From the Department of Chemistry, University of Illinois, Chicago, Illinois 60607-7061
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ABSTRACT |
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The C2 domain of cytosolic phospholipase
A2 (cPLA2) is involved in the
Ca2+-dependent membrane binding of this
protein. To identify protein residues in the C2 domain of
cPLA2 essential for its Ca2+ and membrane
binding, we selectively mutated Ca2+ ligands and putative
membrane-binding residues of cPLA2 and measured the effects
of mutations on its enzyme activity, membrane binding affinity, and
monolayer penetration. The mutations of five Ca2+ ligands
(D40N, D43N, N65A, D93N, N95A) show differential effects on the
membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The
mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A)
in the calcium binding loops show that the membrane binding of
cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully
inserted into the membrane, whereas Phe35 and
Tyr96 are partially inserted. Finally, the mutations of
four cationic residues in a Phospholipases A2
(PLA2s1; EC
3.1.1.4) are a large family of lipolytic enzymes that catalyze the
hydrolysis of the fatty acid ester at the sn-2-position of
phospholipids (1, 2). Among various mammalian PLA2s, 85-kDa
cytosolic PLA2 (cPLA2) can selectively liberate
arachidonic acid from membrane phospholipids, which can be converted to
potent inflammatory lipid mediators, prostaglandins, thromboxanes,
leukotrienes, and lipoxins, collectively known as eicosanoids (3).
Recent genetic studies showed that the deletion of the
cPLA2 gene results in loss of lipid mediator biosynthesis
(4, 5). cPLA2 is therefore an attractive target for
developing specific inhibitors that can be used as novel
anti-inflammatory drugs. cPLA2 is translocated to
endoplasmic reticulum membranes and nuclear envelopes in response to a
rise in intracellular Ca2+ (6, 7). This membrane
translocation of cPLA2 is mediated by its amino-terminal C2
domain, which contains calcium and membrane binding sites (8, 9).
However, the mechanism by which the C2 domain of cPLA2
drives its Ca2+-dependent membrane targeting is
not fully understood. Our recent structure-function study of the C2
domain of protein kinase C- Materials--
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) and
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine were
purchased from Avanti Polar Lipids (Alabaster, AL).
1,2-Di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC) and 1,2-sn-dioleoylglycerol were from Sigma. All
lipids were used without further purification.
1,2-Di-O-hexadecyl-sn-glycero-3-phosphoglycerol (DHPG) was prepared from DHPC by phospholipase D-catalyzed
transphosphatidylation as described by Comfurius and Zwaal (17).
Tritiated POPC ([3H]POPC) was prepared from
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and
[9,10-3H]oleic acid (American Radiolabeled Chemicals)
using rat liver microsomes as described (18, 19). Phospholipid
concentrations were determined by phosphate analysis (20).
1-Stearoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine
([14C]SAPC) (55 mCi/mmol) was from Amersham Pharmacia
Biotech. Styrene-divinylbenzene beads (5.2 ± 0.3-µm diameter)
were purchased from Seradyn (Indianapolis, IN). Fatty acid-free bovine
serum albumin (BSA) was from Bayer Inc. (Kankakee, IL). Restriction
endonucleases and other enzymes for molecular biology were obtained
from either Boehringer Mannheim or New England Biolabs (Beverly, MA).
Kanamycin, Triton X-100, sodium deoxycholate, phenylmethylsulfonyl
fluoride, urea, and guanidinium chloride were from Sigma.
Construction of Expression Vectors and
Mutagenesis--
Baculovirus transfer vectors encoding the cDNA of
cPLA2 with appropriate C2 domain mutations were generated
by the overlap extension polymerase chain reaction (21) using
pVL1393-cPLA2 plasmid as a template (15). Briefly,
appropriate complementary synthetic oligonucleotides introducing the
desired mutation and two other primers at the 5'- and 3'-ends of the
cPLA2 gene, respectively, were used as primers for
polymerase chain reactions, which were performed in a DNA thermal
cycler (Perkin Elmer) using Pfu DNA polymerase (Stratagene,
La Jolla, CA). The method consisted of two steps. In the first step,
two DNA fragments overlapping at the mutation site were generated and
purified on an agarose gel. Then these two fragments were annealed and
extended to generate a full-length mutated cPLA2 gene,
which was further amplified by polymerase chain reaction. The product
was subsequently purified on an agarose gel, digested with
NotI and BglII, and subcloned into the pVL1393
plasmid. The mutagenesis was verified by DNA sequencing of the
cPLA2 gene using a Sequenase 2.0 kit (Amersham Pharmacia
Biotech). The isolated C2 domain constructs (wild type and mutants)
containing amino-terminal 137 residues were generated by polymerase
chain reaction using corresponding pVL1393-cPLA2 vectors as
templates. An NdeI site was incorporated at the 5'-end of
the coding sequence, and a stop codon followed by a HindIII site was introduced after Leu137 at the 3'-end. These
constructs were subcloned into pET28a vector (Novagen, Madison, WI)
using NdeI and HindIII sites. These vectors also
encode a 20-residue amino-terminal tail (MGSSHHHHHHSSGLVPRGSH) containing a His6 tag for affinity purification and a
thrombin cleavage site. The identity of constructs was verified by
restriction digest and DNA sequencing.
