From the Département d'Ingénierie et
d'Etudes des Protéines (DIEP), Commissariat à l'Energie
Atomique, C. E. Saclay, 91191 Gif-sur-Yvette Cedex, France,
¶ Department of Immunology, Umeå University Sweden, and
Laboratory of Molecular Recognition, The Babraham Institute,
Babraham, Cambridge CB2 4AT, United Kingdom
Received for publication, October 10, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Human placental alkaline phosphatase (PLAP) is
one of three tissue-specific human APs extensively studied because of
its ectopic expression in tumors. The crystal structure, determined at
1.8-Å resolution, reveals that during evolution, only the overall
features of the enzyme have been conserved with respect to
Escherichia coli. The surface is deeply mutated with 8%
residues in common, and in the active site, only residues strictly
necessary to perform the catalysis have been preserved. Additional
structural elements aid an understanding of the allosteric property
that is specific for the mammalian enzyme (Hoylaerts, M. F.,
Manes, T., and Millán, J. L. (1997) J. Biol.
Chem. 272, 22781-22787). Allostery is probably favored by the
quality of the dimer interface, by a long N-terminal Alkaline phosphatases (E.C.3.1.3.1)
(APs)1 form a large family of
dimeric enzymes common to all organisms. In man, three out of four AP
isozymes are tissue-specific, one is placental (PLAP), the second is
from germ cell (GCAP), and the third is intestinal. They are 90-98%
homologous, and their genes are clustered on the same chromosome. The
fourth (NSAP) is nonspecific and can be found in bone, liver, and
kidney. It is about 50% identical with the other three, and its gene
is located on another chromosome (1-3). Like the other mammalian
phosphatases, PLAP is post-translationally modified. This includes two
N-glycosylation sites and a glycosylphosphatidylinositol anchor with which these APs are tethered to the membrane. The release
of the enzyme may occur through the action of
phosphatidylinositol-specific phospholipase C.
Irrespective of whether they are of mammalian or bacterial origin, APs
catalyze the hydrolysis of phosphomonoesters (4) with release of
inorganic phosphate and alcohol. PLAP has only low sequence identity
with the Escherichia coli enzyme (30%), but residues
involved in the active site of the enzyme (Ser-92, Asp-91, Arg-166) and
the ligands coordinating the two zinc atoms and the magnesium ion are
conserved. Therefore, the catalytic mechanism, which was deduced from
the structure of the E. coli AP (5), was proposed to be
similar in eukaryotic APs. In E. coli, the mechanism
involves the activation of the catalytic serine by a zinc atom, the
formation of a covalent phosphoseryl intermediate, the hydrolysis of
the phosphoseryl by a water molecule activated by a second zinc atom,
and the release of the phosphate product or its transfer to a phosphate
acceptor (6). However, as judged from various studies using mammalian
APs, PLAP presents novel catalytic characteristics. It is an allosteric
enzyme (7), and it is uncompetitively inhibited by some
L-amino acids, namely L-Phe, L-Trp,
and L-Leu (8-10).
The tissue specificity of the human enzymes has been correlated to an
additional function (3). In NSAP, this specificity involves binding to
collagen (11, 12). In PLAP, evidence has accumulated over the past few
years suggesting a role in cell division in both normal and transformed
cells (13-16). This role probably occurs through its phosphatase
activity against phosphorylated proteins (14, 17). In addition, it has
been suggested that PLAP may be involved in the transfer of maternal
IgG to the fetus (18-20).
PLAP is one of the first proteins found to be ectopically expressed by
cancer cells, leading to the concept that dysregulation of embryonic
genes plays a significant role in the cancer process (21). During the
last 30 years, many clinical reports have been published concerning
PLAP and its use as a tumor marker. However, the results are often
controversial, mainly because the assays cannot distinguish between the
closely related tumor markers GCAP and PLAP (3). The problem of
cross-reactivity in the assays reveals that there is a need to
elucidate the three-dimensional structure of at least one human AP.
