(Received for publication, August 15, 1996, and in revised form, October 24, 1996)
From the Department of Chemistry, University of
Illinois at Chicago, Chicago, Illinois 60607-7061 and
§ Department of Molecular Biophysics and Biochemistry and
the Howard Hughes Medical Institute, Yale University,
New Haven, Connecticut 06511
Recent genetic and structural studies have shed
considerable light on the mechanism by which secretory
phospholipases A2 interact with substrate aggregates.
Electrostatic forces play an essential role in optimizing interfacial
catalysis. Efficient and productive adsorption of the Class I bovine
pancreatic phospholipase A2 to anionic interfaces is
dependent upon the presence of two nonconserved lysine residues at
sequence positions 56 and 116, implying that critical components of the
adsorption surface differ among enzyme species (Dua, R., Wu, S.-K., and
Cho, W. (1995) J. Biol. Chem. 270, 263-268). In an
effort to further characterize the protein residues involved in
interfacial catalysis, we have determined the high resolution (1.7 Å)
x-ray structure of the Class II Asp-49 phospholipase A2
from the venom of Agkistrodon piscivorus piscivorus. Correlation of the three-dimensional coordinates with kinetic data
derived from site-directed mutations near the amino terminus (E6R, K7E,
K10E, K11E, and K16E) and the active site (K54E and K69Y) defines much
of the interface topography. Lysine residues at sequence positions 7 and 10 mediate the adsorption of A. p. piscivorus
phospholipase A2 to anionic interfaces but play little role
in the enzyme's interaction with electrically neutral surfaces or in
substrate binding. Compared to the native enzyme, the mutant proteins
K7E and K10E demonstrate comparable (20-fold) decreases in affinity and
catalysis on polymerized mixed liposomes of
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine and
1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol, while the double mutant, K7E/K10E, shows a more dramatic 500-fold decrease in catalysis and interfacial adsorption. The calculated contributions of Lys-7 and Lys-10 to the free energy of binding of
A. p. piscivorus phospholipase A2 to anionic
liposomes (1.8 kcal/mol at 25 °C per lysine) are additive
(i.e.
3.7 kcal/mol) and together represent nearly half of
the total binding energy. Although both lysine side chains lie exposed
at the edge of the proposed interfacial adsorption surface, they are
geographically remote from the corresponding interfacial determinants
for the bovine enzyme. Our results confirm that interfacial adsorption is largely driven by electrostatic forces and demonstrate that the
arrangement of the critical charges (e.g. lysines) is
species-specific. This variability in the topography of the adsorption
surface suggests a corresponding flexibility in the orientation of the
active enzyme at the substrate interface.
Phospholipases A2 (PLA2; EC
3.1.1.4)1 catalyze the hydrolysis of the
fatty acid ester in the 2-position of 3-sn-phospholipids and
are found both in intracellular and secreted forms (for recent reviews,
see Refs. 1-4). The enzymes act at the lipid-water interface with a
preference for organized lipids (micelles and vesicles) that is often
orders of magnitudes greater than that shown for dispersed substrate.
Calcium-mediated catalysis appears to involve two kinetically and
structurally distinct steps (5, 6). Adsorption of PLA2 to
the interface precedes and is independent of substrate binding to the
active site. Secretory PLA2s are small proteins (14 kDa)
that can be classified into at least four groups based on structural
differences (7, 8). Class I (exocrine pancreas and elapidae and
hydrophodiae snake venoms) and Class II (mammalian nonpancreatic and
crotalidae and viperidae snake venoms) enzymes are highly homologous
(see Fig. 1) and have been extensively studied. Crystallographic and
NMR analyses of multiple secretory PLA2s have implicated a
common interfacial adsorption surface that is structurally distinct
from the active site (2, 9). The proposed interfacial adsorption site
is located on a flat external surface which incorporates a ring of
hydrophobic and cationic residues. Recent genetic studies have shown
that the adsorption of the Class I bovine pancreatic PLA2
to anionic interfaces is driven primarily by a few cationic residues,
particularly Lys-56 and Lys-116 (10). These residues are not conserved
among PLA2s, indicating that many of the key determinants
of interfacial adsorption differ among enzyme species.
The venom of eastern cottonmouth, Agkistrodon piscivorus
piscivorus, contains two cationic monomeric PLA2s,
Asp-49 PLA2 (App-D49) and Lys-49 PLA2, and a
dimeric PLA2. Among these PLA2s, App-D49 has
served as the prototype for a Class II PLA2 (11-13).