Expression of cPLA2 and Mutants in
Baculovirus-infected Sf9 Cells--
Wild type cPLA2
and mutants were expressed in baculovirus-infected Sf9 cells
(Invitrogen, La Jolla, CA). Transfection of Sf9 cells with
mutant pVL1393-cPLA2 constructs was performed using the
BaculoGoldTM transfection kit from Pharmingen (San
Diego, CA). Plasmid DNA for transfection was prepared using an EndoFree
Plasmid Maxi kit (Qiagen, Valencia, CA) to avoid possible endotoxin
contamination. A detailed protocol for expression and purification of
cPLA2 from Sf9 cells is described elsewhere (15).
Protein concentration was determined by the bicinchoninic acid method (Pierce).
Bacterial Expression and Purification of Isolated C2 Domains of
cPLA2 and Mutants--
Escherichia coli strain
BL21(DE3) (Novagen) was used as a host for protein expression. Five
hundred ml of Luria broth supplemented with 50 µg/ml kanamycin was
inoculated with 1 ml of overnight culture grown at 37 °C. Cells were
grown at 22 °C until their absorbance at 600 nm reached ~0.6, and
the protein expression was then induced with 0.5 mM
isopropyl-1-thio- Determination of cPLA2 Activity--
Activity of
cPLA2 was assayed by measuring the initial rate of
[14C]SAPC hydrolysis as described (22). Assay mixtures
contained small unilamellar vesicles of [14C]SAPC (10 µM), 16 µM BSA, 0.1 M KCl, and
varying concentrations of CaCl2 in 60 µl of 20 mM HEPES, pH 8.0. Free calcium concentration was adjusted
using a mixture of EGTA and CaCl2 according to the method
of Bers (23). 12 mol % of 1,2-sn-dioleoylglycerol was added
to [14C]-SAPC vesicles when the free enzyme concentration
was determined by cPLA2 activity assay in vesicle binding
measurements (see below). Reactions were started by adding
cPLA2 to the mixture (to a final concentration of ~20
nM) and quenched by adding 370 µl of
chloroform/methanol/HCl (2:1:0.01) solution after a given period of
incubation (3-5 min) at room temperature. Liberated
[14C]arachidonic acid was separated from the reaction
mixture on small silica gel columns as described by Ghomashchi et
al. (22). Radioactivity of hydrolyzed arachidonic acid was
measured by liquid scintillation counting.
cPLA2-Phospholipid Binding--
The binding of
cPLA2 to phospholipids was measured by a centrifugation
assay using phospholipid-coated hydrophobic styrene-divinylbenzene beads. The beads were coated with DHPC or DHPG as described elsewhere (19). The concentration of phospholipids coated on beads was determined
by measuring the radioactivity of a trace of [3H]POPC
(0.1 mol %) included in all phospholipid mixtures. The bulk
phospholipid concentration of phospholipid-coated beads was typically
50-100 µM. Binding mixtures contained a fixed
concentration of phospholipid-coated beads in 20 mM
Tris-HCl buffer, pH 7.5, 0.1 M KCl, 1 µM BSA,
0.5 mM Ca2+ and varying concentration of enzyme
(20-200 nM). BSA was added to minimize the loss of protein
due to nonspecific adsorption to tube walls. Controls contained the
same mixtures minus beads. After the mixtures were incubated for 15 min, beads were pelleted (12,000 × g for 2 min), and
the enzyme activity of each supernatant toward
[14C]SAPC/1,2-sn-dioleoylglycerol liposomes
was measured (see above). The concentration of bound enzyme
([E]b) was calculated from the difference of
cPLA2 activity in control and binding mixtures. Parameters
n and Kd were determined by nonlinear
least-squares analysis of the bound ([E]b)
versus total enzyme concentration ([E]o) plot using a standard binding equation,
The binding of isolated C2 domains to POPC vesicles was measured by a
centrifugation assay using sucrose-loaded unilamellar vesicles (100-nm
diameter) (25). Sucrose-loaded vesicles were prepared as follows. The
lipid solution of POPC was added to a round-bottomed flask, and organic
solvent was removed by rotary evaporation. The lipid film was suspended
in 20 mM Tris-HCl buffer, pH 7.5, containing 0.2 M sucrose (to a final lipid concentration of ~20
mM) and vortexed vigorously. Unilamellar vesicles were prepared by multiple extrusion through a 0.1-µm polycarbonate filter
(Millipore Corp.) in a Liposofast microextruder (Avestin; Ottawa,
Ontario). For binding measurements, the vesicle solution was diluted
into 20 mM Tris-HCl buffer, pH 7.5, 0.1 M KCl,
and 0.5 mM Ca2+ (or EGTA) to yield a final
phospholipid concentration of 0.5 mM. After adding wild
type or a mutant C2 domain (final concentration of 0.5 µM), binding mixtures were incubated for 15 min at room temperature and centrifuged at 100,000 × g for 30 min
at 25 °C. Supernatants were decanted, and pellets were redissolved
in 15 µl of 10 mM HEPES buffer, pH 7.0, containing 0.1 M KCl and 0.5 mM EGTA. Resuspended pellets were
loaded on 14% polyacrylamide gels, and C2 domains were separated by
SDS-polyacrylamide gel electrophoresis. The amount of protein in each
band was quantified using IS-1000 Digital Imaging System (Key
Scientific, Mt. Prospect, IL). To convert the protein band density to
the protein concentration, a standard curve was constructed from
density values of varying amounts of C2 domain protein samples (1-5
µg).