PLAP is a good representative of all human APs because of the high
sequence homology between these enzymes. The knowledge of its
three-dimensional structure is crucial to elucidating the distinctive
properties of eukaryotic APs, which cannot be predicted with confidence
by analyzing the bacterial AP structure. These properties include the
uncompetitive mechanism of inhibition, the allosteric property related
to catalytic activity, and the elements related to tissue specificity.
In addition, the location of the residues specific to PLAP may help in
the design of new diagnostic tools to eliminate the cross-reactivity
observed in the current ones. We present here the crystal structure of
PLAP at 1.8-Å resolution and a number of structural elements that may explain the specific properties of PLAP and of other mammalian APs.
Purification--
PLAP was purified according to the method
described by Holmgren et al. (22). The frozen placenta, SS
homozygote phenotype, was slowly thawed and cleaned from blood and
membranes and cut into pieces. Phosphate-buffered saline in the same
amount as the weight of the pieces was added, and the mixture was
homogenized. An equal volume of n-butanol was added, and
PLAP was extracted by stirring for 30 min. After centrifugation at
7000 × g for 30 min, the upper butanol phase was
removed, and the aqueous phase was heat-inactivated for 30 min at
56 °C. After a similar centrifugation and filtration, acetone was
added (the same volume as the aqueous phase), and the mixture was
stored at Crystallization--
The protein was concentrated to 10-16
mg/ml in 10 mM Tris, 2 mM MgCl2,
0.02% NaN3, pH 7.0, using an Amicon Centricon 30. Crystallization screening and growth of crystals were carried out using
sitting drop vapor diffusion plates (23). Crystals were obtained from 12% polyethylene glycol 4,000, 2 mM zinc acetate
(essential), 200 mM imidazole malate, pH 5.5-6.5. Other
buffers such as acetate, citrate, and cacodylate gave similar results.
Magnesium is not needed in the reservoir solution because 2 mM MgCl2 is present in the storage buffer.
Large crystals were first grown in the presence of 10 mM
p-nitrophenyl phosphate, which is a substrate used in
colorimetric reactions that is hydrolyzed by PLAP, the solution turning
yellow on mixing with the protein solution. The solubility of the PLAP
substrate/product complex increases with increasing concentrations of
p-nitrophenyl phosphate, whereas the addition of the
product, phosphate, augments precipitation. Solubility variations are
also observed from preparation to preparation, possibly reflecting
variations in the amount and type of glycosylation. Higher solubility
preparations correlate with poor quality crystals or difficulty in
crystallization, suggesting that the presence of longer sugar chains
hinders crystallization. PLAP (0.17 mM) and
p-nitrophenyl phosphate (45 mM) were mixed in
the volumetric ratio 4:1 before setting up the drops. The reservoir
solution consisted of 14% polyethylene glycol 4,000 to 20%
polyethylene glycol 3,350, 100 mM sodium cacodylate, 2 mM zinc acetate, pH 6.5. The crystals grew as long needles
with well defined pyramidal ends, 0.05 × 0.05 mm in
cross-section, often as long as the whole length of the drop.
Cryogenic and Heavy Atom Soaking--
Screening for
cryo-protection conditions was carried out for crystals to be analyzed
with synchrotron radiation. To make up the cryo-protectant, glycerol
was added to the working solution with 0-15% xylitol. A few crystals
were added to this solution. If the crystals dissolved, extra
precipitant was added until the crystals were stable for a prolonged
period of time (at least 20 min). Crystals were manipulated in the
cryo-solution using loops. CryoLoops, mounting pins, and crystal caps
were purchased from Hampton Research (Laguna Hills, Ca). Heavy atom
soaking was carried out using 75-300 µl of soaking solutions in
depression plates.
