|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 46, 30406-30409, November 13, 1998
From the Department of Chemistry and Biochemistry, University of
South Carolina, Columbia, South Carolina 29208
Acid phosphatase activity in the blood serum is
usually separated into tartrate-resistant and tartrate-refractory,
which is reported as the prostatic acid phosphatase level. Human
prostatic acid phosphatase crystals soaked in
N-propyl-L-tartramate were used to collect
x-ray diffraction data to 2.9 Å resolution under cryogenic conditions.
Positive difference electron density, corresponding to the inhibitor,
was found. The quality of the electron density maps clearly shows the
orientation of the carboxylate and N-propyl-substituted amide groups. The hydroxyl group attached to C3 forms two crucial hydrogen bonds with Arg-79 and His-257. Previous crystallographic studies compiled on the tartrate-rat prostatic acid phosphatase binary
complex (Lindqvist, Y., Schneider, G., and Vihko, P. (1993) J. Biol. Chem. 268, 20744-20746)
erroneously positioned D-tartrate into the active site.
Modeling studies have shown that the C3 hydroxyl group on the
D( Human prostatic acid phosphatase
(PAP)1 has been of
significant medical interest ever since tests screening for serum PAP
levels were successfully used to diagnose and stage prostate cancer
(1). Recently, the primary diagnostic protocol for detecting prostate cancer has shifted from evaluating serum PAP levels to utilizing the
prostate specific antigen test. However, accurately detecting serum PAP
levels is still of considerable interest because of its effectiveness
in staging metastatic prostatic cancer and evaluating the progress of
chemotherapy in prostate cancer patients (2).
PAP, which is produced by the prostate gland, is found in the seminal
fluid at concentrations near 1 mg/ml (3). The enzyme is categorized as
an acid phosphatase, because its optimum pH range is between 4 and 7. PAP belongs to the family of high molecular weight phosphatases. Mature
PAP is active as a glycosylated homodimer with
Mr = ~100,000. The enzyme is capable of
hydrolyzing a wide spectrum of substrates including alkyl, aryl, and
acyl orthophosphate monoesters and phosphorylated proteins (4). The
natural substrate for PAP is uncertain, thus, the discovery of the
specific biological function is of great interest and awaits further investigations.
The catalytic mechanism has been intensely studied and it was concluded
that the enzyme should be classified as a histidine phosphatase (5).
The crucial intermediate is phosphoroamidate, namely phosphohistidine.
The rate-limiting step is the breakdown of this covalent phosphoenzyme
intermediate through addition of a nucleophilic water molecule to
phosphoroamidate with concomitant elimination of inorganic phosphate,
via a SN2 mechanism, to form a noncovalent binary
enzyme-inorganic phosphate complex. Extensive studies of chemically
modified enzyme and a series of site-directed mutants allowed Van Etten
and co-workers (5) to propose a sound description of the catalytic process.
L(+)-Tartrate is a fairly good inhibitor of PAP
(Ki = 2.9 × 10 Nontoxic and specific inhibitors of PAP should be a valuable tool in
the search for its natural substrate(s). Structure-based development of
inhibitors can only proceed by first elucidating the accurate molecular
structure of human-PAP and its complexes with lead inhibitors. We
report here the three-dimensional structure of human PAP complexed with
N-propyl-L-tartramate (NPT), which is a
mono-n-propylamide derivative of L-tartrate, at
2.9 Å resolution.
Crystallization and Data Collection--
Human prostatic acid
phosphatase was purified from semen using tartrate affinity
chromatography as described by Van Etten and Saini (9). NPT was
synthesized from L(+)-tartrate according to procedures
previously described by Van Etten and Saini (9). Crystals of native
human PAP were grown from solutions containing 30% PEG 1500, 7% PEG
1000, 6% PEG 400, 100 mM KCl, and 100 mM glycine at pH 10.0. Typical native crystals grew to 0.2 × 0.15 × 0.15 mm in size. The complex with NPT was prepared by
soaking a native human PAP crystal in artificial mother liquor
containing 34% PEG 1500, 7% PEG 1000, 6% PEG 400, 100 mM
KCl, 100 mM glycine at pH 10.0, and 100 mM NPT
for 3 h. Crystal transfers were performed by using cryoloops
appropriately sized to directly pick up the crystal. The crystals were
orthorhombic, space group P212121, with unit cell dimensions a = 119.9 Å,
b = 202.7 Å, and c = 71.1 Å and
contained two homodimers per asymmetric subunit.