Previous work has shown that App-D49 strongly prefers anionic
interfaces to zwitterionic ones and that Lys-7 and Lys-10 are involved
in this selectivity (12, 14-16). Lysines 7 and 10 lie near the
carboxyl end of the amino-terminal helix within a cationic patch that
also includes Lys-11 and Lys-16 (see Fig. 2). This patch lies at the periphery of the proposed interfacial adsorption surface. At least two
other lysines (Lys-54 and Lys-69), which lie on the other side of the
enzyme in close proximity to the substrate binding site, also appear to
play influential roles in interfacial catalysis. In particular, Lys-69,
which is highly conserved among Class II secretory PLA2s,
has been shown to interact with the sn-3 phosphate oxygen of
transition state analogs (17). In an effort to further characterize the
structural determinants underlying interfacial kinetics, we have
determined the crystal structure of App-D49 and analyzed the impact of
selected mutations within the interfacial adsorption surface
(i.e., E6R, K7E, K10E, K11E, and K16E). The side chains at
sequence positions 54 and 69 were also altered to determine the effect
of these substitutions on both interfacial adsorption and head group
selectivity (K54E and K69Y). From the results it is clear that although
the adsorption of both the bovine enzyme and App-D49 to anionic
interfaces is largely dependent on lysine residues, the locations of
these critical residues are quite different. This variability in the
topography of the adsorption surface may have an important role in
determining the specific orientation of the enzyme at the substrate
interface.
1,2-Dioctanoyl-sn-glycero-3-phosphocholine
(diC8PC) and
1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine
(diC8PE) were obtained from Avanti Polar Lipids (Alabaster,
AL);
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (pyrene-PC), -ethanolamine (pyrene-PE), and -phosphoglycerol
(pyrene-PG) were purchased from Molecular Probes (Eugene, OR); and
1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (BLPC) and -glycerol (BLPG) were synthesized as described previously (18). Large unilamellar liposomes of BLPG (or BLPC) were prepared by
multiple extrusions of a phospholipid dispersion in 10 mM
Tris-HCl buffer (pH 8.4) through a 0.1-µm polycarbonate filter in a
Liposofast microextruder (Avestin; Ottawa, Ontario) and then
polymerized in the presence of 10 mM dithiothreitol (19).
Phospholipid concentrations were determined by phosphate analysis (20).
Nonlipid reagents were obtained from the following sources; fatty
acid-free bovine serum albumin, Bayer Inc. (Kankakee, IL); guanidinium
chloride and guanidinium isothiocyanate, Fisher; restriction enzymes,
T4 ligase, T4 polynucleotide kinase, and isopropyl
-D-thiogalactopyranoside, Boehringer Mannheim; and
oligonucleotides, Midland Company (Midland, TX).
The App-D49 gene, synthesized based on the corrected amino
acid sequence of App-D49 (21), was a generous gift from Dr. Gordon Rules of the University of Virginia. The coding region for App-D49 was
subcloned into a pET-21-a vector (Novagen; Madison, WI) and designated
as pSH-app. This synthetic gene carries the Asn to Ser mutation (N1S)
at the amino terminus to facilitate the removal of the initiator Met by
an endogenous methionine aminopeptidase (22). The mutant proteins were
created using a Sculptor in vitro mutagenesis kit (Amersham
Corp.) according to the method of Nakamaye and Eckstein (23) with
modifications. In this method, a phagemid DNA prepared from the pSH-app
vector in the presence of the helper phage R408 (Promega; Madison, WI)
served as a template for mutagenesis, and the mutagenesis protocol was
slightly modified to optimize the mutation efficiency. The
oligonucleotides used for the construction of the mutants were 5-CTT
GAT TAA TTT
AAA CTG GAA CAG-3
(E6R), 5
-TT CTT GAT TAA
CTC AAA CTG GAA CAG-3
(K7E), 5
-CC AGT CAT TTT
GAT TAA TTT CTC-3
(K10E), 5
-T ACC AGT CAT
CTT GAT TAA TTT-3
(K11E), 5
-G CAT TCC GGA
ACC AGT CAT TTT-3
(K16E), 5
-ACA GCC GGT AAC
ACC GTA GCA GCA-3
(K54E), and 5
-GTA GAT ATC CAT
CGG GTT ACA GCC-3
(K69Y), respectively, in which the
underlined bases indicate the location of the mutation. The K7E/K10E
double mutation was performed sequentially; i.e. the K7E
mutation followed by the K10E mutation. After the DNA sequences of the
entire coding regions of mutants were verified by sequence analysis
(24) using a Sequenase 2.0 kit (Amersham), individual recombinant
pSH-app vectors were transformed into Escherichia coli
strain BL21(DE3) (Novagen) for protein expression.