X-ray Data Collection--
A first crystal was shock-cooled at
80 K and used to collect a native data set at 1.9-Å resolution. After
a short soaking (20 min) in a solution containing 3 mM
p-chlormercuri benzene sulphonate (PCMBS) in cryo-solution, a
second crystal was shock-cooled at 80 K. One set of data was collected
at 1.7-Å resolution. Both data sets were collected at
Laboratoire pour l'Utilisation du Rayonnement
Elecrromagnetique, station D41, on a Mar Research image-plate area
detector. Data processing and internal scaling were carried out with
HKL (24) (Table I). The space-group
determination was performed using the autoindexation procedure as
implemented in Denzo, and the enantiomer determination was performed by
observation of the reflections extinction in the output file. The
crystal belongs to the monoclinic space group C2221 with
a = 88.8 Å, b = 114.5 Å, c = 106.9 Å. There is one
monomer per asymmetric unit with a Vm of 2.74, corresponding to
50% solvent in the unit cell.
Structure Resolution--
The structure was solved by molecular
replacement, using the program AMoRe (25), and the coordinates
of one monomer of the wild type E. coli alkaline phosphatase
(ECAP) (entry code 1alk) (5) and the native data set. The best
solution obtained was in the resolution shell of 8.0 to 3.5 Å, with a
weak correlation coefficient (18.1) and a poor contrast between the
first and second solution (Table I). However, when applying the
rotation matrix and translation to the coordinates file, we observed
that the dimer was recreated along a crystallographic 2-fold axis,
which validated this solution as the correct one.
Model Building and Refinement--
The re-building of the model
was limited by the low phasing power of the ECAP model due to the low
(30%) sequence identity between the two APs. Standard sequence
alignment programs gave misleading results and uninterpretable maps.
The method of hydrophobic cluster analysis as coded in the program HCA
(26) was applied to both ECAP and PLAP sequences. The subsequent
hydrophobic patch alignment resulted in a corrected sequence alignment
between the two. The starting model contained most of the ECAP main
chain without loops or insertions, leaving only the side chains for the
conserved residues. The program WARP version 5.0 plus the routine Side
Dock, kindly provided by A. Perrakis (27), was crucial in the
rebuilding of the model as well as the data set collected at 1.7-Å
resolution. The final PLAP model could then be re-built rapidly. In the
active site, the peak height in the Fo Overall Structure--
The overall structure of PLAP is a dimer;
each monomer contains 479 residues, 4 metal atoms, 1 phosphate ion, and
603 water molecules. The two monomers are related by a 2-fold
crystallographic axis. The surface of PLAP is poorly conserved with
that of the E. coli enzyme, with only 8% residues in
common, although the core is preserved. PLAP possesses additional
secondary structure elements comprising an N-terminal
Half of the enzyme surface corresponds to three clearly identifiable
regions whose sequences largely vary among human APs and are lacking in
nonmammalian enzymes (Fig. 3). The first
is the long N-terminal Allosteric Process--
One important property that differentiates
mammalian APs from their bacterial counterpart is the allostery
observed when the enzyme is fully metalized. Mutagenesis studies
reveal that stability and catalytic properties of each monomer are
controlled by the conformation of the second AP subunit (7). The
principle of allostery implies that the binding of the substrate in one
active site will affect the second binding site. The crystal form
studied here requires the presence of the substrate
p-nitrophenyl phosphate to grow. Therefore, the protein
structure described corresponds to a conformation of the enzyme
activated by substrate.
A second item to consider is the hydrophobic character of the dimer
interface of PLAP. Less than 30% of the residues are involved in
hydrogen-bonding interactions, which confer flexibility on the
interface. The surface buried in the dimer interface is slightly larger
in PLAP (4150 Å2) than in ECAP (3900 Å2).
However, the residues involved in dimerization are mainly different, as
is the nature of the interface. In ECAP, 36 out of 82 total residues at
the dimer interface are involved in hydrogen bonds, whereas in PLAP
only 24 out of 83 residues are involved in hydrogen bonding
interactions, and only 9 of these are conserved between the two. It
could be speculated that in PLAP many hydrogen bond interactions have
been replaced by less specific van der Waals contacts, which are more
likely to allow rearrangement of the two monomers.