Monolayer Measurements--
Surface pressure ( Properties of Calcium Ligand Mutants--
According to the
structure of the C2 domain of cPLA2 shown in Fig.
1B, five
Ca2+-binding residues can be classified into three groups:
Asn65, which primarily coordinates CA1; Asp93
and Asn95, which mainly coordinate CA2; and two CBR1
aspartates, Asp40 and Asp43, which coordinate
both Ca2+ ions. Based on this Ca2+ binding
geometry, the mutations of Asn65 and
Asp93/Asn95 would selectively affect the
binding of CA1 and CA2, respectively, whereas the mutations of
Asp40/Asp43 would have effects on the binding
of both Ca2+ ions. Thus, it is possible to systematically
analyze the roles of the two Ca2+ ions in the membrane
binding and activation of cPLA2 by selectively deactivating
their ligands and separately measuring the effects of mutations on
membrane binding and activity. For this purpose, we made five mutants
that contain either Asp to Asn (e.g. D40N) or Asn to Ala
(e.g. N65A) mutations. All five mutants exhibited full
membrane binding affinity and enzyme activity at saturating Ca2+ concentrations (see Figs.
2 and 3),
demonstrating the lack of deleterious conformational changes. We first
measured the binding of wild type and mutants to hydrophobic beads
coated with DHPC, which is a nonhydrolyzable ether analog of
phospholipid, as a function of Ca2+ concentration.
Phospholipid-coated hydrophobic beads have been shown to be useful in
determining the membrane affinity of PLA2s (19). In
particular, this model membrane allows for rapid and accurate
measurement of PC affinity, which is normally difficult to achieve with
PC vesicles due to their low pelleting efficiency at low
concentrations.2 As shown in
Fig. 2, all mutants exhibited increased Ca2+ requirements
for PC binding, and [Ca2+]1/2 values varied from
40 µM for N65A (7-fold increase) to 3.5 mM
for D43N (614-fold increase) (see Table
I). In general, Asp to Asn mutations had
much larger effects than Asn to Ala mutations, because aspartates
provide charged bidentate Ca2+ ligands, whereas asparagines
provide neutral unidentate ones. Comparison among three Asp to Asn
mutations and between two Asn to Ala mutations revealed a common theme.
First, the mutational effect of Asp93 that only coordinates
CA2 is comparable with those of Asp40 and Asp43
that coordinate both Ca2+ ions. This implies that
mutational effects of Asp40 and Asp43 on
membrane binding derive mainly from its coordination to CA2. Second,
between the two Asn to Ala mutants, N95A has slightly higher
[Ca2+]1/2 value than N65A, again suggesting that
CA2 is more important for membrane binding of cPLA2 than is
CA1.
We then measured the cPLA2 activity of wild type and
mutants using [14C]SAPC vesicles as a function of
Ca2+ concentration (Fig. 3). In general, increases in
[Ca2+]1/2 for mutants from activity measurements are uniformly (~3-fold) smaller than those from membrane binding measurements (Table I). This might simply reflect the structural differences between DHPC-coated beads and SAPC vesicles used for binding and activity assays, respectively. Note that, however, Ca2+ dependences of both binding and activity show
essentially the same trend for those mutants that are involved in
coordination to CA2; i.e. the degree of increase in
[Ca2+]1/2 is in the order of D43N > D40N Properties of Mutants of Membrane Binding Residues--
Our
previous study showed that cPLA2 has a unique ability to
partially penetrate into membranes, which is essential for its membrane
binding and interfacial catalysis (15). The Ca2+ binding
loops of cPLA2, CBR1, and CBR3 have a cluster of
hydrophobic residues on the tips of their protruded structures (see
Fig. 1A), suggesting that these residues might be involved
in membrane penetration. We therefore mutated six hydrophobic residues
in these regions to Ala: F35A, M38A, and L39A in CBR1 and Y96A, Y97A,
and M98A in CBR3. To assess the contribution of electrostatic
interactions to membrane binding of the C2 domain, we also mutated four
cationic residues (R57E/K58E/R59E/R61E) that form a cationic patch
along a
To quantitatively assess the effects of mutations on membrane binding,
we measured dissociation constants for the binding of cPLA2
to DHPC-coated beads. Binding isotherms for cPLA2 and selected mutants are illustrated in Fig.
4, and n and
Kd values determined from the curve fitting are
summarized in Table II. Although there are several different ways to
analyze the binding isotherms, we used a simple Langmuir-type equation
(Equation 1) assuming the presence of the enzyme binding sites on the
interface consisting of n phospholipids. Since
Kd is expressed in terms of molarity of these
binding sites, nKd is the dissociation constant in
terms of molarity of lipid molecules, and the relative binding affinity
can be best described in terms of relative values of
(1/nKd) (Table II). The lateral mobility of
phospholipids is not taken into account in this model, because little
is known about the lateral mobility of phospholipids coated on
hydrophobic beads. Since Kd was measured under conditions of enzyme crowding on beads, and because of possible protein-protein interaction, the value obtained might be somewhat different from the Kd obtained under conditions of
low bead coverage.
In general, the relative affinity of mutants for DHPC-coated beads is
consistent with their relative activity toward [14C]SAPC
vesicles. For some mutants, there are changes in both n and
Kd values, indicating that the mutations affect both their membrane affinity and binding mode. This effect is pronounced for
those mutants with lower relative affinity, including F35A, L39A, and
Y96A. Overall, all of the hydrophobic residues except for
Met38 and Met98 appear to have significant and
comparable contributions to hydrophobic membrane binding.