Lyophilized venom from
A. p. piscivorus was purchased from the Miami Serpentarium
(Salt Lake City, UT). The dimeric Asp-49 PLA2, the
monomeric Lys-49 PLA2, and App-D49 were isolated as described elsewhere (11). The pooled App-D49 was rechromatographed on a
CM-Sephadex C-50 column at pH 3.5 and developed with a linear gradient
of 50 to 400 mM ammonium formate. This latter step removed several minor impurities which interfered with crystallization of the
major enzyme species. The chromatographic and catalytic behaviors of
the native App-D49 and the recombinant N1S were indistinguishable (22).
Thus, the recombinant N1S will be referred to as wild type hereafter.
Wild type and mutant proteins were expressed in BL21(DE3) cells
harboring the corresponding pSH-app vectors. A 2-liter culture (Luria
broth containing 50 µg/mL ampicillin) was grown at 37 °C, and
protein expression was induced by the addition of 0.4 mM
isopropyl -D-thiogalactopyranoside when the absorbance of the medium reached 1.0 (
= 600 nm). After the cells were
incubated for an additional 4 h at 37 °C, they were harvested,
resuspended in 50 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 20 mM EDTA and 0.1% Triton X-100, and incubated
for 10 min on ice. Pulse-mode sonication was performed using a Sonifier
450 (Branson) for 10 times at 15 s each. The inclusion body pellet
obtained by centrifugation for 20 min at 10,000 × g/4 °C was resuspended in 5 ml of 0.2 M Tris-HCl buffer, pH 8.0, containing 4 M guanidinium
isothiocyanate and 3 mM EDTA. The insoluble matter was
removed by centrifugation at 35,000 × g for 1 h
at room temperature, and the inclusion body protein was sulfonated
using 2-nitro-5-(sulfothio)-benzoate (10). The reaction mixture was
then loaded onto a Sephadex G-25 column (2.5 × 30 cm)
equilibrated with 50 mM Tris-HCl buffer, pH 8.0, containing
2 M guanidinium chloride and 3 mM EDTA, and the
major protein peak was collected. The sulfonated protein was refolded by slowly adding to the protein solution 200 ml of 25 mM
Tris-HCl buffer, pH 8.0, containing 15 mM
CaCl2, 1 mM EDTA, 2.5 mM reduced glutathion, 0.5 mM oxidized glutathion, and 5 mM dodecyl sucrose. The solution was kept at room
temperature for 40 h, at which point the protein solution was
dialyzed overnight against 4 liters of 25 mM Tris-HCl
buffer, pH 8.0. The solution was filtered through a small (2.5 × 2 cm) Sephadex G-25 column equilibrated with 25 mM HEPES
buffer, pH 8.0, to remove any precipitates and to exchange buffer
solutions. The clear solution was loaded onto a Pharmacia CM-Sepharose
column (2.5 × 20 cm) equilibrated with 25 mM HEPES buffer, pH 8.0. The column was washed extensively with 25 mM HEPES buffer, pH 8.0, until no further peaks emerged,
and then the folded protein was eluted with a gradient of 0 to 1 M NaCl in the same buffer (the native App-D49 eluted with
0.7 M NaCl). The collected protein peak was dialyzed
against water, lyophilized, and stored at
20 °C. Protein purity
was confirmed both by SDS-polyacrylamide electrophoresis and
isoelectric focusing. Protein concentrations were determined by the
micro bicinchoninic acid method (Pierce). The circular dichroism
spectra of proteins were measured in 10 mM phosphate
buffer, pH 7.4, at 25 °C, using a Jasco J-600 spectropolarimeter. Each spectrum was obtained at wavelengths between 195 and 300 nm and
averaged from 10 separate scans.
Large, single crystals of App-D49
suitable for diffraction work were grown by the vapor diffusion method.
Ten-µl droplets containing 30 µg/µl App-D49, 20% v/v ethanol,
and 0.1 M Tris-HCl, pH 7.5, were plated onto plastic
coverslips and inverted over 1-ml reservoirs of 40% v/v ethanol and
0.2 M Tris-HCl, pH 7.5. Crystals, which nucleated within 2 weeks, grew to their maximum size of 0.7 × 0.2 × 0.2 mm
after a variable period ranging from 3 to 6 weeks. Diffraction data for
the App-D49 structure were collected from a single crystal with a dual
panel San Diego Multiwire System detector at 4 °C using graphite
monochromated CuK emission from a RU-300 x-ray
generator. The space group was determined to be P1 with unit cell
dimensions of a = 34.71 Å, b = 39.89 Å, c = 42.85 Å,
= 94.17°,
= 79.10°, and
= 106.27°. The collected data were processed with SCALZO (25) and
yielded a final data set that was 82% complete at 1.5 Å (Table
I). Because of significant degradation of the data at
high resolution, however, a nominal cutoff of 1.7 Å was chosen for
refinement purposes. The calcium-free crystal structure of App-D49 was
solved by molecular replacement with a modified version of the computer
program Merlot 1.5. The search model was created by pruning
nonhomologous side chains from one subunit of the refined coordinates
of the App-D49 dimer (80% sequence homology). The rotation function
yielded two strong peaks consistent with the expected presence of two
independent molecules in the crystallographic asymmetric unit. The
intermolecular translation function was also sharp generating a
solution with excellent packing. Refinement with the computer programs
PROFFT and X-PLOR rapidly reduced the R factor while maintaining
reasonable stereochemistry (Table II). When necessary,
the coordinates were manually rebuilt with the graphics program FRODO.