Biochemical studies performed on PLAP showed that residues Asn-84,
Tyr-367, and Glu-429 are involved in the allostery (7). Our structure
brings additional information concerning the precise role of these
residues. First, Asn-84 is located at the dimer interface with its
In their study, Hoylaerts et al. (7) report that
the mutation E429G affects the affinity of Zn1 for PLAP (7). Glu-429 is solvent-accessible and located at the entrance of the cleft, which leads to the active site where the O
Finally, the N-terminal Specific Uncompetitive Inhibition--
A second kinetic property
of human APs, not shared by their bacterial ancestors, is that they are
stereospecifically inhibited by L-amino acids through an
uncompetitive mechanism. This type of rare inhibition implies that the
inhibitor can bind to the enzyme-substrate complex but not to the
enzyme alone. In the case of a two-step mechanism like that of PLAP,
the complex can be the initial enzyme-substrate or any of the
intermediate complexes. Previous studies on PLAP have shown that the
molecular mechanism of this inhibition involves Arg-166 and Glu-429 and
suggests the presence of a hydrophobic pocket in the active site.
Glu-429 is of particular importance as it is the only active site
residue specific to PLAP. The corresponding residue is a serine in
intestinal AP, a glycine in GCAP, and a histidine in NSAP (8-10). The
structure confirms the involvement of Arg-166 and Glu-429 and also
allows precise description of the hydrophobic pocket, which probably stabilizes the hydrophobic moiety of the inhibitor. Located at the
entrance of the cleft, which leads to the active site, Glu-429 borders
a pocket that extends from the catalytic Ser-92 to the phosphate
product. Arg-166 is found at the edge of a second pocket around the
active site, also bordered by Phe-107, Gln-108, and Tyr-367 from the
second monomer (Fig. 2). In Fig. 5, we
have modeled the inhibition of PLAP by a L-Phe amino acid.
This model shows that the geometry of Glu-429, Arg-166, and the
hydrophobic pocket is ideal to fit an L-amino acid such as
L-Phe. The hydrophobic moiety of the ligand would be
stabilized in the hydrophobic pocket, its carboxyl group by Arg-166 and
its amide group by Glu-429. This topology matches both the
stereo-specificity and uncompetitive character of the inhibition.
The knowledge of the structural organization of these pockets offers a
molecular basis for the design of more specific inhibitors, which may
be useful in drug therapy when involvement of PLAP in tumor growth is
confirmed. Our study of the complexes of PLAP and L-amino
acids should further clarify the role of these two pockets and the
mechanism of uncompetitive inhibition.
Elements Determining Enzyme Specificity--
Although the target
of each human AP remains unknown, the tissue specificity strongly
suggests an adaptation for these enzymes toward specific proteins. For
example, loop 400-430 of NSAP is directly involved in the binding of
collagen (12). Although the case of PLAP is more controversial, our
structural data allow us to speculate that the enzyme may have a
specific target, whereas the bacterial enzyme has none. First of all,
in the active site itself, there are two pockets that may accommodate
residues from the target protein. Should such a protein bind at this
position, Glu-429 would be ideally positioned to stabilize the ester
moiety of the substrate in the initial step of the catalytic process. This residue is probably a key determinant for substrate selection. In
addition, the hydrophobic pocket extends on one side of the active site
into a large surface of about 1700 Å2, which with the
exception of Asp-173, comprises only neutral or hydrophobic residues
from both monomers. The size and character of this region is similar to
that typically observed in protein-protein interactions (32), and it
would be an ideal location to stabilize a protein substrate. As we move
away from the core of the active site, we notice a large valley with an
accessible surface of 4000 Å2, flanked on each side by the
crown and metal binding domains (Fig. 3). The bottom of this
valley is also lined by the loop containing residues 270-285 from the
metal binding domain. This loop interacts with and may be stabilized by
the putative calcium ion. It could therefore be responsible for the
synergism observed between calcium and PLAP activity by intervening in
substrate binding (15). In summary, the organization of the extended
active site would be consistent with PLAP interacting with a large
substrate. Such a substrate is likely to be a phosphorylated protein.