Interestingly, the quadruple mutant of the cationic patch
(R57E/K58E/R59E/R61E) exhibits lower activity and affinity for PC
vesicles, suggesting that these cationic residues are somehow involved
in membrane binding. Because it was unclear how the cationic residues
participate in binding to the zwitterionic membrane surface, we
measured the binding of wild type and mutants including
R57E/K58E/R59E/R61E to anionic DHPG-coated beads. It was previously
shown that both cPLA2 (27) and its isolated C2 domain (28)
have lower affinity for monoanionic phospholipids than for zwitterionic
phospholipids. Consistent with these findings, cPLA2 showed
~7.5-fold lower affinity for anionic DHPG-coated beads than for
DHPC-coated beads. Also, most of the mutations of hydrophobic residues
decreased the binding affinity for DHPG-coated beads less significantly
than that for DHPC-coated ones, suggesting that cPLA2 might
bind to phosphatidylglycerol membranes in a slightly different mode.
Qualitatively, however, similar trends were observed for binding of the
mutants to DHPC- and DHPG-coated beads. Surprisingly,
R57E/K58E/R59E/R61E had a slightly higher affinity for DHPG-coated
beads than the wild type did, demonstrating that these residues are not
involved in binding to anionic membranes. This also suggests that the
low affinity of the quadruple mutant for PC-coated beads might derive
from a secondary effect unrelated to membrane binding.
Properties of Isolated C2 Domain and Its Mutants--
For
hydrophobic residues to contribute to the energetics of membrane
binding, they must penetrate into the hydrophobic core of the membrane.
Thus, mutants of essential hydrophobic residues of the C2 domain are
expected to have significantly altered membrane penetrating ability.
Phospholipid monolayers at the air-water interface serve as a highly
sensitive tool to measure the membrane penetrating ability of protein
(10, 15, 29). Monolayer measurements of mutant proteins were, however,
hampered by generally low protein expression yields of
cPLA2 and mutants from our baculovirus-infected insect
cells. To overcome this difficulty, we prepared the isolated C2 domain
of cPLA2 (residues 1-137) and its mutants. Since these isolated C2 domains can be expressed in E. coli as inclusion
bodies and readily refolded with high yields (~5 mg/liter of
culture), we were able to obtain proteins sufficient for monolayer
measurements. This also allowed us to compare the membrane penetrating
ability of the C2 domain versus the whole cPLA2
molecule. We first measured relative membrane binding affinity of
isolated C2 domain mutants to see if these mutants behave similarly to
their parent proteins. For these measurements, we used sucrose-loaded
POPC vesicles at a high enough concentration (i.e. 0.5 mM) to ensure complete pelleting, and the relative affinity
was estimated from the amount of protein bound to POPC vesicles. As
listed in Table III, relative binding affinity of isolated C2 domain mutants is comparable with that of
cPLA2 mutants (see Table II), indicating that the
mutational effects are essentially the same for the isolated C2 domain
and the whole protein. Then we measured the penetration of these C2 domains into POPC monolayers. In these studies, a POPC monolayer of a
given Differential Roles of Ca2+ Ions and Their
Ligands--
Our previous structure-function study on the C2 domain of
protein kinase C- Hydrophobic Residues--
Both electrostatic and hydrophobic
interactions are involved in the membrane binding of most peripheral
membrane binding proteins and their relative contributions vary with
the type of proteins (see, for example, Refs. 30-33). This study shows
that the binding of C2 domain of cPLA2 is mainly driven by
hydrophobic interactions that are achieved by the penetration of
hydrophobic residues in the calcium binding loops (CBR1 and CBR3) into
the membrane core. To our knowledge, the C2 domain of cPLA2
has the highest membrane penetrating power than any other C2 domain
studied, including that of protein kinase
C-
Based on these findings, we propose a membrane binding mode of the C2
domain of cPLA2 as illustrated in Fig.
7. This model is different from one
proposed by Williams and co-workers (12) in that both CBR1 and CRB3 are
involved in membrane penetration, and the cationic cluster does not
make direct contact with the membrane surface. The finding that the
isolated C2 domain of cPLA2 has higher penetrating power
than cPLA2 indicates the difference in membrane penetrating
mode between the isolated C2 domain and the C2 domain in the intact
cPLA2 molecule. Presumably, the carboxyl-terminal domain of
cPLA2, which contains the active site, interferes with the
maximal membrane penetration of the amino-terminal C2 domain. We
previously showed that the partial membrane penetration of cPLA2 is essential for its interfacial catalysis (15).
Thus, it is possible that any membrane factors, such as
diacylglycerols, which make up for the compromised membrane penetrating
power of cPLA2 may induce its membrane translocation and
activation.