Two hundred and sixty-six water molecules were assigned based on
density in the Fourier difference maps, hydrogen-bonding potentials,
and refined temperature factors of less than 45 Å2 at full
occupancy. A second group of 52 less well defined water molecules
refined to temperature factors ranging from 46 to 65 Å2.
The distal portions of solvent-exposed side chains (e.g.
lysines) accounted for the majority of the protein disorder.
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PLA2-catalyzed hydrolysis of polymerized mixed liposomes was performed at 37 °C in 2 ml of 10 mM HEPES buffer, pH 7.4, containing 0.1 µM pyrene-containing phospholipids (1 mol %) inserted into 9.9 µM BLPG, 2 µM bovine serum albumin, 0.16 M NaCl, and 10 mM CaCl2. Enzyme concentrations were adjusted to keep the half-life of the reaction below 5 min (e.g. [wild type] = 1-10 nM and [K7E/K10E] = 0.1-1 µM). Progress of the reaction was monitored as an increase in fluorescence emission at 378 nm using a Hitachi F4500 fluorescence spectrometer with an excitation wavelength of 345 nm. The spectral bandwidth was set at 5 nm for both excitation and emission. The pseudo-first-order rate constant was determined from the nonlinear least squares analysis of the reaction progress curves (10, 18, 19). This was divided by the enzyme concentration to obtain the apparent second-order constant, (kcat/Km)app. The kinetics of PLA2-catalyzed hydrolysis of diC8PC and diC8PE micelles were measured in the presence of 0.5 mM phospholipid, 0.16 M NaCl, and 10 mM CaCl2 with enzyme concentrations between 1-10 nM. The time course of the phospholipid hydrolysis was monitored with a computer-controlled Metrom pH-stat (Brinkmann) in a thermostated vessel. Under these conditions, the hydrolysis of diC8PC (and diC8PE) followed first-order kinetics, since the substrate concentrations remained lower than the apparent Km values.
Liposome-Protein Binding MeasurementsThe adsorption of the
wild type and mutant enzymes to polymerized liposomes was examined
fluorometrically. Typically, the fluorescence emission (excitation at
= 280 nm) of the protein solution (0.5 µM) at 345 nm
(F345) was measured at equilibrium in 2 ml of 10 mM HEPES buffer, pH 7.4, containing 0.1 to 100 µM BLPG polymerized liposomes and varying concentrations
of NaCl and CaCl2. An initial decrease in protein
fluorescence was observed due to the adsorption of the protein to the
cuvette wall (less than 5% of the total signal) (26). Protein
concentrations were, therefore, corrected for this loss and the
liposome solution was added only after the protein signal stabilized.
To minimize potential artifacts due to both the inner filter effect and
the scattering by BLPG polymerized liposomes, each observed
F345 value was background-corrected against the
F345 value observed in the presence of the same
concentration of BLPC polymerized liposomes. BLPC polymerized liposomes
have the same spectroscopic properties as BLPG polymerized liposomes but App-D49 has an extremely low affinity for them (apparent
dissociation constant > 1 mM) (14). The relative
fluorescence change (Frel) for each phospholipid
concentration was calculated as (Fmax
F345)/(Fmax
Fmin) where Fmax and
Fmin represent the protein fluorescence at 345 nm in the absence of BLPG and in the presence of an excess amount of
BLPG (e.g. 0.2 mM), respectively. Values of
n and Kd were determined by the nonlinear
least squares analysis of the Frel
versus [BLPG]o data using Equation 1,
![]() |
(Eq. 1) |
![]() |
App-D49 is a typical Class II PLA2 with high
sequence homology to other members of this class (Fig.
1). The enzyme was crystallized in the absence of added
calcium ion and, as expected, both independent molecules in the
crystallographic asymmetric unit lack discernible metal ions (Fig.