Data for the binary complex were collected by flash freezing the
crystal at 123 K in a stream of nitrogen generated by a Molecular Structure Corp. low temperature X-Stream attachment. A Rigaku rotating
anode source at 50 kV and 100 mA with mirror optics and an R-Axis IV
area detector at a distance of 150 mm were used to collect data as
1o oscillation frames over 180o range yielding
a redundancy factor of 5 and an Rmerge = 13.8%. A total of 224,797 reflections with I > 1 Molecular Modeling--
The coordinates for NPT were generated
in SYBYL using the interactive model building protocol Sketch Molecule
(11). The resultant structure was then subjected to 100 cycles of
energy minimization using the Powell method and a gradient termination. Torsion angles of NPT for atoms O1-C1-C2-C3 ( Map Interpretation and Refinement--
Initial difference
electron density maps for the binary complex were calculated with
X-PLOR (online) with phases based on the refined structure of native
human PAP solved at 3.1 Å resolution. Visual examination of the maps
and model building were done with the interactive graphics program
CHAIN. NPT was manually fitted into the appropriate
Fo Inhibitor Binding in PAP--
Initial difference Fourier maps of
the binary complex, which was phased with the nonliganded native
structure, revealed interpretable positive electron density within the
active site for three of the four monomers present in the asymmetric
subunit. NPT was manually positioned within the observed density as a
rigid body for subunits A, B, and D. The asymmetric nature of the
electron density, because of the mono-n-propyl moiety,
facilitated the correct positioning of the tartrate pharmacophore.
The L-tartrate PAP binary complex reported by Lindqvist
et al. (8) suffered from incorrect modeling of tartrate, due
in part to the symmetrical nature of tartrate and ambiguous electron density. Also, their tartrate model (Protein Data Base access code:
1rpa) corresponds to D-tartrate (2S,
3S) and does not accurately portray the structure of the
used inhibitor (Fig. 2). D-Tartrate does not inhibit PAP although its enantiomer,
L-tartrate (2R, 3R), is a moderate
(Ki = 10
The major difference between modeled D-tartrate and
observed L-tartrate within the active site stems from the
position of the C3 hydroxyl group. In D-tartrate, the C3-OH
group points away from His-257 (distance = 4.57 Å) and lies
underneath and parallel to the plane of the guanidino group of Arg-79
(Fig. 2). Thus, the C3-OH group of D-tartrate is not in a
geometrically favorable position to form strong hydrogen bonds with
either Arg-79 or His-257. In contrast, the C3-OH group of NPT is
positioned in between His-257 and Arg-79 and is in a favorable location
to form hydrogen bonds with His-257 (distance = 2.67 Å), Arg-79
(distance = 2.20 Å) (Fig. 2). Furthermore, the C1 carboxylate
group of NPT is directed away from the carboxylate group of Asp-258 and
into the vicinity of His-12, Arg-11, and Arg-15, thereby decreasing
repulsive electrostatic interactions. Conversely, the C1 carboxylate
group of D-tartrate is directed toward the carboxylate
group of Asp-258 and is in a position to generate unfavorable
stereoelectronic interactions.
The guanidino group of Arg-127, which is thought to participate in the
substrate specificity of PAP, is approximately 11 Å away from the
amide group of NPT and is not in a position to interact with the bound
substrate. The hydroxyl group of Tyr-123 is in a geometrically
favorable position to form a hydrogen bond with the amide oxygen (O41)
of NPT, however, the distance between Tyr-123's hydroxyl group and the
O41 atom of NPT is 4.16 Å, thus denying a strong hydrogen bond. A
bridging water molecule could afford an indirect hydrogen bonding
interaction between Tyr-123 and NPT, however, we did not see electron
density to support this hypothesis.
