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Structure
of an Acidic Phospholipase A2 from the Venom of Deinagkistrodon
acutus in a New Crystal Form
GU
Li-Chuan, ZHANG Hai-Long , SONG Shi-Ying, ZHOU Yuan-Cong1, LIN
Zheng-Jiong*
(
National Laboratory of Biomacromolecules, Institute of Biophysics, the Chinese
Academy of Sciences, Beijing 100101, China;
1Institute
of Biochemistry and Cell Biology, the Chinese Academy of Sciences, Shanghai 200031, China )
Abstract The three-dimensional structure of an acidic
phospholipase A2 purified from the venom of Deinagkistrodon
acutus (Agkistrodon acutus) was determined in a new crystal form by
molecular replacement at 0.28 nm resolution with a crystallographic R
factor of 21.9% (R-free=25.7%) and reasonable stereochemistry. Being similar to the previous reported
crystal form, a significant
conformational adaptation of segment 14―23 at the
dimer interface was observed. This
segment was related to the “interface recognition site” (IRS).
It was found that a positively charged residue at position 34 seems to
be a common feature for most of hemolytic PLA2s belonging to group
II. Structural comparison between
the two crystal forms showed that NaCl had significant effects on the crystal
packing, thus leading to dramatic
changes of the unit cell parameters.
In the new crystal form,
MPD (2-methyl-2, 4-pentanediol) molecules exist in the hydrophobic
channel of the enzyme.
Key words phospholipase A2; X-ray
crystallography; three-dimensional structure; hemolysis; interfacial catalysis
Phospholipase
A2 (PLA2s,
EC 3.1.1.4) catalyzes the hydrolysis of the fatty acid ester at the sn-2
position of phospholipids in a calcium-dependent reaction. The low molecular mass PLA2s
are classified into two main groups based on sequence and structural
homology. It is well known that in
addition to being a catalyst for the hydrolysis of phospholipids, PLA2 from snake venom
possesses a wide variety of pharmacological activities[1, 2] such as
neurotoxicity,
cardiotoxicity,
myotoxicity, and
hemolytic, anticoagulant and
antiplatelet activities.
It
has long been observed that the hydrolysis rate catalysed by phospholipase A2
increases dramatically while the substrates present as multimolecular
aggregates and the increase depends on the NaCl concentration. Biochemistry research suggested that
NaCl promotes affinity between PLA2 and the phospholipid interface[3].
Some
of PLA2s from snake venom show hemolytic activity, such as the CMS-9 from Naja nigricollis
venom[4] and basic PLA2 from Agkistrodon halys
Pallas[5]. Studies of
the hemolytic mechanism of PLA2 have been done by chemical
modification[6―8]. The first structure of hemolytic PLA2, the crystal structure of the basic PLA2
from Agkistrodon halys Pallas was determined[9]. According to the structure and the fact
that most PLA2s with hemolytic activity are basic PLA2s, Zhao et al.[10] proposed that a basic residue cluster
in C-terminus region might be responsible for the hemolytic activity.
Four
kinds of PLA2 were obtained from the venom of Dienagkistrodon
acutus through gene engineering[11, 12]. All these PLA2 were
characterized and their amino acid sequences were determined using cDNA
method. They were designated as
acidic phospholipase A2I (aPLA2I), acidic phospholipase A2II
(aPLA2II), basic
phospholipase A2 (bPLA2) and Lys49-phospholipase
A2 (Lys49-PLA2) according to their isoelectric
points, which are 4.49, 5.05, 9.49 and 9.3,
respectively. It was shown
that aPLA2I has an inhibiting effect on platelet aggregation and all
phospholipase A2s except aPLA2II have hemolytic
activities. An acidic PLA2
recently purified from the venom of Dienagkistrodon acutus and
characterized by Zhang et al.[13] displays the same isoelectric
point and pharmacological activities as aPLA2I.
This
PLA2 enzyme was crystallized in two crystal forms with the same
space group but different unit cell parameters [P21(I) and P21(II)][13]. In contrast with P21(I)
form, which was grown in solution
containing NaCl, P21(II)
form was grown in the absence of NaCl.
Crystal structure of this enzyme in P21(I) has been
determined and the relationship between structure and activity of inhibiting
platelet aggregation was discussed[14]. In this paper,
we report crystal structure determination of P21(II)
at 0.28 nm resolution and structural comparison with P21(I)
crystal form, and explore the
hemolytic site based on a comparison of the amino acid sequences and the
three-dimensional structures.
1
Materials and Methods
1.1
Crystallization and data collection
The
PLA2 enzyme provided for crystalliztion was extracted from the venom
of Dienagkistrodon acutus (Agkistrodon acutus, collected from Jiangxi Province, China)according to the procedure
outlined previously[13].
