(Received for publication, October 9, 1996)
From the The outer membrane phospholipase A (OMPLA) of
Escherichia coli is present in a dormant state in the cell
envelope. The enzyme is activated by various processes, which have in
common that they perturb the outer membrane. Kinetic experiments,
chemical cross-linking, and analytical ultracentrifugation were carried
out with purified, detergent-solubilized OMPLA to understand the
underlying mechanism that results in activation. Under conditions in
which the enzyme displayed full activity, OMPLA was dimeric. High
detergent concentrations or very dilute protein concentrations resulted
in low specific activity of the enzyme, and under those conditions the
enzyme was monomeric. The cofactor Ca2+ was required for
dimerization. Covalent modification of the active site serine with
hexadecylsulfonylfluoride resulted in stabilization of the dimeric form
and a loss of the absolute calcium requirement for dimerization. The
results of these experiments provide evidence for dimerization as the
molecular mechanism by which the enzymatic activity of OMPLA is
regulated. This dimerization probably plays a role in vivo
as well. Data from chemical cross-linking on whole cells indicate that
OMPLA is present in the outer membrane as a monomer and that activation
of the enzyme induces dimerization concurrent with the appearance of
enzymatic activity.
Outer membrane phospholipase A
(OMPLA1; also known as detergent-resistant
phospholipase A or PldA protein) is one of the few enzymes present in
the outer membrane of Gram-negative bacteria. OMPLA hydrolyzes the acyl
ester bonds in (phospho)lipids and has Ca2+ as an essential
cofactor (1, 2). Initially, the Escherichia coli enzyme was
purified and characterized (3), and its structural gene, designated
pldA, was subsequently cloned and overexpressed (4-7).
Recently, Brok et al. (8) reported the cloning of the pldA genes of several other Enterobacteriaceae
species. Comparison of the OMPLA amino acid sequences revealed a high
degree of homology within this family, but no clear homology exists
with sequences of water-soluble (phospho)lipases. A Although OMPLA is embedded in its own substrate in the cell envelope,
no enzymatic activity can be detected in normally growing cells (14,
15). Because OMPLA is expressed constitutively, genetic regulation
cannot explain the absence of enzymatic activity. Moreover, the
expressed protein is correctly transported to and inserted into the
outer membrane, where it resides in a dormant state. Activity is
induced by various processes that perturb the membrane, such as
phage-induced lysis (16), temperature shock (4), and colicin secretion
(17, 18). Similar results have been reported in vitro after
reconstitution of OMPLA in lipid vesicles (19). Membrane-perturbing
peptides, such as polymyxin B, melittin, or cardiotoxin, activate the
enzyme. In purified, detergent-solubilized OMPLA, the enzymatic
activity depends strongly on the detergent concentration (2, 5).
The present study was undertaken to determine the molecular mechanism
responsible for the activation of OMPLA. The results of enzymatic
activity assays, chemical cross-linking, and analytical ultracentrifugation experiments suggest that OMPLA is active as a
dimer. The effects of the detergent concentration, the cofactor calcium, the covalent modification of the active site, and the enzyme
concentration on dimerization were studied in detail. Data from
chemical cross-linking on whole cells indicate that OMPLA is present in
the outer membrane as a monomer and that activation of the enzyme
induces dimer formation.
n-Dodecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate
(12-SB) and glutaraldehyde were purchased from Fluka and
octylpolyoxyethylene glycol (octyl-POE) from Bachem. All other
chemicals were of the highest purity commercially available.
E. coli OMPLA was overproduced from the
plasmid ppL7.5 as inclusion bodies and folded in vitro and
purified as described previously (12). Protein concentrations were
determined spectrophotometrically using an
A280 nm1% of 29.2 (20). OMPLA (1 mg/ml) was modified with 1 mole equivalent of hexadecylsulfonylfluoride
(from a stock solution in acetonitrile) in buffer (2.5 mM
12-SB, 20 mM Tris-HCl, pH 8.3, 5 mM
CaCl2) for 16 h at room temperature (20).