Comparison with the Other Human Alkaline Phosphatases--
An
important issue in the use of PLAP as a cancer marker is the assessment
of the potential to differentiate it from the other human alkaline
phosphatases present in serum. All mammalian APs have closely related
sequences, conserving all the three specific elements: the four
cysteines involved in the disulfide bridges, the glycosylation sites,
and the residues that coordinate the fourth metal ion. Hence, it is
likely that the same ion and glycosylation sites are present in all
APs. The core is thus largely preserved, and the divergence between the
four proteins is concentrated in the elements of specificity. PLAP and
GCAP are the closest 2, with only 10 substitutions between them (33).
Eight of these positions have sufficient solvent exposure to
differentiate between the surfaces of these two enzymes (Fig.
6). There are 58 substitutions between
PLAP and intestinal AP, corresponding to about 12% of all residues
(Fig. 6). Half of these are situated on the specific elements that
encompass less than one-third of the protein residues. The greatest
diversity exists between PLAP and NSAP, with 209 substitutions (Fig.
6). 56% of the 161 residues in the specific elements are substituted
in NSAP, consistent with their involvement in the mediation of
specificity and modulation of activity. Finally, three epitopic
surfaces distinguish PLAP from the other APs. One corresponds to the
active site pocket in proximity to Glu-429, the second corresponds to
the metal-binding site, and the third corresponds to the region
comprising Glu-15 and Ile-67 (Fig. 6).
Conclusion--
The structure of human PLAP, the first structure
of a eukaryotic example of this family of enzymes, gives some hints
regarding the evolution of this enzyme. Compared with the bacterial
homologues, the catalytic requirement has helped the preservation of
the overall core of the enzyme, the main secondary structure, and the
residues necessary for the catalysis. However, the development of a
placenta in mammals has been accompanied by the emergence of tissue
specificity and the coadaptation of the enzyme to a specific protein
target. The acquisition of allostery and the uncompetitive inhibition imply that this enzyme can be regulated in vivo. These new
properties have led to extensive structural modification both in the
active site and at the enzyme surface, in agreement with the hypothesis that the target of PLAP may be a phosphorylated protein.
-helix from one
monomer that embraces the other one, and similarly by the exchange of a
residue from one monomer in the active site of the other. In the
neighborhood of the catalytic serine, the orientation of Glu-429, a
residue unique to PLAP, and the presence of a hydrophobic pocket close
to the phosphate product, account for the specific uncompetitive
inhibition of PLAP by L-amino acids, consistent with the
acquisition of substrate specificity. The location of the active site
at the bottom of a large valley flanked by an interfacial crown-shaped
domain and a domain containing an extra metal ion on the other side
suggest that the substrate of PLAP could be a specific phosphorylated protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
40 °C for 1 h. After centrifugation at 11000 × g for 40 min, the pellet was dissolved in 10 mM Tris, 2 mM MgCl2, pH 7.0, by
stirring and by dialyzing extensively against the same solution. The
solution was then applied to a DEAE-Sephadex column and washed with
Tris-buffered saline, pH 7.0. PLAP was eluted with a gradient from
Tris-buffered saline to Tris-buffered saline + 0.2 M NaCl;
the A280 and the catalytic phosphatase activity were determined. The
pooled active fractions were dialyzed against 0.1 M glycine
and concentrated by DIAFLO ultrafiltration to 3 ml. This fraction was
applied to a preparative isoelectric focusing column (Ampholytes pH
3-10, Amersham Pharmacia Biotech), eluted 48 h later and assayed
for catalytic activity. The active fractions were pooled and
gel-filtered on a Sephadex G-200 column in Tris-buffered saline.