2). Instead, a single water molecule replaces each of
the primary calcium ions and makes comparable hydrogen bonds to a
carboxylate oxygen of Asp-49 and three backbone carbonyls (Tyr-28,
Gly-30, and Gly-32). Least squares superimposition of the -carbon
traces of App-D49 and its dimeric counterpart from A. p.
piscivorus venom reveals the anticipated conservation of the
homologous core of tertiary structure (Fig. 3). The
root-mean square deviation of the homologous cores of App-D49 and the
dimer is 0.66 Å, whereas the comparable value for the non-core regions is 1.42 Å. The differences between the enzymes, as well as the differences that may be attributed to the effects of pH and
crystallization media on the structure of App-D49, are largely confined
to those regions whose conformations have been previously noted to be
highly variable (e.g. the calcium binding loop (residues
26-34), the
-wing (residues 74-84), and the carboxyl terminus
(residues 123-131)). The residues focused on in this study (Glu-6,
Lys-7, Lys-10, Lys-11, Lys-16, Lys-54, and Lys-69) are all well
resolved in the electron density map as they reside in stable regions
with little protein disorder. The side chains of Lys-7, Lys-10, Lys-16,
and Lys-54 can be positioned to their respective C
atoms, whereas
Arg-6, Lys-11, and Lys-69 can be visualized in their entirety.
The dimensions of the crystalline App-D49 are approximately 22 × 30 × 42 Å with 50% of the structure -helix and 10%
-sheet. The two long anti-parallel
-helices that form the backbone of the
molecule (residues 37-54 and 90-109) are securely braced by a series
of disulfide bridges. The active site residues (His-48, Asp-49, Tyr-73,
and Asp-99) occupy the base of a short internal hydrophobic cavity or
cleft that opens onto the external surface. The disposition of
productively bound substrate within the active site has been shown
through the co-crystallization of several PLA2s with
transition state and substrate analogs (17, 27-29). The structural
work (9, 30), along with extensive biochemical (31, 32), genetic (10,
33-36), and electrostatic data (37), suggests that the interfacial
adsorption surface surrounds and incorporates the external opening of
the hydrophobic channel.
All of the mutant proteins were expressed and refolded as efficiently
as the wild type (data not shown), indicating similar thermodynamic
stability. Retention times for the refolded wild type App-D49 and the
native enzyme were identical on a CM-Sepharose column. The mutant
enzymes, including the double mutant (K7E/K10E), migrated as expected
for their altered surface charges. The CD spectra of the native, wild
type, and the mutant proteins were indistinguishable (Fig.
4) as were estimates of -helical content based on
222 values (~50%), indicating that the mutations did not induce any gross structural change in protein conformation. The
preservation of tertiary structure is consistent with the surface
locations of the altered side chains.
Kinetic Properties of App-D49 and Mutants
A major advantage of the polymerized mixed liposome system over other PLA2 kinetic systems is that it allows an unambiguous distinction between phospholipids interacting with the active site of PLA2 (i.e., hydrolyzable inserts) and ones interacting with the interfacial binding site of PLA2 (i.e. polymerized matrix) (18). Thus, it is possible to separately determine the head group specificity of the interfacial adsorption surface and the active site. Furthermore, it is also possible to separately analyze the effects of protein and phospholipid modification on interfacial adsorption and substrate binding (10, 18, 33). Using this approach, we measured the kinetic properties of wild type and mutants toward a wide variety of polymerized mixed liposomes. Two zwitterionic phospholipids, pyrene-PC and pyrene-PE, and anionic pyrene-PG were used as inserts in the anionic BLPG polymerized matrix to determine the phospholipid head group specificity of the active site of each enzyme. Apparent second-order constants, (kcat/Km)app, determined for these proteins are summarized in Table III. Although (kcat/Km)app has no obvious physical meaning, it is a useful parameter for comparing overall interfacial activity of wild type and mutants (10, 33). In agreement with a previous report (22), recombinant wild type App-D49 showed essentially the same activity and head group specificity toward polymerized mixed liposomes as native App-D49. The active sites of both enzymes prefer anionic phospholipids to zwitterionic ones by ~3.5-fold. This is a modest value when compared to the high (up to 5,000-fold) adsorption preference of App-D49 for anionic interfaces (18). Of note, App-D49 has comparable activities toward pyrene-PC and pyrene-PE substrates indicating that the enzyme cannot distinguish between the two zwitterionic head groups. Thus, the ratio of enzyme activity (pyrene-PG/pyrene-PC) can be used to describe the anionic phospholipid preference of each protein (see Table III).
