context of the full-length
enzyme. All of the C2 domain constructs used for in vitro
binding assays were monomers as analyzed by gel filtration (data not
shown) and correctly folded as judged by intrinsic fluorescence spectra
(Fig. 3). The membrane binding properties
of these mutants were compared with those of the D43N mutant, in which
the acidic residue shown crystallographically to bind both
Ca2+ ions was replaced by a neutral analogue.

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Fig. 3.
Analysis of purified C2 domain mutants.
A, analysis of the wild-type (WT) and mutant
cPLA2 C2 domains by SDS-polyacrylamide gel electrophoresis.
Samples of purified wild-type and mutant cPLA2 C2 domains
were analyzed by electrophoresis on 20% SDS-polyacrylamide Phast gels.
Molecular mass markers (47, 30, 19, 15, 6, and 3 kDa) are shown in the
right lane. B, comparison of intrinsic
fluorescence spectra of the refolded wild-type and M38N/L39A C2 domains
and solubly expressed D43N and Y96S/V97S/M98Q mutants with the spectrum
of the urea-denatured wild-type C2 domain. The denatured protein has a
peak at 348 nm, nearly identical to that of a free tryptophan. The
refolded and solubly expressed proteins have an identical emission
wavelength maximum at 326 nm.
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The ability of C2 domain mutants to bind Ca2+ was first
evaluated using a rapid gel-shift assay, which is based on a large
difference in the overall charge between a Ca2+-bound and
Ca2+-free form of cPLA2-C2 and, consequently,
differential migration on a native gel. Neither the mutations in CBR1
nor CBR3 significantly affected Ca2+ binding at the high
concentration of free Ca2+ (0.5 mM) that was
used in the Ca2+-dependent gel-shift assay,
whereas, as expected, the D43N mutant did not bind Ca2+
(Fig. 4A). The calcium binding
affinities of the CBR1 and CBR3 mutants were further examined by
isothermal titration calorimetry and compared with those of the
wild-type C2 domain. For wild-type cPLA2-C2, a
Kd of 2.4 µM was determined, in
excellent agreement with the Kd of 1.7 µM that was determined previously for a similar
cPLA2-C2 construct (residues 1-138) by isothermal titration calorimetry under the same experimental conditions (20). Mutations in CBR1 and CBR3 had only a modest effect on calcium binding
affinities, with Kd values of 9.2 and 7.6 µM, respectively. Despite their abilities to bind
Ca2+, the CBR1 and CBR3 mutants did not bind PC vesicles at
saturating Ca2+ concentrations as shown by both
multilamellar large vesicle sedimentation assays (Fig. 4B)
and FRET assays (Fig. 4C). Using the FRET analysis, an
apparent Kd for PC binding of 11 ± 2 µM was obtained for the wild-type C2 domain, in agreement
with previously reported affinities (25, 29). In contrast, none of the
mutants showed any measurable FRET at the highest lipid concentration
used (250 µM) and 1 mM Ca2+.
Higher phospholipid concentrations could not be tested due to an inner
filter effect of the lipid vesicles. As a consequence of its impaired
Ca2+ binding, the D43N mutant also showed no detectable
binding to PC vesicles (Fig. 4, B and C). All
three mutations that inhibited in vitro vesicle binding also
abolished in vivo membrane translocation of the full-length
protein in response to Ca2+ (Fig.
5).

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Fig. 4.
Calcium and phospholipid binding properties
of C2 domain mutants. A, calcium binding was qualitatively
assessed by mobility shift of the domains in the presence of
Ca2+ on native 20% polyacrylamide Phast gels. The
cPLA2 C2 domain has an overall negative charge in the
absence of calcium and migrates farther toward the anode than when it
is bound to calcium. The position of the bands was visualized by
staining with Coomassie Brilliant Blue R-250. B, shown is
the binding of wild-type (WT) and mutant cPLA2
C2 domains to large multilamellar vesicles (MLV). Proteins
were incubated with large multilamellar vesicles composed of PC in the
presence or absence of 0.2 mM free CaCl2 for 5 min, and the vesicles were then sedimented by centrifugation. Protein
bound to the sedimented vesicles was analyzed by SDS-polyacrylamide gel
electrophoresis followed by Coomassie Blue staining. C,
shown is the binding of wild-type ( ) and mutant cPLA2 C2
domains including the CBR1 ( ), CBR3 ( ), and D43N (+) mutants to
large unilamellar vesicles measured by fluorescence energy transfer.