Subsequently, the protein solution was dialyzed twice against 100 volumes of buffer (2.5 mM 12-SB, 20 mM
Tris-HCl, pH 8.3, 2 mM EDTA) and stored at Outer membrane
phospholipase activity was determined spectrophotometrically using
2-hexadecanoylthioethane-1-phosphocholine (21) as substrate (5). The
assay buffer contained 50 mM Tris-HCl, pH 8.3, 5 mM CaCl2, 0.1 mM
dithiobis(2-nitrobenzoic acid), 0.2 mM Triton X-100, and
0.25 mM substrate. Routinely, 50 ng of OMPLA was assayed
and activities were calculated from the absorbance at 412 nm; the
activities are given as µmol of substrate hydrolyzed per min and per
mg protein (units/mg).
OMPLA was diluted to a
final concentration of 0.05 mg/ml in buffer (20 mM
Tris-HCl, pH 8.3, 2 mM EDTA) containing various concentrations of detergent. 12-SB was used in the range of 1.25-22.75 mM, and octyl-POE was used in the range of 0.05-5% (v/v).
The solutions were incubated for 16 h at room temperature, after
which samples were assayed for enzymatic activity.
OMPLA was incubated at 0.18 mg/ml in
buffer (50 mM Hepes, pH 8.3, 100 mM KCl,
containing either 20 mM CaCl2 or 20 mM EDTA) in the presence of various concentrations of 12-SB
(2-25 mM) in a total volume of 100 µl. After
preincubation of the solutions for 16 h at room temperature, 5 µl of a 1% stock solution of glutaraldehyde in 2.5 mM
12-SB was added, and the reaction was allowed to proceed for 4 h
at room temperature. Subsequently, 100 µl of gel loading buffer (0.1 M Tris-HCl, pH 6.8, 3% SDS, 15.4% glycerol, 7.7%
Sedimentation coefficients
and protein molecular weights of the detergent-protein complexes were
determined by velocity and equilibrium centrifugation experiments in a
Beckman Optima XL-A analytical ultracentrifuge as described by
Ludwig et al. (22). OMPLA (1 mg/ml) was dialyzed at
room temperature for 16 h against buffer (100 mM NaCl,
20 mM Tris-HCl, pH 8.3, containing either 0.5, 1.5, or 5%
octyl-POE and either 2 mM EDTA or 20 mM
CaCl2). Samples of inhibited OMPLA were prepared in an
analogous manner. After dialysis, the protein concentration was
determined spectrophotometrically, and dilutions were prepared in
dialysate. Velocity measurements were carried out with concentrated
protein samples (0.4 mg/ml), whereas sedimentation equilibrium
measurements were done with more dilute samples (0.1 mg/ml). The
partial specific volume of the protein was calculated (23) from the
amino acid composition to be 0.73 ml/g. Sedimentation velocity
experiments yielded the sedimentation coefficient according to the
Svedberg equation, using a density of 1.006 g·cm A stock solution of OMPLA (5 mg/ml)
was diluted in buffer (20 mM Tris-HCl, pH 8.3, 2 mM EDTA, 2.5 mM 12-SB). The solutions were
incubated overnight at room temperature, and subsequently the enzymatic
activities were measured. The total volume of enzyme preincubation
solution added to the assay was kept below 20 µl to prevent
interference with the activity measurement. At the high protein
concentrations, 100 ng was assayed, whereas at the lower protein
concentrations, down to 0.6 ng of OMPLA was used in the enzymatic
assay.
The pldA mutant E. coli strain CE1348 (8) containing pldA plasmid pRB1 (8)
was grown overnight at 37 °C in 100 ml of L-broth supplemented with
100 µg/ml ampicillin. Cells were collected by centrifugation and
resuspended in ice-cold buffer (100 mM NaCl, 50 mM Hepes, pH 8.3, 20 mM CaCl2) to
an A600 of 20. Enzymatic activities of whole
cells and of cell lysates, obtained by sonication at 0 °C, were
measured in the chromogenic assay on a double-beam spectrophotometer.
The reference cuvette did not contain dithiobis(2-nitrobenzoic acid).