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The cluster of lysines lying adjacent to the carboxyl end of the
amino-terminal -helix is geographically remote from the catalytic
site (e.g., >15 Å from His-48). Mutation of these residues to Glu had no appreciable effect on the enzyme's head group
preference. These mutations did, however, significantly decrease
adsorption and catalysis on polymerized mixed liposomes. When compared
to the wild type, K7E and K10E demonstrated an approximately 20-fold drop in activity toward pyrene-PC/BLPG polymerized mixed liposomes. In
contrast, K11E and K16E displayed modest 3.8- and 2.6-fold decreases,
respectively. The rate decrease for the double mutant (K7E/K10E) was
500-fold on polymerized mixed liposomes and approximated the product of
the decreases for the individual mutations. To investigate the effect
of mutations on the binding of App-D49 to electrically neutral
interfaces, we measured the activities of wild type and mutants using
zwitterionic diC8PC micelles. These micelles were chosen
because they provide neutral interfaces and yet are a good substrate
for App-D49 due to their loose interfacial packing density. When
assessed using these micellar substrates, none of the amino-terminal
mutations caused a significant drop in activity. Even the K7E/K10E
mutant showed 83% of wild type activity. This suggests that Lys-7 and
Lys-10 play an essential role in binding to anionic interfaces but are
dispensable in adsorbing to electrically neutral interfaces or in
substrate binding.
Many secretory PLA2s contain a cationic residue at position 6 in addition to those at position 7 and 10. If the enzymatic activity of App-D49 toward substrate aggregates is simply proportional to its electrostatic affinity for the interface, one would expect that the E6R mutant with additional positive charge in the vicinity of Lys-7 would be more active on anionic polymerized mixed liposomes. Surprisingly, the E6R enzyme displayed a 4-fold decrease in activity toward all polymerized mixed liposomes. This inhibition was not due to a disruption of the catalytic apparatus, since the activity of E6R toward electrically neutral diC8PC micelles was only modestly affected (i.e. a 13% decrease). The decrease in activity suggests, however, that this mutant binds to the BLPG polymerized mixed liposomes in a suboptimal mode. The activity of PLA2 toward anionic interfaces is, therefore, not merely proportional to the net charge of the interfacial adsorption surface.
To identify the origin of App-D49's small, but definite, preference
for anionic head groups, two lysines (Lys-54 and Lys-69) that are
located close to the active site were mutated, and the resulting
proteins' activities were measured on polymerized mixed liposomes and
micelles. If the active site of App-D49 prefers pyrene-PG to pyrene-PC
because of electrostatic repulsion between the lysine side chain(s) and
the choline head group, one would expect that K54E and K69Y would have
enhanced activities toward pyrene-PC while showing the same activities
toward pyrene-PG as the wild type. On the other hand, if the preference
for pyrene-PG derives from favorable interactions (e.g.
hydrogen bonding) between the lysine(s) and the glycerol head group,
the mutants should show reduced activity on pyrene-PG but not on
pyrene-PC. As it turned out, the K54E mutation had little effect on the
enzyme activity toward pyrene-PC (15% decrease), pyrene-PG (20%
decrease), or micellar diC8PC (17% decrease). This
mutation did, however, result in a 2-fold enhancement of activity on
pyrene-PE in both polymerized mixed liposomes and micellar
diC8PE. In the case of diC8PE, the
(kcat/Km)app
values for wild type and the K54E mutant were 4.5 × 105 M1 s
1 and
1.0 × 106 M
1
s
1, respectively. These results suggest that Lys-54 does
not directly interact with productively bound phospholipids, but a
glutamate side chain at this position might potentially form a
favorable electrostatic contact with the ammonium ion of
phosphatidylethanolamine. The activity of the K69Y mutant toward both
pyrene-PC and pyrene-PE lipids in polymerized mixed liposomes was
identical to the wild type, indicating that the Lys-69 side chain has a
negligible effect on the productive binding of zwitterionic head
groups. The mutation did, however, decrease the enzyme activity toward
pyrene-PG/BLPG by approximately 3-fold, rendering K69Y nonselective for
the phospholipid head group. The modest nature of these changes
underlines the equivalent abilities of the tyrosine and lysine side
chains in coordinating the sn-3 phosphate of a productively
bound phospholipid. At the same time, the decrease in activity toward
the pyrene-PG substrate suggests that the
-ammonium group of Lys-69
may form hydrogen bond(s) with the glycerol head group which are not
achievable with the phenolic hydroxyl of Tyr-69 (Fig.
5).
Binding of App-D49 and Mutants to BLPG Polymerized Liposomes
To delineate the relationship between the enzyme
activity and the interfacial adsorption affinity of mutants, we
measured their binding to BLPG polymerized liposomes. We previously
showed that nonhydrolyzable BLPG polymerized liposomes could be used to
determine the dissociation constant for PLA2-liposome
complex (18, 19). The n and Kd values
(Table IV) determined for the wild type and mutant
enzymes were derived from curve fittings of the respective isotherms
(Fig. 6). All of the proteins showed analogous
saturation binding curves, indicating similar binding modes. Under the
conditions in which the kinetic measurements were performed (10 mM Ca2+ and 0.16 M NaCl), the wild
type enzyme showed a high affinity for anionic BLPG liposomes
comparable to that reported for the native App-D49
(n·Kd = 0.7 µM) (19).