In summary, this report represents the second part of our systematic
structure-function analysis on the C2 domain of membrane-binding proteins. These studies reveal similarities and differences between the
C2 domains of protein kinase C--strand (R57E/K58E/R59E/R61E) have
modest and negligible effects on the binding of cPLA2 to
zwitterionic and anionic membranes, respectively, indicating that they
are not directly involved in membrane binding. In conjunction with our
previous study on the C2 domain of protein kinase C-
(Medkova, M.,
and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552),
these results demonstrate that C2 domains are not only a membrane
docking unit but also a module that triggers membrane penetration of
protein and that individual Ca2+ ions bound to the calcium
binding loops play differential roles in the membrane binding and
activation of their parent proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
showed that the C2 domain not only
brings the protein to the membrane surface but also triggers the
membrane penetration of protein (10). Also, the study revealed that
individual Ca2+ ions bound to the C2 domain play
differential roles in membrane binding and activation. Tertiary
structures of the C2 domains of protein kinase C (11) and
cPLA2 (12, 13), despite their overall similarity, have
noticeable differences; i.e. they have different
-strand
connectivity (14), and, more importantly, the C2 domain of
cPLA2 contains two clusters of exposed hydrophobic residues
in the calcium binding loops (see Fig. 1A) (12). Consistent with this unique structure, both cPLA2 (15) and its
isolated C2 domain (16) have been shown to be able to penetrate into the membrane. In this study, we performed an extensive
structure-function analysis of the C2 domain of cPLA2 to
assess the roles of six hydrophobic residues and five Ca2+
ligands located in the calcium binding loops in the membrane binding
and activation of cPLA2. We also determined the role of a
cluster of cationic residues in a
-strand. In conjunction with our
previous study on protein kinase C-
, this study reveals interesting similarities and differences between the two C2 domains.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (Research
Products, Mount Prospect, IL). After overnight incubation, cells were
harvested by centrifugation at 5000 × g and 4 °C
for 10 min. Cells were resuspended in 50 ml of 50 mM
Tris-HCl buffer, pH 8.0, containing 50 mM NaCl, 2 mM EDTA, 0.4% (v/v) Triton X-100, 0.4% (w/v) sodium
deoxycholate, 1 mM phenylmethylsulfonyl fluoride. After the
suspension was sonicated, the inclusion body pellet was obtained by
centrifugation at 50,000 × g for 15 min at 4 °C. The pellet was resuspended in 50 ml of 50 mM Tris-HCl
buffer, pH 8.0, containing 50 mM NaCl, 2 mM
EDTA, 0.8% (v/v) Triton X-100, 0.8% (w/v) sodium deoxycholate. After
centrifugation at 50,000 × g for 15 min at 4 °C,
the pellet was resuspended in 50 ml of 50 mM Tris-HCl
buffer, pH 8.0, containing 5 M urea and 5 mM
EDTA. The pellet was stirred at room temperature for 15 min, and then the suspension was centrifuged at 100,000 × g for 10 min at 4 °C. The washed inclusion body was resuspended in 10 ml of
50 mM Tris-HCl buffer, pH 8.0, containing 8 M
guanidinium chloride. Insoluble matter was removed by centrifugation at
100,000 × g for 10 min at 4 °C, and the supernatant
was loaded onto a Sephadex G-25 column (2.5 × 35 cm) equilibrated
with 50 mM Tris-HCl buffer, pH 8.0, containing 5 M urea and 5 mM EDTA. The first major peak was
collected (35 ml) and dialyzed against 50 mM Tris-HCl, pH 8.0, 1.5 M urea and then against 50 mM
Tris-HCl, pH 8.0. The refolded C2 domain was purified using a Ni-NTA
column (Qiagen) according to the manufacturer's instructions. Purity
of protein samples was higher than 90% electrophoretically. Aliquots
of purified proteins were stored at
20 °C.
where [PL]o represents total phospholipid
concentration. This equation assumes that each enzyme binds
independently to a site on the interface composed of n
phospholipids with a dissociation constant of Kd. To
determine the Ca2+ dependence of binding, cPLA2
(~60 nM) was incubated for 15 min with
phospholipid-coated beads (60 µM) and 1 µM
BSA in 150 µl of 10 mM HEPES (pH 7.0) containing 100 mM KCl and varying concentrations of Ca2+ (or
EGTA) (see Fig. 2). After centrifugation (12,000 × g
for 2 min), [E]b was determined by the activity
assay (see above). The binding of Ca2+ ions to the isolated
C2 domain was shown to be consistent with the cooperative Hill model
(24). The concentration of Ca2+ giving rise to half-maximal
binding (or activity) ([Ca2+]1/2) was thus
determined from curve fitting of data to a Hill equation,
(Eq. 1)
where y, a, h, and
[Ca2+] are relative binding (or activity), arbitrary
normalization constant, Hill coefficient, and free Ca2+
concentration, respectively.
(Eq. 2)
) of solution
in a circular Teflon trough was measured using a Wilhelmy plate
attached to a computer-controlled tensiometer (Nima Technology,
Coventry, United Kingdom). The trough (4-cm diameter × 1-cm
depth) has a 0.5-cm-deep well for a magnetic stir bar and a small hole
drilled at an angle through the wall to allow an addition of protein
solution. Five to ten ml of phospholipid solution in chloroform was
spread onto 10 ml of subphase (10 mM HEPES, pH 7.0, 0.1 M KCl, and 0.1 mM of free Ca2+) to
form a monolayer with a given initial surface pressure
(
0). The subphase was continuously stirred at 60 rpm
with a magnetic stir bar. Once the surface pressure reading of
monolayer had been stabilized (after ~5 min), the protein solution
(typically 50 µl) was injected to the subphase, and the change in
surface pressure (
) was measured as a function of time at
23 °C. Typically, the
value reached a maximum after 20 min.