The N-propyl moiety from the amide portion of NPT is in a
trans geometry typical of trans-peptide bonds observed in
proteins. This geometry extends the propyl group of NPT into a
hydrophobic region formed by Trp-174, Phe-171, Ser-175, and Ile-18. The
additional noncovalent interactions most likely account for the small
decrease in Ki for NPT (Ki = 2.5 × 10 Overall Structure and Tartrate Inhibition--
The overall
topology of human PAP is consistent with rat PAP with the active site
located at the C-terminal end of the
The
The sequence motif of RHGXRXP, which is conserved
in rat and human prostatic acid phosphatases and in human lysosomal,
rat liver lysosomal, yeast, and Escherichia coli acid
phosphatases (20), has similar conformation among PAP (RHGDRSP), SPM
(RHGESEL), and RFB (RHGQSEW) (Fig. 3).
However, the sequence corresponding to the
~XRXP portion in acid phosphatases differs from
those present in glycolytic phosphatases. Arg-15 of PAP, corresponding
to the second arginine in RHGXRXP, is in a
position to donate a single hydrogen bond to the carboxylate group of
L-tartrate in PAP. However, SPM and RFB do not have the
second arginine found in the motif and instead have a serine (Ser-11).
Modeling shows that this serine is too far from L-tartrate
and in either case, Ser-11 of SPM and RFB most likely does not hydrogen
bond to L-tartrate.
Moreover, the conserved proline residue in the sequence motif
RHGXRXP has a significant impact on the
conformational differences observed between PAP, SPM, and RFB. The
absence of proline (Pro-17 (PAP), Leu-13 (RFB), and Trp-13 (SPM)) in
phosphoglycerate mutase and fructose-2,6-bisphosphatase leads to the
formation of a short
In addition to the sequence motif RHGXRXP,
residues Arg-79, His-257, and Asp-258 are involved in binding
L-tartrate. Residues corresponding to Arg-79 and His-257 in
PAP are conserved in RFB (Arg-57 and His-142) and SPM (Arg-59 and
His-181). However, Asp-258, which is conserved in the known acid
phosphatases, is not conserved in the SPM or RFB. Asp-258 in PAP
participates in two hydrogen bonds to L-tartrate. One of
the interactions originates from the main-chain and involves the amide
nitrogen of Asp-258 donating a hydrogen bond to the C1 carboxylate
group of L-tartrate (Asp-258-N ... NPT-O2
distance 3.07 Å). The second involves the side-chain carboxylic acid
oxygen of Asp-258 and the C2 hydroxyl group of L-tartrate
(Asp-258-OD2 ... NPT-O2 distance 2.83 Å). The residues, which
would correspond to Asp-258 in SPM and RFB, are Gly-182 and Gln-143,
respectively. The main chain hydrogen bond to L-tartrate would be conserved because of the similar folds of SPM and RFB, however, the side chain of Gly-182 in SPM is not capable of forming a
hydrogen bond and the side chain of Gln-143 in RFB, which is one carbon
unit longer than Asp-258, would most likely be out of position. Thus, a
strong binding of L-tartrate to SPM and RFB is unlikely,
even though the phosphate binding site of these enzymes is structurally
similar to PAP. Therefore, tartrate resistance is not only a function
of differing topology and active site structure, but also is dependent
upon the residues within structurally similar active sites.
*
This work was supported by the National Science Foundation
(Grants MCB 9604004 and BIR 9419866) and the Department of Energy (Grant DE-FG-95TE00058).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 structure factors (access code 2hpa)
have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, NY.
The abbreviations used are:
PAP, prostatic
acid phosphatase; NPT, N-propyl-L-tartramate; PEG, polyethylene glycol; SPM, S. cerevisiae (yeast)
phosphoglycerate mutase; RFB, rat liver
fructose-2,6-bisphosphatase.