Crystals were grown at 18 ℃
by the hanging-drop method with 10 g/L of protein, 0.05 mol/L HEPES buffer (pH 7.5) and 35% MPD in 8 ml
drops equilibrated against 0.5 ml solution of 0.1 mol/L HEPES buffer (pH 7.5)
and 70% MPD. The data were collected
with a Mar345 research area detector using a single crystal. The data were scaled and merged using
the HKL suite[15]. The
crystal belongs to space group P21 with cell dimensions a=4.348
nm, b=7.149 nm, c=4.385 nm and b=116.32°. There are two protein molecules in the
asymmetric unit according to the Matthews coefficient[16]. The data set contains 5 253 unique
reflections with R-merge of 8.4% and completeness of 88.5% (78.6% for
the high resolution shell) at 0.28 nm.
The details of purification,
crystallization and preliminary X-ray analysis of P21(II)
have been published earlier[13].
1.2
Structure determination and refinement.
The
structure was solved by the molecular replacement method using the AMoRe
package[17]. The search
model used in the cross-rotation function calculation was the dimer from the
previously reported P21(I) structure (Dienagkistrodon
acutus aPLA2, 1IJL
in the Brookhaven Protein Data Bank).
The cross-rotation function search was calculated using the data in the
resolution range 0.8―0.4
nm with an integration radius of 1.9 nm.
The top 4 peaks of the cross-rotation function were used to calculate
the translation functions. The
translation function showed that the first peak was the correct solutions judged
by their significance of correlation coefficients. The rigid body refinement of Amore resulted in an R
factor of 39.8% and a correlation value of 47.7%. The initial model, oriented and positioned according to
the molecular replacement solution,
was examined on a Silicon Graphics O2 workstation using the program
FRODO/TURBO, and the molecular
packing in the crystal was confirmed to be reasonable.
The
refinement was carried out using the CNS program[18] with 10% of the
data reserved to calculate the free R factor[19]. A total of 4 511 reflections in the
resolution range 0.8―0.28
nm were used in the refinement.
Model building was carried out using the program FRODO/TURBO. The main-chains and side-chains of the
molecules were adjusted according to the 2Fo-Fc and Fo-Fc
electron density maps. During the
course of the refinement, the R
factor was gradually reduced.
One calcium ion, one
sulfate anion, three MPD
(2-methyl-2, 4-pentan-ediol) molecules and 80 water molecules were included in
the model based on the appearance of corresponding electron densities and their
environments. After several cycles
of atomic position and group B factor refinements and model
rebuilding, R value and R-free
value were converged, the Fo-Fc
map showed no obviously uninterpretable features.
2
Results
2.1
Model quality
The
final model gives an R value of 21.9% and an R-free value of 25.7% in
the resolution range 0.8―0.28
nm. The 2Fo-Fc
electron density is well defined for both the backbone and the side-chains
except for several residues on the molecular surface (Fig.1). The model has good stereochemistry with
r.m.s. deviations from
ideal values being 0.0012 nm for bond lengths, 2.61°
for bond angles and 27.06°
for torsion angles. Calculations
by the program PROCHECK[20] indicated that most of non-glycine and
non-proline residues in the asymmetric unit were located in the most favored
regions (163 residues, 81.5%) or
the additional allowed regions (33 residues, 16.5%), and
only four (2%) in the general allowed regions. The refinement results are listed in Table 1.
Fig.1 The fitting of the proline riched
C-terminus of molecule A with 2Fo-Fc electron density map
contoured at the 1.0 s
level
2.2
Dimer structure
The
sequence of the PLA2 enzyme has four different residues (Ala102, Ala103, Pro131 and Pro133)
and one inserted residue (Pro122) in comparison with that of the
aPLA2I. The final model
consists of all (1 912) non-H protein atoms from two crystallographically
independent molecules, one calcium
ion, one sulfate anion, three MPD molecules and 80 water molecules. All these atoms occupied 56.8% of the
unit cell volume.
Like
P21(I), the two
molecules (A and B) in the asymmetric unit form a dimer with approximate
two-fold symmetry and the dimer is stabillized by large amounts of hydrophobic
interactions formed by residues Leu2, Ile3,
Ile9, Met19, Phe20, Trp21, Ala24, Trp31 and Trp119
and five intermolecular hydrogen bonds.
The surface area buried by the formation of the dimer is 9.05 nm2 (program CCP4), corresponding to 14.6% of the monomer
surface area, a value close to
that of P21(I) as a typical dimer[21].