Samples (50 µl aliquots) were added to the sample as well as to the
reference cuvette to compensate for turbidity. To 100-µl aliquots of
the whole cell suspension or the cell lysates 5 µl of a 10%
formaldehyde solution in water was added, and the reaction was
incubated for 2 h at room temperature. Thereafter, 100 µl of
SDS-PAGE loading buffer was added and the samples were heated for 10 min at 60 °C. Of the resulting solution, 25 µl were analyzed by
SDS-PAGE followed by Western blot analysis using a mouse polyclonal antiOMPLA serum as described (8).
After preincubation of OMPLA in 2.5 mM
12-SB, which is slightly above the critical micelle concentration (1.3 mM), the enzyme displays full activity in the enzymatic
assay (Fig. 1A). At concentrations below the
critical micelle concentration, the enzyme loses its activity,
presumably due to precipitation upon losing bound detergent (19). With
increasing concentrations of detergent present in the preincubation,
the specific activity was significantly lowered. The dependence of
enzymatic activity on the concentration of detergent is not a specific
feature of the zwitterionic detergent 12-SB. Similar results were
obtained with several other detergents, such as
OMPLA was
preincubated with various concentrations of detergent (2-25
mM 12-SB) and subsequently incubated with glutaraldehyde. The products of the cross-linking reaction were analyzed by SDS-PAGE. After preincubation in 2 mM 12-SB and subsequent
cross-linking, only high molecular weight aggregates were visible on
the gel (Fig. 2A, lane 3). OMPLA that was
preincubated at 2.5 or 3 mM 12-SB was efficiently
cross-linked into a defined oligomeric form with an apparent molecular
weight of 42,000 (Fig. 2A, lanes 4 and 5). OMPLA
preincubated at higher 12-SB concentrations was not cross-linked
(Fig. 2A, lanes 6-9). OMPLA displays
"heat-modifiability" (8), a property that is shared by many
bacterial outer membrane proteins (25); because it is rather resistant
to denaturation in SDS, the folded protein runs at 27,000. Only after
boiling of the samples, the denatured OMPLA migrates at the expected
31,500 position. The cross-linked oligomeric form migrated as a
Mr 63,000 protein after boiling of the sample
(results not shown). Therefore, the Mr 42,000 form detected in the unboiled samples most likely represents a folded
dimer.
Inhibition of OMPLA with hexadecylsulfonylfluoride leads to the
irreversible inactivation of the enzyme due to modification of the
active center residue Ser-144 (20). The modified enzyme can be
considered a mimic of the acyl enzyme that is thought to occur during
catalysis. Therefore, it was of interest to investigate how this
modification would influence the dimerization process. Interestingly,
even without cross-linking, a considerable amount of the dimer was
visible on the gel (Fig. 2B, lane 2). After
preincubation in 2.5 or 3.0 mM 12-SB, inhibited OMPLA
was completely cross-linked into the dimer (Fig. 2B, lanes 4 and 5). At higher concentrations of 12-SB, inhibited OMPLA
could be cross-linked as well (Fig. 2B, lanes 6-9). This
finding is in sharp contrast to the results obtained for the unmodified
enzyme (Fig. 2A). Apparently, the modification of the active
site leads to stabilization of the dimer. The amounts of dimer formed
after cross-linking at high detergent concentrations were considerably
higher when compared to the amount of dimer already present in the
sample of inhibited OMPLA before cross-linking.
Because Ca2+ is an essential cofactor for the enzymatic
hydrolysis of (phospho)lipids by OMPLA (1, 2), we studied the role of
this cofactor in dimerization. OMPLA was preincubated in 3 mM 12-SB in the presence of Ca2+. After
reaction with glutaraldehyde, the protein appeared to be cross-linked
efficiently (Fig. 2C, lane 2). In the presence of EDTA,
little cross-linking was observed (lane 3). In contrast, the
inhibited OMPLA was efficiently cross-linked, independent of the
presence of Ca2+ or EDTA (Fig. 2C, lanes 4 and
5).