Calcium ion had no effect on this binding (Fig. 6). When compared to
the wild type, the mutant enzymes displayed a wide range of
Kd values with only minor variation in n
(n 40). In general, the relative binding affinity
of mutants to BLPG polymerized liposomes paralleled their relative
enzymatic activity toward pyrene-PC/BLPG polymerized mixed liposomes.
For example, K7E, K10E, and K7E/K10E had 50-, 45-, and 500-fold
increases in Kd, respectively. In the case of
K7E/K10E, for which the binding saturation could not be reached with
BLPG concentration up to 200 µM, the
Kd value was determined from a curve fitting
assuming n = 40. Also, K11E and K16E showed 2-fold
increases in Kd, whereas K54E and K69Y had
essentially the same Kd as wild type. For mutants of
amino-terminal lysines, these results indicate that their reduced
activities are primarily due to the decreases in interfacial adsorption
affinities and again underscore the essential roles of Lys-7 and Lys-10
in the interfacial adsorption of App-D49. For K54E and K69Y, the
binding data confirm that their altered substrate selectivity is solely
due to changes in substrate binding. From these kinetic and binding
data, it appears that wild type and mutants all bind to anionic
BLPG-based polymerized mixed liposomes in the same productive mode and
that tighter interfacial binding will lead to higher interfacial
activity of enzyme. A notable exception was E6R which showed 3-fold
increase in binding affinity despite its 4-fold decrease in activity.
For this mutant, the enhanced binding to anionic interfaces is
consistent with an increase in net positive charge in the vicinity of
the critical Lys-7 and Lys-10. Therefore, the decrease in enzyme
activity for this mutant again suggests that it might bind to the BLPG
polymerized mixed liposomes in a suboptimal mode. Finally, we measured
the effect of ionic strength of the medium on the adsorption of wild type and mutants to BLPG polymerized liposomes. As summarized in Table
IV, the binding of wild type was significantly diminished in the
presence of 1 M NaCl, whereas the mutants with lower
interfacial binding affinities were less sensitive to the ionic
strength. In the presence of 1 M NaCl, the wild type
protein and the K7E and K10E mutants showed comparable binding
affinities for BLPG polymerized liposomes. Furthermore, the
n·Kd values for these enzymes were
smaller than that of the K7E/K10E mutant only by an order of magnitude,
demonstrating the electrostatic nature of the interfacial adsorption of
App-D49 to anionic BLPG polymerized liposomes.
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The
three-dimensional structure of App-D49, crystallized in the absence of
calcium ion, shows the structural features typical of other Class II
PLA2s. The catalytic site, calcium-binding apparatus, and
hydrophobic channel are identical to those described previously. Based
on analogy with other PLA2s, the interfacial adsorption surface of App-D49 surrounds the opening of the hydrophobic channel and
includes residues 2 (Leu), 3 (Phe), 6 (Glu), 19 (Met), as well as
portions of the calcium-binding loop. App-D49 is notable for its
relatively large number of cationic residues with a correspondingly high pI (9.5). These charges are arranged asymmetrically with maximal
positive charge potential in the region of the putative interfacial
adsorption surface (Fig. 7B). This
distribution of positive charge potential is in sharp contrast to that
observed for bovine pancreatic PLA2. The residues chosen
for mutation (Glu-6, Lys-7, Lys-10, Lys-11, Lys-16, Lys-54, and Lys-69)
lie on the perimeter of the proposed interfacial adsorption surface
(Fig. 7A). Mutations at these positions do not significantly
affect the tertiary structure as determined by CD. With the exception of Glu-6 and Lys-54, all of the side chains are solvent-exposed lysines
facing the same side of the molecule. Lys-7 and Lys-10 are located on
the same side of the amino-terminal -helix, whereas Lys-11 and
Lys-16 are both located further from the hydrophobic channel with their
side chains remote from the plane of the proposed interfacial
adsorption surface (Fig. 8).
Interfacial Adsorption of App-D49
App-D49 normally shows high
enzymatic activity toward anionic phospholipid aggregates but extremely
low activity toward densely packed zwitterionic phospholipid monolayers
and bilayers (12, 14, 38). As a result, the activation of App-D49 on
zwitterionic aggregates generally requires the formation of anionic
lipid domains containing a reaction product such as a fatty acid (38).