Binding reactions contained 0.5 µM protein, 1 mM EDTA with or without 2 mM CaCl2,
and increasing concentrations (from 2.5 to 250 µM) of
PC/dansyl-PE vesicles in 2 ml of buffer containing 20 mM
Tris, pH 7.5, and 150 mM NaCl at 20 °C. The solid
line represents the nonlinear least-squares fit to the data
obtained for the wild-type cPLA2 C2 domain. The apparent
Kd for PC binding in the presence of 1 mM CaCl2 for the wild-type cPLA2 C2
domain was 11 ± 2 µM. Data for the mutants were not
fitted since no detectable binding was observed for any of them.
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Fig. 5.
Mutations of the C2 domain prevent
translocation of GFP-cPLA2 in PtK2 cells. Mutants of
the full-length GFP-cPLA2 construct (amino acids 1-749)
were assayed for translocation in vivo. The left
panel for a given mutant illustrates fluorescence of the GFP
fusion for a field of transfected cells. The right panel
illustrates the same field 3-5 min after addition of 4-bromo-A23187
calcium ionophore at a concentration of 20 µM. All three
of the C2 domain mutants abolish translocation.
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Mapping the Phospholipid-binding Surface by Shifts in Intrinsic
Fluorescence Maxima--
A series of mutants were constructed to
replace single surface-exposed hydrophobic residues (usually
phenylalanine) with tryptophan. By monitoring the intrinsic
fluorescence of these mutants, it is possible to infer which of these
residues enters a more non-polar environment upon vesicle binding.
These Trp replacements were constructed in the context of a mutant
enzyme in which the only endogenous Trp residue in the wild-type enzyme
(Trp-71) was replaced by phenylalanine. The positions at which Trp
replacement mutants were created are illustrated in Fig.
6 and include F35W in CBR1, F49W at the
end of strand
2, F63W at the beginning of CBR2, and Y96W and V97W in
CBR3. In the presence of PC vesicles, the addition of Ca2+
to the CBR1 or CBR3 Trp mutants resulted in a blue shift of the fluorescence spectrum (Fig. 7A
and Table I), whereas the wild type and
F49W showed no significant change in the emission wavelength maximum,
and the F63W mutant had its maximum slightly red-shifted. The large
blue shift for the CBR1 (F35W) and CBR3 (Y96W and V97W) Trp mutants
indicates that these residues are in an environment that is much more
protected from solvent when the domain is bound to vesicles. This would
be consistent with these residues being directly involved in vesicle
binding. For the wild-type enzyme, the single Trp present (Trp-71) is
deeply buried in the core of the domain and shows only a minimal shift
upon addition of Ca2+ and PC vesicles. The F49W mutant also
showed no blue shift, and F63W showed a slight red shift, suggesting
that these residues do not enter a more non-polar environment upon
vesicle binding. The maximum emission wavelength of F63W upon vesicle
binding is nearly the same as that of free tryptophan, suggesting an
extreme solvent exposure. The red shift and the pronounced change in
fluorescence intensity displayed by the CBR2 mutant (F63W) may be
indicative of conformational changes in the domain or may indicate
interaction with the polar region of the membrane. Taken together, the
results show that residues in CBR1 and CBR3 insert into the hydrophobic portions of the membrane, whereas residues 49 and 63 remain in a polar
environment.

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Fig. 6.