OMPLA (4 mg) was cross-linked on a preparative scale in 3 mM 12-SB, which resulted in the formation of over 80%
dimer. The enzyme was purified from the reagents by DEAE cellulose ion
exchange chromatography (yield: 3 mg). In this step, the residual
amount of monomeric OMPLA present after cross-linking could not be
completely separated from the covalent dimer, resulting in a 10%
contamination of monomer in the dimer preparation (data not shown).
After purification, the cross-linked OMPLA was still enzymatically
active, albeit at a reduced level (50%). This observation opened the
possibility of studying the enzymatic activity of this covalent dimer
under conditions in which native OMPLA loses activity. The cross-linked dimer was preincubated at various concentrations of 12-SB. In contrast
to native protein, the specific activity of cross-linked OMPLA was
independent of the detergent concentration present during preincubation
(Fig. 3).
The results of the chemical
cross-linking experiments described above suggest that the dimeric
state of OMPLA is the active form of the enzyme. To test the stability
of the dimer, dilutions of OMPLA to very low protein concentrations
were prepared. These dilutions were preincubated to reach equilibrium,
and subsequently the enzymatic activities were measured. At high
protein concentrations in the preincubation, the specific activity
remained constant, but below 1 µg/ml the specific activity was
strongly reduced (Fig. 4), underscoring the assumption
that the active state is an oligomeric form. The reduction in specific
activity is 8-fold for the lowest enzyme concentration tested,
indicating that the monomer of OMPLA has only low activity or no
activity at all. Experiments at lower protein concentrations were not
feasible due to limitations in sensitivity of the kinetic assay. The
dilution inactivation data were fitted to a saturation curve. A
dissociation constant of 3.4 nM was derived, assuming a
simple equilibrium between an active dimer and an inactive monomer. The
dilution inactivation experiment described above for the native enzyme
was also carried out with the purified, cross-linked dimer (Fig. 4).
Below 1 µg/ml, only a slight decrease in activity was observed,
presumably due to the 10% monomer contamination in the dimer
preparation. The fact that the dimeric enzyme was active over this
whole protein concentration range demonstrates that the reduction in
activity for the native enzyme is due to dissociation and not to
nonspecific losses.
Analytical ultracentrifugation
was applied to study the monomer-dimer equilibrium in further detail.
For membrane proteins, the determination of the molecular weight by
this technique is complicated by the presence of detergents and/or
lipids, which are needed for solubilization of the protein. The
determined molecular weight of the complex is the sum of the molecular
weight of the protein and of the adhering detergent/lipid molecules.
However, when a detergent is chosen with the same density as the
buffer, the contribution of the detergent is canceled out. Octyl-POE
has a density of near unity and has been successfully applied in the molecular weight determination of various membrane proteins (22, 26).
Analytical ultracentrifugation experiments were carried out with OMPLA
in the presence of octyl-POE at concentrations of 0.5, 1.5, and 5%, at
which OMPLA has optimal, intermediate, or strongly reduced enzymatic
activity, respectively (compare Fig. 1B). In sedimentation
velocity experiments, OMPLA behaved as a homogenous complex at the
three octyl-POE concentrations. Sedimentation equilibrium experiments
were carried out to determine the molecular weight of OMPLA in the
protein-detergent complex, and the results are summarized in Table
I. A molecular weight of 55,000 was derived at optimal
detergent concentration in the presence of the cofactor
Ca2+. This value is intermediate between the calculated
masses of the monomer (31,500 Da) and the dimer (63,000 Da) and
indicates the existence of an equilibrium, with 86% of the protein in
the dimeric form. At higher octyl-POE concentrations, the molecular weight decreased to values close to that of the monomer. In the presence of EDTA the observed molecular weight agrees within
experimental error with that of the monomeric form, showing that
Ca2+ is essential for stabilization of the dimer of native
OMPLA. The effect of the covalent modification of the active site on dimerization was also studied in analytical ultracentrifugation experiments. The molecular weight of inhibited OMPLA corresponds to the
dimeric state, irrespective of the detergent concentration (Table I).