The maximal
value depended on the protein concentration at the
low concentration range and reached a saturation when the protein
concentration was higher than 2 µg/ml. Protein concentrations were
therefore maintained above 3 µg/ml for cPLA2 and 6 µg/ml for C2 domains to ensure that the observed
represented a
maximal value. The critical surface pressure (
c) was
determined by extrapolating the
versus
0 plot to the x axis (26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of the C2 domain of cPLA2.
A, ribbon diagram of the C2 domain of
cPLA2. Locations of two calcium binding loops (CBR1 and
CBR3), two calcium ions (CA1 and CA2), mutated hydrophobic residues,
and cationic residues are shown with their residue numbers (12).
B, a schematic representation of Ca2+ binding
loops of cPLA2. Five mutated calcium ligands are
illustrated as well as two ligands from the peptide backbone. The
numbers indicate distance in Å.
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Fig. 2.
Ca2+ dependences of bead binding
of cPLA2 and Ca2+ ligand mutants. Proteins
(60 nM) include wild type ( ), D40N (
), D43N (
),
N65A (
), D93N (
), and N95A (
). Bulk concentration of DHPC on
beads was 60 µM. Each data point represents an average of
triplicate measurements. The solid lines
represent theoretical curves constructed from parameters determined
from the nonlinear least-squares fit using Equation 2.
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Fig. 3.
Ca2+ dependences of enzyme
activity of cPLA2 and Ca2+ ligand mutants.
Proteins include wild type ( ), D40N (
), D43N (
), N65A (
),
D93N (
), and N95A (
). Assay mixtures contain 10 µM
[14C]SAPC, 20 nM enzyme, 16 µM
BSA, and varying concentrations of Ca2+ in 20 mM HEPES, pH 8.0. Each data point represents an average of
duplicate experiments. The absolute value of maximal activity was 0.30 nmol/(µg·min). The solid lines represent
theoretical curves constructed from parameters determined from the
nonlinear least-squares fit using Equation 2.
[Ca2+]1/2 values of calcium ligand mutants of
cPLA2
D93N
N95A, and their relative
[Ca2+]1/2 values were nearly identical for
binding and activity. In striking contrast, the mutation of CA1 ligand
Asn65 to Ala exhibited a larger effect on activity than on
binding. This unique effect is best illustrated by comparing the
[Ca2+]1/2 values of N65A and N95A for binding and
activity. The [Ca2+]1/2 value of N65A is about
half of that of N95A for binding but three times larger for activity. These results imply that CA1 might be more important for
cPLA2 activity than CA2. Taken together, it appears that
the two calcium ions bound to the C2 domain of cPLA2 appear
to play differential roles in its membrane binding and activation.
-strand and are implicated in interaction with anionic
phospholipid head groups. First, we measured the relative enzyme
activity of these mutants using [14C]SAPC vesicles as a
substrate. The relative activity determined from the initial rates of
hydrolysis is listed in Table II. All mutants have modestly lower activity than does wild type with two
mutants in the CBR3, Y96A and V97A, showing the lowest activity. This
indicates that some of these residues are important in interfacial catalysis of cPLA2 but that none is critical. Since
mutations of C2 domain residues would not significantly affect the
catalytic efficiency of the enzyme, the relative activity of mutants
should reflect their relative membrane binding affinity.
Kinetic and membrane binding affinity of C2 domain mutants of
cPLA2
A is calculated from
the accessible surface area of each amino acid determined from a
tripeptide model by Chothia (32).
G0 is
calculated from the relative affinity of mutants using the equation,
G0 =
RT ln(relative affinity).
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Fig. 4.
Binding isotherms of cPLA2 and
selected hydrophobic mutants to DHPC-coated beads. Proteins
include wild type ( ), F35A (
), L39A (
), Y96A (
), V97A
(
), and R57E/K58E/R59E/R61E (
). Binding mixtures contained 20 mM Tris-HCl, pH 7.5, 0.1 M KCl, 1 µM BSA, 0.5 mM Ca2+. Bulk DHPC
concentration varied from 5 to 50 µM, depending on the
membrane affinity of protein. Data shown here are from one
representative measurement for each mutant. Triplicate measurements
were made for each mutant for n and Kd
determination. The solid lines represent
theoretical curves constructed from parameters determined from the
nonlinear least-squares fit using Equation 1.
0 was spread at constant area, and the
was
monitored after the injection of the protein into the subphase. In
general,
is inversely proportional to
0 of the
phospholipid monolayer, and an extrapolation of the
versus
0 plot yields the
c, which
specifies an upper limit of
0 of a monolayer that a
protein can penetrate into (26). Fig. 5
shows that the isolated C2 domain of cPLA2 has high
intrinsic ability to penetrate into densely packed monolayers with
c of 34 dyne/cm. In fact, the isolated C2 domain has much
higher monolayer penetrating power than the whole cPLA2
molecule (
c = 21 dyne/cm). The isolated C2 domain, however,
shows no detectable penetration in the absence of Ca2+,
demonstrating that the penetration is a Ca2+-dependent
process. All three mutations of hydrophobic residues in CBR 1 significantly lower
values at a given
0. As a
result,
c values decrease by 5-7 dyne/cm, with F35A and
L39A having slightly lower
c values than M38A, which is
consistent with their relative membrane binding affinity. Differential
effects of the mutations on the monolayer penetration are more
pronounced for CBR3 mutants illustrated in Fig.