The superposition of the dimer in P21(II)
and P21(I) gives an r.m.s. deviation of 0.09 nm for all the Ca
atoms. This shows the overall
similarity between the two dimers from different crystal forms (Fig.2). If the molecules A from the two forms
were superimposed, the two
molecules B would deviate with each other only slightly, resulting from minor structural
differences in two segments around b-wing
and C-terminal ridge, i.e. 76―93, 124―133
for molecule A and 74―90, 124―134
for molecule B (r.m.s.
deviation>0.1 nm for the Ca
atoms). The comparison between the
two crystal forms of the enzyme is important as it indicates the stability of
the dimer structures.
Fig.2 Superposition of Ca
traces of dimers form different crystal forms, P21(II) (thick
line) and P21(I) (thin line)
The two molecules in
the asymmetric unit of P21(II) greatly differ from each other
at some local regions,
particularly the segment consisting of residues 14―23
(0.825 nm for Ca
atom at residue 19), located at
the dimer interface. In molecule
A, the segment includes a short a-helix
(17 to 22), while in molecule B it
shows an unusual irregular turn conformation. This observation is consistent with that previously reported
for P21(I)[14]. The different conformation of segment 14―23
seen in P21(I) is present once again in this new crystal
form. Since segment 14―23
involves several residues at the “interface
recognizing site”
(IRS)[22, 23], that the
segment conformation can adapt when it binds to a contacted molecule implies
the flexibility of the IRS as suggested previously[14]. This work reinforces this
viewpoint. The conformational
flexibility of the IRS may be required by the “allosteric
activity”
of the enzyme-oil/water interface interaction.
2.3
Positively charged residue 34 and hemolytic activity
Dienagkistrodon acutus
aPLA2I is the only known member of acidic PLA2s which
exhibits hemolytic activity.
Careful inspection shows that aPLA2I does not contain as many
basic residues at putative hemolytic site as proposed for Agkistrodon halys
Pallas bPLA2. This implies that not all of the basic residues in the
cluster are responsible for the hemolytic activity and only a few residues out
of this cluster may be pivotal in exerting the hemolytic activity.
The amino acid
sequence comparison of hemolytic group II PLA2 with non-hemolytic
group II PLA2 reveals distinct difference at the residue 34 (Table
2). At this position, all of hemolytic group II PLA2
enzymes have a basic residue and all non-hemolytic group II PLA2s
have non-basic residues. His34 of Dienagkistrodon
acutus aPLA2 points out of the molecular surface.
Table 2 The N-terminus amino acid sequence comparison
of the group II PLA2,
showing characteristic differences between hemolytic and non-hemolytic
PLA2
The top five enzymes are all
hemolysins, and the remaining are
non-hemolytic. It is very clear
that all of the PLA2s with hemolytic activity possess a basic
residue at 34 position. The blanks
in the sequences are based on a homology numbering scheme in the large family
of PLA2 molecules,
derived from the bovine pancreatic PLA2 sequence[24―26].
The suggestion that
the basic residues at position 34 may play a pivotal role in the hemolytic
activity was supported by point mutational study of Agkistrodon halys
Pallas bPLA2, in which
when the positively charged Arg34 was changed to a negatively
charged residue or a neutral residue,
the hemolytic activity was significantly decreased (see accom-panying
paper[36]).
2.4
Ca2+ ions and MPD molecules
Calcium is an
essential cofactor for the catalytic activity of phospholipase A2.
In the present structure, calcium
ion is missing in the molecule B,
but evidently occupies the expected position around the calcium binding
loop of molecule A, although we
did not add Ca2+ to the crystallization solution. A structural comparison of the calcium
binding loops for both molecules shows no remarkable difference in their
overall conformation. This implies
that the presence of a calcium ion does not influence the loop structure
significantly.
Deinagkistrodon acutus
aPLA2 was crystallized in P21(II) from MPD with
high concentration (35%). Three
MPD molecules are visible in the structure. Two MPD molecules (MPD 1 and MPD 2) are located at the
bottom of the hydrophobic channel leading to the active site of molecule
A. The other one is positioned at
the hydrophobic channel of molecule B.
The MPD molecules bind in the hydrophobic channel mainly through
hydrophobic contacts. Hydrogen
bonds to the protein molecules also contribute to these interactions. One MPD molecule in molecule A seems to
be coordinated by the calcium ion and being more stable than the other two MPD
molecules (Fig.3).
Fig.3 Two MPD molecules (MPD 1 and MPD 2)
located at the bottom of the hydrophobic channel (molecule A)
MPD
1 is coordinated by the calcium ion.