Whereas for native OMPLA the dimerization was absolutely dependent on
calcium, the monomer-dimer equilibrium was only partly shifted toward
the monomer in the absence of calcium in the case of the inhibited
enzyme.
Molecular weight of OMPLA under various conditions as determined by
analytical ultracentrifugation
Department of Enzymology and Protein
Engineering,
Biophysical Chemistry
and ** Microbiology, Biocenter, University of Basel,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland
-barrel
structure has been proposed for OMPLA (8), analogous to the outer
membrane porins, of which the x-ray structures have been solved
(9-11). Recently, we succeeded in the overproduction, in
vitro refolding, and subsequent purification of the enzyme on a
large scale (12), which allowed its crystallization (13).
Chemicals
20 °C.
Activity measurements revealed that 94% of the protein was modified.
For analytical ultracentrifugation experiments, a sample of OMPLA was
prepared in octyl-POE. To this end, 8 mg of OMPLA was loaded to a 1-ml
fast-flow Q Sepharose column (Pharmacia), which was preequilibrated
with buffer (10 mM Tris-HCl, pH 8.3, 2.5 mM
12-SB). The column was washed with 25 ml of buffer (10 mM
Tris-HCl, pH 8.3, 0.5% octyl-POE) and eluted with 10 ml of this buffer
containing 1 M KCl. OMPLA was subsequently dialyzed twice
at 4 °C against 20 volumes of 10 mM Tris-HCl, pH 8.3, containing 0.5% octyl-POE. In a similar manner, a sample of
inhibited-OMPLA in octyl-POE was prepared.
-mercaptoethanol, and 0.008% bromphenol blue) was added, and 20 µl of this solution (corresponding to 1.8 µg of OMPLA) was analyzed
by SDS-PAGE. The gels were stained with Coomassie Brilliant Blue for
visualization of the protein bands. On a preparative scale, OMPLA was
cross-linked at 3 mM 12-SB as described above, but after
the incubation with glutaraldehyde, the cross-linking was stopped by
the addition of ethanolamine to a final concentration of 50 mM, followed by extensive dialysis against buffer (20 mM Tris-HCl, pH 8.3, 2.5 mM 12-SB).
Cross-linked OMPLA was purified by DEAE ion exchange chromatography
similar to the purification procedure for the recombinant protein
(12).
3 for
the solvent. Sedimentation equilibrium data were plotted as the
logarithm of the concentration versus r2.
Molecular weights of the complexes were calculated with a computer program similar to that of Chernyak et al. (24) using a
floating baseline algorithm.
Dependence of the Enzymatic Activity on Detergent
Concentration
-octylglucopyranoside, dodecylphosphocholine, Triton X-100, several
alkylsulfobetaines, and several alkylpolyoxyethylene
glycols.2 As an example, the results
obtained with octyl-POE are shown in Fig. 1B. Previously,
the dependence of the enzymatic activity on the detergent concentration
has been interpreted in terms of a slow reversible conformational
transition of the enzyme (5, 19). So far, this process has not been
understood at the molecular level. In this study, we have
investigated whether oligomerization is responsible for the observed
differences.
Fig. 1.
The specific activity of OMPLA as a function
of the concentration of detergent present in the preincubation
solution. OMPLA was incubated at 0.05 mg/ml in buffer containing
detergent (12-SB or octyl-POE) at various concentrations. After
overnight incubation, 50 ng of protein was assayed for enzymatic
activity in the chromogenic assay. In panels A and
B, the data obtained for 12-SB and octyl-POE, respectively,
are given. The arrows indicate the critical micelle
concentration of 12-SB (5) and of octyl-POE (43).
[View Larger Version of this Image (20K GIF file)]
Fig. 2.
SDS-PAGE analysis of samples of OMPLA after
cross-linking with glutaraldehyde. In panel A, the
results for the cross-linking of OMPLA at various concentrations of
12-SB are shown. Lane 1, molecular weight marker; lane
2, reference of untreated OMPLA; lanes 3-9,
cross-linking of OMPLA at 12-SB concentrations of 2 mM
(lane 3), 2.5 mM (lane 4), 3.0 mM (lane 5), 4.0 mM (lane
6), 6.0 mM (lane 7), 10 mM
(lane 8) and 25 mM (lane 9).