Tryptophan replacement mutants used for
intrinsic fluorescence quenching studies. The schematic
illustrates the positions of the exposed hydrophobic residues that were
selected for Trp replacement mutants. Each mutation was done in the
context of a W71F mutant of the wild-type cPLA2 C2 domain
so that each mutant would have a single Trp residue. The
Ca2+ sites observed structurally are shown as black
spheres. The three loops at one end of the domain that are
involved in calcium binding are referred to as CBR1, CBR2, and CBR3
(19, 47). The figure was prepared with BOBSCRIPT (48).
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Fig. 7.
Intrinsic fluorescence spectra of the
wild-type and Trp replacement mutants. A, the intrinsic
fluorescence (arbitrary units) of the wild-type (WT) and
mutant cPLA2 C2 domains with large unilamellar PC vesicles
either in the absence (solid lines) or presence
(dashed lines) of 1 mM free CaCl2.
B, intrinsic fluorescence of the cPLA2 C2
domains with 1 mM free CaCl2 in the presence of
large unilamellar vesicles of 100% PC (solid lines), 90%
PC and 10% 12-doxyl-PC (dotted lines), or 90% PC and 10%
7-doxyl-PC (dashed lines).
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Table I
Probing membrane binding and penetration of single tryptophan
cPLA2 C2 domain mutants by fluorescence measurements
For the cPLA2 C2 domains containing a single tryptophan residue
at a different position on the surface of the C2 domain, maximum
emission wavelengths corresponding to the spectra presented in Fig. 7
are listed. The fluorescence was measured first in the presence of PC
vesicles and 2 mM EDTA and then following addition of 3 mM CaCl2. The integrated values for the
fluorescence spectra of the mutants in the presence of calcium and
vesicles containing 100% PC were compared with the spectra obtained in
the presence of calcium and vesicles composed of 90% PC and 10%
7-doxyl- or 12-doxyl-PC. The percent quenching in the presence of
doxyl-labeled PC relative to the 100% PC vesicles is listed.
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Both Loops CBR1 and CBR3 Become Deeply Immersed in the Hydrophobic
Portion of the Membrane--
We had previously shown that the
cPLA2 C2 domain penetrates into membranes upon binding
(28). The change in intrinsic fluorescence upon vesicle binding
suggests that CBR1 and CBR3 are interacting with the membrane. To
determine the extent to which the domain penetrates into the membrane
and its orientation on the membrane, we examined quenching of the
intrinsic fluorescence of the Trp mutants by doxyl probes covalently
attached at either position 7 or 12 of the sn-2-acyl chain.
Chapman and Davis (30) had employed similar methodology on SytI-C2A and
found that Phe-234 and, to a lesser extent, Phe-231 (both in CBR3)
penetrate into the phospholipid membrane. Fig. 7B and Table
I show that in cPLA2, both CBR1 and CBR3 Trp mutants have
their fluorescence quenched by the doxyl-PC lipids. In contrast, the
wild-type (Trp-71), F49W, and F63W C2 domains were unaffected by the
presence of doxyl-PC. Because both 7- and 12-doxyl-PC show equivalent
fluorescence quenching, the residues of CBR1 and CBR3 must be immersed
so that they are <10-11 Å from both positions 7 and 12 of the
sn-2-acyl chain (31). Our results indicating that CBR3 and
CBR1 of cPLA2-C2 penetrate into the membrane whereas CBR2
does not are consistent with the character of these loops. In all C2
domains of known structure, there is at least one exposed hydrophobic
residue at the tip of CBR3. In cPLA2-C2, CBR1 has a much
more hydrophobic character than in other C2 domains. Our results with
doxyl-PCs indicate that CBR2 does not penetrate into the membrane,
consistent with the red shift observed for the F63W mutant observed
upon binding PC vesicles.