Structural Origins of L(+)-Tartrate Inhibition of
Human Prostatic Acid Phosphatase*
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
)-stereoisomer of tartrate, which does not
significantly inhibit prostatic acid phosphatase, does not form strong
hydrogen bonds with Arg-79 or His-257. The structure of human prostatic
acid phosphatase, noncovalently bound in
N-propyl-L-tartramate, is used to develop
inhibitors with higher specificity and potency than
L(+)-tartrate.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
5 M at
pH 5.0). It is specific for acid phosphatases; in addition to PAP, it
also inhibits homologous lysosomal acid phosphatases and acid
phosphatases isolated from such tissues as liver, bone, and kidney,
which are usually not present in the blood serum (6). The inhibition is
a stereospecific property because D()-tartrate and
mesotartrate are only very poor inhibitors. On the other hand, other
-hydroxycarboxylic acids have been tested, and it was concluded that
the inhibitor must possess an -hydroxyl group in the
D-configuration. The 
-carbon must be part of a carboxyl
group or be attached to a carboxyl or hydroxyl group (7). The
specificity of L(+)-tartrate inhibition was further
investigated by Lindqvist et al. (8) who determined the
crystal structure of rat PAP with noncovalently bound
L(+)-tartrate. Unfortunately, these studies were based on data at 3 Å resolution and yielded only poor density for the ligand. In addition, a wrong stereoisomer of the tartrate model was used.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
(I) was measured and reduced to 39,548 unique
reflections using the HKL suite of programs (10). The data set is
98.5% complete to 2.9 Å resolution and contains 60% reflections with
I > 2(I) in the 2.98-2.88 Å resolution shell.
1),
C1-C2-C3-C4 (2), and C2-C3-C4-O4 (3) were
manually adjusted to 131o ( = 12o),
174o ( = 1.0o), and 118o
( = 12o), respectively, within the graphics program
CHAIN (12). The values indicate the standard deviations calculated
from small molecule data. Initial torsion angle values were based on
the average values observed for L(+)-tartrate using small
molecule crystallography on L(+)-tartrate and obtained from
the Cambridge Structural data base (13).
Fc electron density initially contoured at a 2.3 level. The agreement between the NPT
model and the initial difference density was excellent (Fig. 1). The complex was refined using
simulated annealing, positional (conventional least squares) and
temperature factor refinement with noncrystallographic symmetry
restraints imposed on the four PAP subunits present in the asymmetric
part of the unit cell, but not on the ligands or solvent molecules
(14-16). The final Rfactor is 21.1%
(Rfree = 30.8%) in the resolution range of
8-2.9 Å with root mean square deviations from ideal bond length = 0.014 Å and bond angles = 1.7o.
View larger version (24K):
[in a new window]
Fig. 1.
Stereoview of the positive F-Fmap contoured
at the 2.3 level within the active site of human PAP. The
inhibitor (NPT), which was omitted during initial map calculations, was
modeled into the electron density. The electron density around the C1
carboxyl group is clearly planar when viewed down the axis of the
C1-C2 bond bisecting the O1-C1-O11 angle (orientation not
shown).
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
5 M) and specific
PAP inhibitor (9). Torsion angle measurements for the final tartrate
model reported by Lindqvist et al. (8) were 147o
(
= 82o), 83o ( = 91o) and
18o ( = 224o) for 1,
2, and 3, respectively, with indicating angular deviation from average small molecule values
calculated for L-tartrate. Our final tartrate model in
subunit A has torsion angles of 125o ( = 6o) 178o ( = 8o) and
134o ( = 16o) corresponding to
1, 2, and 3, respectively.
Average torsion angle values and sample standard deviations
(s) for all three NPT molecules included in the final model
are 1 = 123o (s = 3o) 2 = 178o (s = 0o), and 3 = 135o
(s = 3o). Although the torsion angles of
NPT deviate minimally outside the range calculated for small
molecule data on L-tartrate, both NPT and
L-tartrate have a similar conformational profile. In both
cases, 1 and 3 are mobile whereas
2 remains conformationally rigid. This suggests that the
hydroxyl groups attached to C2 and C3 in NPT and L-tartrate
remain relatively rigid, whereas the carboxylate groups adjust to
optimize intermolecular interactions.