Panel B, cross-linking of inhibited OMPLA at various
concentrations of 12-SB. Lane 1, molecular weight marker;
lane 2, inhibited OMPLA before cross-linking; lanes
3-9, inhibited OMPLA after cross-linking (the 12-SB
concentrations were identical to the preincubation concentrations used
for OMPLA in panel A). Panel C, cross-linking of
OMPLA and of inhibited OMPLA at 3 mM 12-SB in the presence
of 20 mM Ca2+ or 20 mM EDTA.
Lane 1, molecular weight marker; lane 2, OMPLA + Ca2+; lane 3, OMPLA + EDTA; lane 4,
inhibited OMPLA + Ca2+; lane 5, inhibited OMPLA + EDTA. In all cases, samples were not boiled before electrophoresis.
M, monomer; D, dimer; A, aggregates. The numbers at the left indicate the molecular weights of
the marker proteins (in thousands).
[View Larger Version of this Image (46K GIF file)]
Fig. 3.
The specific activity of cross-linked OMPLA
as a function of the concentration of detergent present in the
preincubation solution. Cross-linked OMPLA () or native OMPLA
(
) was preincubated at 0.05 mg/ml in buffer containing 12-SB at
various concentrations. After overnight incubation, 50 ng of protein
was assayed for enzymatic activity in the chromogenic assay.
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
Dilution inactivation of OMPLA. OMPLA
() or cross-linked OMPLA (
) was incubated at various enzyme
concentrations. The solutions were incubated overnight at room
temperature, followed by measurement of the enzyme activity.
[View Larger Version of this Image (22K GIF file)]
Protein
Octyl-POE
MW (+ Ca2+)
MW (+ EDTA)
%
OMPLA
0.5
55,000a
31,800a
OMPLA
1.5
35,400a
30,700a
OMPLA
5.0
31,800a
28,500a
Inhibited
OMPLA
0.5
61,300b
47,000a
Inhibited
OMPLA
1.5
60,600b
ND
Inhibited
OMPLA
5.0
60,000b
ND
a
18,000 rpm.
b
14,000 rpm.
In normally growing
cells, OMPLA is present in the outer membrane, but enzymatic activity
is only detectable after perturbation of the membrane. We investigated
whether or not activation in vivo can be correlated with
dimerization. For these experiments, we used the expression plasmid
pRB1, which carries the pldA gene encoding OMPLA under
control of its own promoter (8). CE1348 cells with or without plasmid
pRB1 were grown overnight and the enzymatic activity of OMPLA present
in intact cells was measured. OMPLA activities of cells carrying the
plasmid (5 milliunits/ml of cell culture) did not deviate significantly
from that of CE1348 cells without plasmid. Possible explanations for
this lack of activity are: (i) the substrate cannot reach the enzyme,
(ii) the enzyme is not expressed, or (iii) the enzyme is in an inactive state. The first possibility is unlikely because the active site of the
enzyme is supposedly present at the cell surface (8) and because the
substrate is small enough to diffuse into the periplasmic space. The
second option was tested by Western blotting, which showed that in
cells carrying the plasmid, OMPLA was produced in a heat-modifiable,
correctly folded form (data not shown). We conclude that the enzyme
apparently resides in the outer membrane in a dormant state. After
activation of OMPLA by sonication an activity of 110 milliunits/ml of
the plasmid-containing cell culture was measured, whereas the activity
of the CE1348 cells without plasmid remained low (5 milliunits/ml).
After chemical cross-linking of OMPLA with formaldehyde in the
nonactivated cells, the enzyme was detected on Western blots as the
folded monomeric form at 27,000 and also partially as unfolded protein
at 31,500 (Fig. 5, lane 1). The appearance of
the unfolded form is presumably due to the 10-min incubation at
60 °C in loading buffer before SDS-PAGE needed to obtain clear gels.