2.5
Crystal packing
Both crystal forms of Deinagkistrodon
acutus aPLA2 belong to P21 space group, however their unit cell parameters are considerably
different. The unit cell
parameters of P21(II) (a=4.348 nm, b=7.149 nm, c=4.385 nm and b=116.32°)
are in contrast with those of P21(I) (a=4.871 nm, b=3.800 nm, c=6.990 nm and b=99.35°). In the two crystal forms, the dimer structures are similar, whereas
the patterns of the dimer packing differ greatly.
Fig.4 represents dimer
packing of the two crystal forms.
In the unit cell of P21(I), the two dimers extend along the c axis and the two non-crystallographic two-fold
axes of the two dimers run parallel with each other and deviates 13.1°
from c axis. While in P21(II)
the two dimers extend along the b axis, the two-fold axes of the two dimer are approximately
perpendicular to each other and one of them deviates 14.4°
from b axis.
Fig.4 Comparison of the two dimer packing
(A) P21(I), the non-crystallographic two-fold axes
of the dimers run parallel to each other.
(B) P21(II),
the non-crystallographic two-fold axes of the two dimers are
approximately perpendicular to each other.
In P21
(I) form, one Zn2+ ion was found to connect two His34
residues from two adjacent dimers while in P21(II) form, such
metal-mediated interactions are absent.
The different crystal
packing leads to different dimer-dimer contact regions in the two crystal forms. In P21(I), the dimers contact with each other
tightly through jagged
interlock. The molecule A
in a dimer only forms tight interactions(hydrogen bonds and hydrophobic
interactions) with molecule B in a neighboring dimer and vice versa. The contact regions mainly involve
segments 86―91, 113―126, 131―134
of molecule A and 1―5, 114―132
of molecule B. In P21(II), molecule A forms extensive interactions
only with molecule B from adjacent dimers; however,
molecule B has extensive interactions with both molecules B and A from
adjacent dimers. The contact
regions here mainly involve segments 30―43, 71―85, 111―118
of molecule A and 85―92, 130―134
of molecule B.
3
Discussion
It has long been
observed that the increased hydrolysis rate of phospholipase A2 on
substrates presenting as multimolecular aggregates depends on NaCl
concentration. The interfacial
rate enhancement was attributed to three factors: (a) promotion of PLA2 binding by net anionic
charge of the interface; (b)
enhancement of substrate affinity of PLA2 at the interface; and (c) stimulation of the
rate-limiting chemical step[3]. The structural analysis and comparison of the two crystal
forms of Deinagkistrodon acutus aPLA2 correlate with this
viewpoint.
The differences
between the structures of P21(I) and P21(II)
can be summarized into three main points:
calcium ion’s occupancy in the calcium binding loop, the presence or absence of MPD
molecules in the hydrophobic channel and the pattern of dimer’s packing. These differences may reflect the
effect of the presence of NaCl as well as the concentration of MPD.
This work shows the
stability of the dimer of Deinagkistrodon acutus aPLA2 in
different crystallization conditions and the dependence of the dimer packing on
the crystallization conditions.
Careful examination indicates that the intra-dimer interactions of Deinagkistrodon
acutus aPLA2 mainly consist of hydrophobic interactions, whereas the inter-dimer interactions
mainly involve hydrophilic interactions (hydrogen bonds). It implies that the presence of NaCl or
the change of ionic strength has significant influence on the hydrophilic
interactions between dimers,
resulting in different crystal forms.
In P21(I)
form both molecules in the asymmetric unit bind calcium ions, but in P21(II) form
only the molecule A binds a calcium ion.
This suggests that NaCl may improve affinity of PLA2 to its
cofactor. The molecule A has high
catalytic activity during interfacial catalysis. The molecule B with unusual conformation for segment 14―23,
however has low catalytic activity.
The molecule B may bind the calcium ion only in the presence of
NaCl.
Unlike P21(II), there is no MPD molecule in the P21(I)
structure. The MPD molecules
locate at the hydrophobic channel and might mimic the fatty acid chains of a
substrate analogue bound in the active site. The presence of MPD in the hydrophobic channel in one form
but not in the other indicates that NaCl may affect the charge’s environment of
the hydrophobic channel and then affect the affinity of PLA2 to its
substrate.
The influence of NaCl
on the interactions between PLA2 molecules implies that the ion
strength will affect the distribution of charges on the protein surface, and thus affect the interactions
between the enzyme and the oil-water interface.
Acknowledgements We thank Dr. ZHAO Xu-Dong for his help during data
collection and processing.
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Received:
October 22, 2001Accepted: December 6, 2001
This
work was supported by the Chinese Academy of Sciences (No.39970174)
*Corresponding
author: Tel, 86-10-64888513; Fax, 86-10-64877837; e-mail，[email protected]