Data collection, phasing, and refinement statistics
Fc map calculated from the native date set allowed
us to unambiguously model a zinc at M1 and M2 and a magnesium at the M3
position, as in the E. coli structure. The peak
height in the Fo
Fc map
calculated from the PCMBS data was high at M3, but the geometry
was in agreement with a magnesium. In addition, after refinement with a
magnesium at M3, the B-factor of this atom was equivalent to those of
its ligands, and no residual density was observed in the final
Fo
Fc map. The statistics of
the final model as given by XPLOR (36) are summarized in Table
I. PROCHECK (28) was used to analyze the structure geometry. The
three-dimensional structures of PLAP and ECAP were superimposed with
the iterative program ALIGN (29). The coordinates have been deposited
to the PDB at Brookhaven (entry code 1EW2).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-helix
(residues 9-25), forming an arm that embraces the other monomer; an
-helix and a
-strand in a highly divergent region (residues
208-280) and a different organization of the small
-sheet in domain
365-430. PLAP has two glycosylation sites at Asn-122 and Asn-249 that
vary in their degree of glycosylation depending of the placental sample
(Fig. 1). Two disulfide bridges are
located at different positions from those in the E. coli
enzyme. One is close to the first glycosylation site and may rigidify
the loop that carries the carbohydrate chain. The other is located near
to Asp-481, where the glycosylphosphatidylinositol anchor is attached,
and may stabilize the orientation of PLAP relative to the membrane. In
the active site, only the residues that are essential to the catalysis
are preserved, i.e. the catalytic serine, the three metal
ion sites (Zn1, Zn2, and Mg3) and their ligands, whereas most of the
surrounding residues are different (Fig.
2). The upper part of the active site
contains a large number of basic residues, and the lower part contains
mainly aliphatic or hydrophobic residues. Among these hydrophobic
residues, we observe Tyr-367, which belongs to the second monomer. The
consequences of such a pattern of residues are typical for mammalian
APs and will be discussed below.
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Fig. 1.
Electron density for carbohydrates and fourth
metal ion. a and b, glycosylation site
connected to Asn-122 corresponding, respectively, to the reported
1.8-Å structure (a) and to a 2.1-Å data set still under
refinement (b) (Table I). c, carbohydrate
connected to Asn-249 in stacking with Trp-248 and coordination of the
fourth metal ion with Glu-216, Phe-269-CO, Glu-270-O 2, Asp-285, and
one water molecule. In each case, the 2 Fo
Fc map is shown in blue and is contoured
at the 1.2
level. The Fo
Fc
map is shown in green and contoured at 10
level. This
figure was made with TURBO (34).
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Fig. 2.
Active site of PLAP. A
Corey-Pauling-Koltun representation of the active site pocket of PLAP
is shown. The residues conserved with ECAP are represented in
white. The substituted residues are colored according to the
residue type, Phe, Trp, and Tyr are violet; Asn, Cys, Gln,
Met, Ser, and Thr are green; Arg, His, and Lys are
blue; Asp and Glu are red; Ala, Gly, Ile, Leu,
Pro, and Val are yellow. Residues from the second monomer
are pink. This figure was made with TURBO.
-helix described above. The second, an
interfacial "crown domain," is formed by the insertion of a
60-residue segment (366) from each monomer. It consists of two
small interacting
-sheets, each composed of three parallel strands
and surrounded by six large and flexible loops containing a short
-helix. Third, a "metal binding domain," comprised of 76 residues (209) and folded into two
-strands flanked by two
-helices. It includes the glycosylation site at Asn-249, stabilized
by a stacking interaction with Trp-248, and an additional metal ion. In
the Fo
Fc map, calculated from
the native data set, the peak for this metal corresponds to about 10 electrons. Its height and the crystallization conditions suggest the
presence of a magnesium, but its coordination by carboxylates from
Glu-216, Glu-270, and Asp-285 by Phe-269-CO and a water molecule is
also in agreement with a calcium ion (30) (Fig. 1c). The
crown domain and the metal binding domain correspond to novel folds,
since a search for other structurally related domains using the program
DALI (31) yielded no significant matches.
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Fig. 3.
Overall structure. The overall structure
of PLAP is shown in ribbon representation with the residue
side chains of the three extra domains in ball and stick
representation. The monomer I is shown in pale green,
monomer II in blue, N-terminal -helix in red,
crown domain in orange, and metal binding domain in yellow.