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DISCUSSION |
The importance of translocation to membranes as an activating
mechanism for various enzymes in cells is becoming increasingly apparent. A variety of ubiquitous protein modules function as mediators
of translocation. For example, the role of pleckstrin homology domains
in recognizing specific phospholipids and bringing about translocation
to membranes has been demonstrated for a range of signaling proteins
(reviewed in Ref. 32). Although C2 domains that are present in a great
number of proteins have been implicated in membrane binding and vesicle
fusion (33, 34), the contribution of these domains to cellular
localization has been established for only a few proteins. For example,
it has been shown by deletion analysis that the C2 domain is necessary
for calcium-dependent translocation of protein kinase C
and Nedd4 to plasma membranes (35, 36) and of cPLA2 to
nuclear membranes and the endoplasmic reticulum (13). However, we have
demonstrated here that the cPLA2 C2 domain alone is
sufficient for calcium-dependent translocation in intact
cells. We have furthermore demonstrated that the epitopes of the
cPLA2 C2 domain that penetrate into the lipid bilayer are essential for membrane binding and translocation. Other parts of the
intact cPLA2 beyond the C2 domain may modify lipid
specificity, calcium sensitivity, and enzyme activity at the target
site (18, 29). In particular, the ability of the intact enzyme to bind phosphatidylinositol 4,5-bisphosphate at low calcium concentrations (18, 37) has been attributed to the presence of a pleckstrin homology
domain. The binding of the anionic phosphatidylmethanol to the intact
enzyme in the absence of calcium, but not to the C2 domain alone (29),
also indicates that there are multiple sites of membrane binding on the
enzyme. Although our results show that the C2 domain is sufficient for
membrane translocation in response to calcium ionophore, other pathways
of activation of cPLA2 may rely on these additional sites
of membrane interaction.
Our mutagenesis results show that membrane interaction for the
cPLA2 C2 domain is driven largely by hydrophobic forces. We have demonstrated that the CBR1 and CBR3 mutants, which replace hydrophobic residues with polar ones, M38N/L39A and Y96S/V97S/M98Q, have functional Ca2+ binding, but show no detectable
binding to phospholipid membranes in vitro or in
vivo. Our results are consistent with the observation that
cPLA2 C2 domain binding to PC vesicles is enhanced at high ionic strengths (38). Calcium binding is clearly another critical factor in membrane interaction. The importance of Asp-43, which makes
interactions with each of the two Ca2+ ions bound to
cPLA2-C2, is confirmed by the loss of membrane binding by
the D43N mutant. By preventing Ca2+ neutralization of the
negatively charged residues, there may be an electrostatic repulsion
between the protein and negative charges in the membrane. For the
protein kinase C
II C2 domain, it was shown that replacing acidic
calcium-binding residues with basic residues did not result in
calcium-independent membrane binding (39), suggesting that charge
neutralization is not calcium's only role in membrane binding. Another
role for Ca2+ ions might be acting as direct ligands of the
phosphate groups in the membrane such as seen for annexin V (40).
Given the importance of the hydrophobic residues in CBR1 and CBR3 for
membrane binding, we sought to determine whether these residues
penetrate into the hydrophobic portions of the lipid bilayer and, if
so, to what extent they are immersed in the membrane. The ability of
the doxyl labels at positions 7 and 12 of the sn-2-acyl chains to quench fluorescence from residues in CBR3 indicates that this
loop is immersed in the hydrophobic core of the membrane. The CBR3
loops in SytI-C2A (30) and in protein kinase C
(41) have also been
shown to penetrate into lipid bilayers, suggesting that C2 domains in
general may be similarly oriented when bound to membranes. A more
surprising result from the doxyl-PC quenching is that CBR1 is immersed
in the hydrophobic portion of the membrane to a similar depth as CBR3.
Although CBR1 in cPLA2-C2 is longer and more hydrophobic
than in most C2 domains, other C2 domains also have hydrophobic
residues in CBR1, and it may be that membrane penetration by this loop
is a general feature of C2 domain-membrane interaction. For
cPLA2, it has been recently shown that
calcium-dependent membrane penetration plays a critical
role in the enzyme activity and greatly contributes to membrane binding
and arachidonate specificity of the enzyme (42).