6. Y96A, with the lowest binding affinity
for PC-coated beads and PC vesicles, shows a dramatic decrease in
c. The order of decrease in
c (Y96A > V97A > M98A) is again consistent with their relative affinity for
PC membranes. In contrast to all hydrophobic mutants, the quadruple
mutant of cationic residues has essentially no effect on monolayer
penetration, thereby demonstrating the specific nature of reduced
monolayer penetrating power seen for the mutations of hydrophobic
residues.
Properties of isolated C2 domain of cPLA2 and its mutants
c values.
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Fig. 5.
Penetration of cPLA2, C2 domain,
and CBR1 mutants into the POPC monolayer as a function of its initial
surface pressure. Proteins include cPLA2 ( ),
isolated C2 domain (
), F35A (
), M38A (
), and L39A (
). The
subphase contained 20 mM HEPES buffer, pH 7.0, 0.1 mM free Ca2+, and 6 µg/ml of C2 domains (or 3 µg/ml of cPLA2). The penetration of wild type C2 domain
was also measured in the presence of 0.1 mM EDTA in the
subphase (
). Each data point is from duplicate measurements.
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Fig. 6.
Penetration of C2 domain and CBR3 mutants
into the POPC monolayer as a function of its initial surface
pressure. Proteins include isolated C2 domain ( ), Y96A (
),
V97A (
), M98A (
), and R57E/K58E/R59E/R61E (
). Experimental
conditions were the same as described for Fig. 5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
showed that individual Ca2+ ions bound
to the C2 domain have different roles in the membrane binding and
activation of protein kinase C-
(29). Specifically, it was shown
that a calcium ion bound close to CBR1 brings the protein to the
membrane surface, whereas calcium ions bound to CBR3 induce the
membrane penetration of the protein, resulting in enzyme activation.
Results from the mutational analysis of the C2 domain of
cPLA2 also suggest differential roles of calcium ions bound
to its C2 domain. As far as the specific role of each calcium is
concerned, however, a major difference seems to exist between the two
C2 domains. It should be noted here that there are considerable
differences in stoichiometry and geometry of calcium coordination
between the two C2 domains. Thus, comparison of the role of a calcium
ion in cPLA2 with that of its counterpart in protein kinase
C-
should be made with caution. For cPLA2, the following
results indicate that CA2 bound close to CBR3 is important for membrane
binding and that CA1 bound close to CBR1 is involved in
cPLA2 activation. First, mutations of Asp40,
Asp43, Asp93, and Asn95, which
coordinate CA2, have larger effects on membrane binding than that of
Asn65, which coordinates CA1. Second, the mutation of
Asp43, which is closest to CA2 (see Fig. 1B),
has the largest impact on membrane binding. Third, the mutations of CA2
ligands show comparable effects on membrane binding and enzyme
activity, whereas the mutation of Asn65 affects enzyme
activity much more significantly. Notice that this assignment of roles
is distinct from that given to calcium ions bound to protein kinase
C-
. This also implies that the two C2 domains interact with
membranes in different modes. A recent study of the isolated C2 domain
of cPLA2 showed that two Ca2+ ions bind to the
C2 domain sequentially with positive cooperativity, which in turn
induces intradomain conformational changes and membrane translocation
(24). Since the membrane binding of cPLA2 precedes its
interfacial catalysis, it is likely that CA2 binding takes place first,
which then promotes CA1 binding although the reverse order of calcium
addition cannot be precluded. The 10-fold increase in
[Ca2+]1/2 for the membrane binding of N65A (see
Table II) indicates that CA1 binding also contributes to membrane
binding, albeit to a lesser degree. Thus, it appears that the binding
of both Ca2+ ions to the C2 domain takes place prior to the
membrane binding of cPLA2. Since the calcium affinity of
cPLA2 is much higher in the presence of membranes, it is
reasonable to assume that binding of both calcium ions occurs in the
vicinity of the membrane surface. Thus, it is likely that the binding
of two Ca2+ ions and the membrane binding occur essentially
simultaneously. In the case of protein kinase C-
, it was
demonstrated that the binding of calcium ion(s) to CBR3 induces the
membrane penetration of protein, which leads to activation. Because the
measurements of membrane penetration and/or conformational changes for
Ca2+ ligand mutants were hampered by low protein expression
yields, it is unclear at present how exactly the two Ca2+
ions play their specific roles. Based on the coordination geometry of
the two Ca2+ ions (see Fig. 1B) and the membrane
binding properties of cPLA2, we speculate that CA2 induces
the local conformational changes in the calcium binding loops to expose
hydrophobic residues for hydrophobic membrane binding (see below),
whereas CA1 binds to one or more membrane phospholipids to properly
orient the cPLA2 molecule for interfacial catalysis. This
notion is based on the fact that CA2 has six coordinations to both CBR1
and CBR3, whereas CA1 has only four coordinations to CBR1 and CBR2 (see
Fig. 1B). Thus, the higher affinity binding of CA2 to its
ligands would alter the conformation of essential hydrophobic side
chains in CBR1 and CBR3 and possibly promote the lower affinity binding of CA1 (hence the order of addition). On the other hand, CA1, due to
its incomplete coordination sphere in the C2 domain, should still be
capable of coordinating to membrane phospholipids even after binding to
the C2 domain. Since the membrane binding of cPLA2 is
driven mainly by hydrophobic interactions (see below), the binding of
CA2, if it indeed exposes all hydrophobic residues, would be more
important for membrane binding than that of CA1, which would contribute
less to conformational changes and thus primarily contribute to less
important electrostatic interactions. The calcium-induced
conformational changes of calcium binding loops containing essential
hydrophobic residues are supported by a recent NMR study of the
isolated C2 domain of cPLA2 (13). Also, the notion that CA1
binds to phospholipids and thus contributes to electrostatic
interactions is supported by our finding that N65A has a considerably
higher [Ca2+]1/2 value for anionic DHPG-coated bead binding than for DHPC-coated bead binding, whereas N95A shows the
essentially the same calcium dependence for both types of beads (data
not shown). Further investigation is needed to elucidate exact roles of
the two calcium ions and determine the temporal and spatial sequences
of calcium and membrane binding in the interfacial catalysis of
cPLA2.