Only small amounts of the dimeric form were detected. After
cross-linking in sonicated cells, large amounts of the dimeric form
with an apparent molecular weight of 42,000 were detected (Fig. 5,
lane 2). The results of the in vivo cross-linking
experiments indicate that OMPLA is present in an inactive monomeric
form in the outer membrane of resting cells and that the enzyme
dimerizes upon activation.
In the present study, we have investigated whether the activity of OMPLA is regulated by oligomerization. The results of kinetic experiments, chemical cross-linking and analytical ultracentrifugation provide experimental support for a dimerization model in which the enzyme is in equilibrium between an inactive monomeric and an active dimeric state. The dimer of OMPLA was present at low detergent concentrations, whereas at high detergent concentrations the enzyme was monomeric. In the kinetic experiments, up to a 15-fold reduction in activity upon dimer dissociation was observed. This figure represents an upper limit for the activity of the monomeric species, and a more precise determination was hampered by sensitivity limitations of the assay at extremely low protein concentrations. Based on our observations, we cannot exclude the possibility that the monomer has some low enzymatic activity. However, the absence of enzymatic activity of OMPLA in both lipid vesicles (2) and in the outer membrane (14, 15) suggests that the monomer is inactive.
Analytical ultracentrifugation has the advantage that the ratio of dimers and monomers can be determined under equilibrium conditions. The dimeric form was present at low detergent concentrations, whereas at intermediate or high detergent concentrations, the monomer was predominant. The dimerization equilibrium was also studied by chemical cross-linking. The cross-linking proved to be very efficient, and the results were in full agreement with the data obtained in the analytical ultracentrifugation experiments. Analytical ultracentrifugation and chemical cross-linking identified the dimer of OMPLA only at low detergent concentrations, and preincubation of OMPLA under these conditions resulted in full activity. At intermediate detergent concentrations, the results of the preincubation experiments deviate from those obtained by the other techniques. Under those conditions, the preincubation experiments still gave considerable activities, suggesting that significant amounts of dimer were present. Several factors could account for this discrepancy. First, the enzyme was preincubated in 12-SB, but for the activity measurement, the enzyme was transferred to a solution containing the detergent Triton X-100. Second, the presence of substrate in the assay results in the formation of the acyl enzyme intermediate. Our results obtained with the inhibited OMPLA indicate a strong stabilization of the dimeric state for the acyl enzyme analog, suggesting that under assay conditions the monomer-dimer equilibrium could be shifted toward the dimer. Third, the presence of the cofactor calcium could also influence the monomer-dimer equilibrium. All of these possibilities could contribute to the overestimation of the amount of dimer.
Dilution inactivation of oligomeric enzymes is a process in which the protein concentration is decreased below the dissociation constant for oligomerization. OMPLA, being a membrane protein, is present in the micellar phase. We accomplished dilution inactivation by varying the protein/detergent ratio. This ratio was varied in two ways. In the preincubation experiment, the protein concentration was kept constant, and increasing the detergent concentration resulted in dimer dissociation. In the dilution inactivation experiment, decreasing the protein concentration at a fixed detergent concentration resulted in dimer dissociation. Final proof for the dimer model came from our kinetic measurements with cross-linked OMPLA, which retained enzymatic activity. Changing the protein/detergent ratio in this case did not affect the enzymatic activity, confirming our dimerization model.
Oligomeric regulation of enzyme activity has been reported for many water-soluble enzymes (reviewed in Ref. 27). Dilution inactivation has been described for several of these enzymes (28-30). There is to our knowledge only one membrane enzyme for which oligomeric regulation has been described before, i.e. Ca2+-ATPase (31, 32). This enzyme is regulated by dimerization, and its activity is dependent on both the detergent concentration and the protein concentration (31). Moreover, the dimerization was Ca2+-dependent (32). OMPLA is the second example of a membrane protein for which dimerization is the mechanism of activity regulation.