The contributions of Lys-7 and Lys-10 to the free energy of binding of
App-D49 to anionic BLPG polymerized liposomes can be estimated using
the equation G0 = R·T ln (n·Kd
for wild type/n·Kd for mutant). Under
standard conditions with the concentration of free phospholipid set at
1 M, each lysine contributes approximately
1.8 kcal/mol at 25 °C. This calculated value compares well with the known value (
1 to
3 kcal/mol) for electrostatic interactions between two oppositely charged residues in proteins (39) and with our previous mutational experiments with the bovine pancreatic PLA2
(10). Based on these structural and functional data, it is probable that the enzyme's helical surface containing Lys-7 and Lys-10 lies
parallel to the membrane during interfacial adsorption. Since the
contributions of Lys-7 and Lys-10 to the free energy of interfacial binding/adsorption appear additive (
3.7 kcal/mol at 25 °C), there is, at most, limited synergy in the binding of the two lysines to
anionic interfaces (40). The strong electrostatic effect on interfacial
adsorption is supported by the striking correlation between adsorption
and the ionic strength of the medium. Other contributions to the
binding energy presumably come from a number of weaker electrostatic
and hydrophobic interactions (for instance, see Maliwal et
al. (32)). The presence of calcium ion does not appreciably affect
the interfacial adsorption of App-D49, consistent with the minor effect
that an additional calcium ion has on the protein's global
electrostatics (37).
Lys-7 and Lys-10 are clearly the dominant residues directing the
interfacial adsorption of App-D49. This is in contrast to our previous
finding (10) for the bovine pancreatic PLA2 where Lys-56
and Lys-116 are critical residues confirming that the determinants of
interfacial adsorption are specific to the species of PLA2 being studied. Our results also dispute the notion that the interfacial adsorption of secretory PLA2 is driven by a large number of
weak interactions which cannot be specifically blocked. Whether the variability in interface topology translates into orientational differences at the interface remains unclear since hydrophobic groups
also play key roles in optimizing the interaction. The introduction of
an additional charge to the amino-terminal -helix by the E6R
mutation modestly (
G0 =
1.1 kcal/mol at
25 °C) increases the affinity of App-D49 for anionic interfaces.
This enhanced adsorption, however, does not facilitate productive-mode
substrate binding to the active site since E6R has lower activity
toward anionic polymerized mixed liposomes than the wild type. This
reduction in activity may stem from electrostatic repulsion between
Arg-6 and Lys-7 leading to a nonoptimal orientation of the lysine side
chain. Alternatively, the side chain of Arg-6 may mechanically
interfere with the proper orientation of the enzyme at the
interface.
Residues 53 through 58 lie at the
carboxyl terminus of the first anti-parallel -helix. This section of
the enzyme lies under the calcium-binding loop and adjacent to the
region occupied by the head group of productively bound substrate. The
charge and orientation of side chains in this region have been shown to
influence the head group selectivity of PLA2. For example,
Lys-53 of the bovine pancreatic PLA2 (10) and Arg-53 of the
porcine pancreatic PLA2 (41) appear to be responsible for
the anionic phospholipid preferences of these enzymes. Glu-56 of the
Class II human nonpancreatic enzyme makes a direct stabilizing contact
with the ammonium ion of the PE head group in the x-ray structure of
the transition-state analog complex (17). The side chain of App-D49's
Lys-54 points toward bulk solvent and, consistent with this
orientation, its mutation to Glu did not significantly change the
enzyme activity toward PC or PG substrates. The small (2-fold) increase
in activity of K54E toward PE substrates either in polymerized mixed
liposomes or in micelles indicates that the
-carboxylate of Glu-54
may weakly interact with the ethanolamine head group. The calculated decrease in substrate binding energy due to this putative electrostatic interaction is correspondingly small; 0.4 kcal/mol at 25 °C
(
G0 =
R·T ln
((kcat/Km)app for
mutant/(kcat/Km)app for wild type)).
The crystal structures of several transition-state and substrate analog
complexes have shown that both the Tyr-69 of Class I PLA2s
and the Lys-69 of Class II enzymes form a hydrogen bond with the pro-S
nonbridging oxygen of the sn-3 phosphate (17, 29) (see Fig.
5). We suggest that the -ammonium group of Lys-69 may be able to
form additional hydrogen bonds with phospholipid head groups,
especially with PG whose hydroxyl groups can function as hydrogen bond
acceptors (Fig. 5B). Such an interaction would not be
achievable by the phenolic oxygen of Tyr-69 or with PC and PE as
substrate. This would explain why K69Y has the essentially the same
activity as wild type toward PC and PE substrates but shows a 3-fold
drop in activity toward PG substrate. Our recent data indicate that the
Lys-69 of App-D49 and of the human nonpancreatic PLA2 also
interacts favorably with other anionic phospholipids (e.g.
phosphatic acid and phosphatidylserine) that can serve as strong
hydrogen bond acceptors (42). Class II secretory PLA2s presumably show a modest degree of substrate selectivity due to the
ability of Lys-69 to form a hydrogen bond(s) with anionic phospholipid
head groups.