,3 presumably due to its
unique structure, containing a number of hydrophobic residues in the
calcium binding loops. A combined contribution of six hydrophobic
residues to binding energy, which can be estimated using the equation
Go =
RT ln(relative affinity)
(see Table II), is 9.1 kcal/mol at 25 °C, assuming the additivity of
individual contributions. This suggests that the hydrophobic
interactions are strong enough to drive the membrane binding of
cPLA2. Our monolayer data show that Ca2+ is
essential for the membrane penetration of the C2 domain. As described
above, this is mainly because Ca2+ triggers local
conformational changes in the calcium binding loops, which exposes
hydrophobic residues and thereby elicits their membrane penetration.
Mutational effects of hydrophobic residues on activity, membrane
binding, and monolayer penetration indicate that four hydrophobic
residues, Phe35 and Leu39 of CBR1 and
Tyr96 and Val97 of CBR3, are directly involved
in membrane penetration and hydrophobic interactions. Although these
data apparently indicate that Tyr96 makes the largest
contribution to the membrane penetration, it should be taken into
account that the Tyr to Ala mutation involves a larger change in
accessible surface area than any other mutation. To assess more
quantitatively the contribution of each amino acid to membrane
penetration and hydrophobic interactions, one thus has to determine the
change in free energy of binding per change in accessible surface area
(
G0/
A) caused by each
mutation. These values determined for individual mutations are given in
Table II. It was shown that an average
G0/
A value for transferring
an amino acid from ethanol to water is ~24 cal/mol/Å2
(34). Thus, one can estimate from this value the degree of penetration
of each hydrophobic residue in the calcium binding loops. For instance,
Leu39 and Val97 the mutations of which yield
high
G0/
A values of 30 and
40 cal/mol/Å2, respectively, should be fully inserted into
the hydrophobic core of membranes, whereas Phe35 and
Tyr96 with smaller
G0/
A values would partially
penetrate into the membrane during membrane binding. This notion is
also consistent with a general observation that aromatic side chains
have a high tendency to reside in the membrane-water interface (35). If
the membrane binding of cPLA2 is driven primarily by
hydrophobic interactions, then its affinities for PC and
phosphatidylglycerol membranes should be comparable. Thus, the PC
preference of cPLA2 and its C2 domain (28) suggests the
presence of either a specific PC binding site(s) or an anionic patch on
the membrane binding surface of the C2 domain. While the answer to this
question entails further investigation, the PC preference of
cPLA2 makes it less likely that four cationic residues in
the
3 strand are directly involved in membrane binding of
cPLA2. This is also consistent with the finding that wild
type cPLA2 and the quadruple mutant (R57E/K58E/R59E/R61E) have comparable affinities for DHPG-coated beads. The modestly lower
binding affinity of the mutant for PC membranes might be due to local
structural changes that affect PC binding more significantly than
phosphatidylglycerol binding. Similar differential effects of mutations
on PC and phosphatidylglycerol binding are also seen with
mutations of hydrophobic residues (see Table II).
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Fig. 7.
A proposed membrane binding mode of the C2
domain of cPLA2. The backbone of the C2 domain is
shown in a ribbon diagram, and mutated residues
are illustrated in a space-filling representation. Leu39
and Val97, whose side chains are fully inserted, are shown
in red, and Phe35, Met38,
Tyr96, and Met98, whose side chains are
partially inserted, are shown in pink. Four cationic
residues are shown in blue.
and cPLA2 in terms of
their mechanisms of membrane binding and activation. The two C2 domains are similar in that they are not only a membrane docking unit but also
a module that triggers membrane penetration of protein and in that
their individual Ca2+ ions bound to the calcium binding
loops play differential roles in membrane binding and activation. On
the other hand, individual Ca2+ ions of the two C2 domains
have different specific roles, and the membrane penetration of
hydrophobic residues plays a more important and direct role in the
membrane binding of the C2 domain of cPLA2 than in that of
protein kinase C.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM52598 and GM53987.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.
These authors equally contributed to this work.
§ To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 W. Taylor St., Chicago, Illinois 60607-7061. Tel.: 312-996-4883; Fax: 312-996-0431; E-mail: wcho{at}uic.edu.
2 L. Bittova and W. Cho, unpublished observation.
3 M. Medkova and W. Cho, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; BSA, bovine serum albumin; CBR, calcium binding region; DHPC, 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine; DHPG, 1,2-di-O-hexadecyl-sn-glycero-3-phospholycerol; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; SAPC, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine.
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