This figure was made with MOLMOL (35).
oxygen within hydrogen-bonding distance of Asp91-N (3.2 Å) and
probably intervenes through this residue in the stabilization of the
catalytic Ser-92. On the same loop, Val-85 and Asp-86 interact with
Ala-1 and Ile-2 from the other monomer, and Lys-87 interacts with
Leu-369 and Tyr-367, also from the other monomer. Therefore, Asn-84 may
affect the enzyme activity through its interaction with Asp-91 and may
intervene in the allostery process because of its involvement in the
dimerization (Fig. 4). Second, Tyr-367 is
located at the entrance of the active site of the second monomer. Its
hydroxyl is located 6.1 Å from the phosphate and 3.1 Å from His-432,
which in turn chelates the zinc atom Zn1. The network consisting of
Tyr-367 (monomer1)-His-432 (monomer2)-Zn1 (monomer2)-PO4
(monomer2) might affect the step of phosphoseryl hydrolysis by
modulating the reactivity of Zn1 (Fig. 4). The presence in the active
site of a residue from the second monomer and its probable involvement
in the enzyme reactivity is a new structural feature that could not
have been postulated from the ECAP structure, as the loop containing
residues 366 to 375 in PLAP is an insertion with respect to ECAP.
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Fig. 4.
Residues involved in the allostery.
Ribbon representation of the active site is shown, with
monomer 1 in green, and monomer 2 in magenta. The
network Glu-429-His-320-Zn1 is circled in blue, the
network Tyr-367(#2)-His-432(#1)-Zn1 in yellow, and the
residues involved in the stabilization of the N-terminal from the
second monomer are circled in brown.
1 oxygen interacts with the N
nitrogen of His-320 (distance = 3.4Å). In turn, His-320-N
interacts with Zn1 (distance = 2.4 Å). Therefore, the network consisting of Glu-429-His-320-Zn1-PO4 might also affect the
hydrolysis of the phosphoseryl moiety through the destabilization of
Zn1 (Fig. 4). Therefore, Glu-429 may not be directly involved in the allostery process but probably affects the activity and the metals in the enzyme.
-helix described above is removed from the
rest of its own monomer, but it interacts with the second monomer with
a buried surface area of 555 Å2, suggesting an involvement
in enzyme dimerization. Residues of the N-terminal extremity interact
with Arg-370 from loop 366-375 and with Asn-106 and Arg-117 from the
second monomer. Therefore, the interaction of the N-terminal
-helix
with the second monomer may help stabilize Tyr-367 and, therefore,
affect the enzymatic activity (Fig. 4).
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Fig. 5.
Uncompetitive inhibition. Shown is
modeling of L-Phe uncompetitive inhibitor in the active
site of PLAP. The L-Phe amino acid is in stick
representation colored in yellow. PLAP is in
Corey-Pauling-Koltun representation with one monomer colored in
white and the second colored in pink. Residues of
PLAP interacting with L-Phe are colored in residue type:
acidic in red, basic in blue, neutral in
green, aromatic in violet. The metal ions are
colored in orange. This figure was made with TURBO.
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Fig. 6.
Comparison of human alkaline
phosphatases. a, surface representation of the overall
structure of the dimer of PLAP colored in ivory with the
residues differing from the sequences of GCAP in color. Residues unique
to PLAP are blue, and residues conserved with at least one
other human AP are orange. b, sequence alignment
of PLAP, GCAP, intestinal AP, and NSAP. The first line of the alignment
corresponds to the consensus sequence for human APs. Also included are
residues unique to PLAP, shown as stars highlighted in
blue. Residues that are not conserved with PLAP are
highlighted in green, and residues homologous with PLAP are
highlighted in yellow.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr. J. C. Boulain and Dr. J. B. Charbonnier for fruitful discussion during the course of this study. We also thank Dr. A. Poupon for help in the use of the HCA program and A. Bentley for help in the data collections at LURE station W41.
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FOOTNOTES |
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* Work at the Babraham Institute was supported by a grant from the Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom (to M. J. T.), and work at University of Umeå was supported by Cancerfonden.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1EW2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence should be addressed. E-mail: mhledu@cea.fr (for M. H. L.-D.) or estura{at}cea.fr (for E. A. S.).
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M009250200
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ABBREVIATIONS |
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The abbreviations used are: AP, alkaline phosphatase; PLAP, placental AP; GCAP, germ cell AP; NSAP, non-specific AP; ECAP, E. coli AP.
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