Calcium is an essential cofactor of OMPLA. One can envisage two
possible roles for calcium: a structural role and a functional one. For
the water-soluble phospholipase A2, the Ca2+
ion plays a functional role in polarizing the carbonyl moiety of the
scissile ester bond and in binding the phosphate group of the substrate
(33, 34). Although OMPLA does not share sequence homology with
water-soluble phospholipase A2, calcium could play a
similar role in the catalytic machinery of OMPLA. However, a clue for
the role of calcium in OMPLA might come from the structure of several
porins in which it has been reported that Ca2+ is required
to maintain the quaternary structure. In the crystal structure of the
trimeric porin from Rhodobacter capsulatus, a shared
Ca2+-binding site is located at the subunit interface (10,
11), and Ca2+ is also essential to maintain the trimeric
structure of the R. sphaeroides porin (35). OMPLA has been
proposed to be structurally related to the outer membrane porins,
having a -barrel topology in common (8). Therefore, we suggest that
Ca2+ plays a structural role in the stabilization of the
dimeric state.
Previously, it has been suggested that conformational changes were responsible for the activation of OMPLA (2, 5, 19, 20). The reactivity of the active site and the number of active sites per molecule have been determined as a function of the detergent concentration by the covalent modification of the active site serine with hexadecylsulfonylfluoride (20). The number of active sites was one per molecule of OMPLA, and this number was constant, irrespective of detergent concentration. The reactivity with the inhibitor was the highest near the critical micelle concentration, and the reactivity was strongly diminished at high detergent concentrations. The authors of that study concluded that the enzyme could adopt different conformations and that there exists a slow reversible equilibrium between these states. In view of our current dimerization model, the reduced reactivity can be explained in terms of decreasing concentrations of dimer upon increasing the detergent concentration. The fact that even at high detergent concentrations all of the available enzyme still can be modified, albeit with reduced reactivity, suggests that the dimer that has reacted with the inhibitor is withdrawn from the monomer-dimer equilibrium. Apparently the association of monomers into the active dimer is slow compared to the chemical reaction with the inhibitor. Our results show that dimerization is an important factor in the activation process. Dimerization of the enzyme could induce conformational changes of the protein due to the formation of the subunit interface. Such a conformational change might then lead to the formation and/or optimization of the active site. Besides conformational changes, dimerization could induce activation by the formation of shared active sites by complementarity, as is the case for several other oligomeric enzymes, such as citrate synthetase (36), glutamine synthetase (37), aspartate aminotransferase (38), triose phosphate isomerase (39), and human immunodeficiency virus protease (40). At this moment we do not have experimental support in favor of shared active sites. It is hoped that the x-ray analysis will provide insight into the mechanism by which the activity in the dimer is induced. In this respect, it is noteworthy that OMPLA crystallizes in space group P3121 (13) with one molecule per asymmetric unit. The presence of a crystallographic two-fold symmetry axis in this space group would be consistent with a dimer being the building block of the crystal.3
In vivo, OMPLA is present in the outer membrane in a dormant state. The results of the cross-linking experiments with nonactivated cells showed that OMPLA is monomeric under these conditions. The absence of efficient cross-linking is a negative result, but it is probably not an artifact of the technique that was used. Cross-linking with formaldehyde has been applied successfully for protein complexes in the outer membrane and in the periplasm of whole cells (41, 42). OMPLA can be activated by various treatments that have in common the fact that they perturb the membrane and finally lead to cell lysis. OMPLA was activated by sonication, which is a crude method resulting in the breakage of the cells. After this step, the enzymatic activity was easily detectable, and the dimeric state of the enzyme was identified by formaldehyde cross-linking. Apparently OMPLA is present in the intact outer membrane as a monomeric protein, and the enzyme is capable of dimerization only after perturbation of the outer membrane. These results support the model for dimerization as a mechanism for OMPLA activation in vivo. This finding raises the question of what the mechanism is by which dimerization is normally prevented in the outer membrane. One explanation could be that in the outer membrane the conformation of OMPLA is restricted due to the tight lipid packing. Perturbation of the membrane then presumably allows the protein to change its conformation, allowing dimerization to occur. Another possible explanation comes from the asymmetry of the outer membrane. Perturbation of the membrane is likely to result in the presence of phospholipids in the outer leaflet of the outer membrane, which might favor dimerization. Our future research aims to understand the regulation and the molecular mechanism of the activation process.