The critical interaction distance between a tryptophan and a
membrane-embedded nitroxide spin-labeled phospholipid at which a
quenching can be observed has been estimated to be 10-11 Å (31). This
enables us to place Phe-49 (at the end of strand
2) and Phe-63 (at
the beginning of CBR2) outside the hydrophobic core of the lipid
bilayer. Consistent with this, there is no
calcium-dependent blue shift in the emission maximum upon
liposome binding for either of these two mutants. Nevertheless, there
is a pronounced change in the intensity of fluorescence for residue 63 upon binding PC vesicles. This could mean that CBR2 is forming
interactions with the polar head groups of the membrane or that its
environment is changing due to a conformational change in the protein
induced by lipid binding. Either of these interpretations would be
consistent with the observation that micelle binding by
cPLA2-C2 induces NMR chemical shift changes for residues in
CBR2 (20) and similar observations for short-chain phosphatidylserine
(di-C6-phosphatidylserine) binding to the SytI C2A domain
(43).
A model of the cPLA2 C2 domain bound to a lipid membrane is
presented in Fig. 8. This model, based on
our fluorescence quenching results, requires that Phe-35 in CBR1 and
Tyr-96 and Val-97 in CBR3 are immersed to approximately the same depth
in the hydrophobic core of the membrane and that Phe-49 and Phe-63 are
at a very different level, i.e. outside the hydrophobic
region. A further requirement was to place the C2 domain in such a
position that the phosphates of the phospholipids could make direct
interaction with the exposed coordination sites of the Ca2+
ions in the crevice formed by CBR1 and CBR3. This constraint may be
reasonable in light of other protein-phospholipid interactions that
have been characterized such as for annexin V, but has no experimental
evidence in the context of C2 domains. In the model shown in Fig. 8,
residues in CBR2 would lie just at the interface between the solvent
and the head group region of the membrane. This arrangement with CBR1
and CBR3 penetrating and CBR2 at the interface is somewhat different
than what was proposed by Chae et al. (43) for the SytI C2A
domain based on NMR chemical shifts caused by binding to short-chain
phosphatidylserine. These workers proposed that a hydrophobic ridge
consisting of residues from CBR2 and CBR3 forms the
phospholipid-interacting surface of the domain, with CBR1 directed away
from the membrane. The apparent lack of involvement of CBR1 in membrane
binding by SytI-C2A may mean that cPLA2-C2 and SytI-C2A
bind membranes very differently, given that we observe CBR1 to
penetrate into the membrane to a similar degree as CBR3, but the
difference may also be related to the use of soluble short acyl chain
phospholipids for the NMR study. A heteronuclear single quantum
correlation NMR study of cPLA2-C2 in the presence and
absence of dodecylphosphocholine micelles (20) has shown that residues
in all three CBRs undergo the greatest changes in
15N/NH chemical shifts.

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Fig. 8.
Model of the cPLA2 C2 domain
bound to a phospholipid membrane. A, shown is the chemical
structure of spin-labeled phospholipids used in fluorescence quenching
experiments
(1-palmitoyl-2-stearoyl-(n-doxyl)-sn-glycero-3-phosphocholine)
with a doxyl group at position 7 or 12 of the sn-2-acyl
chain. Values for the depths of doxyl groups are taken from Ref. 49.
B, the crystallographically observed cPLA2 C2
domain structure was placed onto the crystal structure of a
dimyristoyl-PC monolayer (one layer from the bilayer crystal structure
is shown) (50). The crystal structure of the dimyristoyl-PC has the
lipids arranged in two staggered levels. Positions 7 and 12 of the
sn-2-acyl chains in each of the two levels are indicated by
red and blue arrows. The domain was positioned so
that Trp residues at positions 96, 97, and 35 (rendered as
green CPK models) would be immersed into the membrane to
approximately equal depths and <10 Å from positions 7 and 12 of the
sn-2-acyl chains. The domain was also positioned so that
residues 49 and 63 (purple CPK models) were farther than 10 Å from position 7 of the sn-2-acyl chains in the lower of
the two staggered PC levels. The solvent-exposed areas of the bound
Ca2+ ions (cyan) were positioned to form a
direct interaction with the phosphate of the phospholipid head group.
The figure was prepared with BOBSCRIPT and Raster3D (51).
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