View larger version (17K):
[in a new window]
Fig. 2.
Superposition of the rat PAP-tartrate complex
(thin lines) onto the human PAP-NPT complex (residues
labeled) shows that the C3 hydroxyl group in L-tartrate
(TAR1345) is positioned to form strong hydrogen bonds with Arg-79 and
His-257, whereas the C3 hydroxyl group in D-tartrate
(TAR343) is not. Substrate-protein hydrogen bonding interactions
in subunit A of human PAP:
Arg-15N 
h-TAR1345O1 (2.30 Å),
His-12N
2-TAR1345O1 (2.64 Å),
Asp-258N-TAR1345O11 (2.92 Å),
Arg-11N-TAR1345O11 (2.81 Å),
Arg-79Nh4-TAR1345O3 (2.20 Å),
His257N
1-TAR1345O3 (2.67 Å).
5 M) versus that of
L-tartrate (Ki = 2.9 × 105 M) (9). The NPT-PAP binary complex
provides a good lead inhibitor enzyme model to initiate structure-based
inhibitor design.
-sheet in the / domain
(17). Furthermore, human PAP does not experience a significant
structural change upon binding the inhibitor. Superposition of the
-carbons from a single subunit of native human PAP, vanadate-rat-PAP and tartrate-rat-PAP complexes onto the -carbons of human-PAP-NPT complex yielded root mean square deviations of 0.384 Å, 0.786 Å, and
0.714 Å, respectively. Most of the structural differences in the
active site occurred among arginine and histidine residues and involved
positional shifts of the side groups to accommodate optimal hydrogen
bonding to the inhibitor.
/ domain of human PAP has a similar fold compared with the
/ domains found in Saccharomyces cerevisiae (yeast)
phosphoglycerate mutase (SPM) (Protein Data Bank access code: 4pgm) and
the recombinant C-terminal (bisphosphatase activity) domain of rat liver fructose-2,6-bisphosphatase (RFB) (Protein Data Bank access code:
1fbt) (18, 19). Superposition of / domains of SPM and RFB onto
the / domain of human PAP, utilizing 123 and 121 equivalence
points (CA atoms), respectively, yielded corresponding root mean square
deviations of 2.85 Å and 3.14 Å.
View larger version (15K):
[in a new window]
Fig. 3.
A superposition of the active sites from
human PAP (residues labeled), SPM (single lines), and RFB (double
lines) shown with the PAP inhibitor NPT (in center).
-helix-coil loop structure. The loop narrows
the active site and functions to impart specificity for small molecule
phosphate esters such as phosphoglycerate and fructose-2,6-bisphosphate (18). Conversely, Pro-17 in PAP does not allow for the formation an
-helix-coil loop structure to close in on the active site. Instead,
the large random coil loop structure, which is structurally conserved
in rat PAP, extends downward into the -domain causing the active
site to be open and accessible to large substrates such as
phosphorylated proteins.
FOOTNOTES
To whom correspondence should be addressed. Tel.: 803-777-2140;
Fax: 803-777-9521; E-mail: lebioda{at}psc.sc.edu.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Wang, Z. Wei, Q. Bian, Z. Cheng, M. Wan, L. Liu, and W. Gong Crystal Structure of Human Bisphosphoglycerate Mutase J. Biol. Chem., September 10, 2004; 279(37): 39132 - 39138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Shakarian, M. B. Joshi, E. Ghedin, and D. M. Dwyer Molecular Dissection of the Functional Domains of a Unique, Tartrate-resistant, Surface Membrane Acid Phosphatase in the Primitive Human Pathogen Leishmania donovani J. Biol. Chem., May 10, 2002; 277(20): 17994 - 18001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-Q. Zhang, M.-S. Lee, S. Zelivianski, and M.-F. Lin Characterization of a Prostate-specific Tyrosine Phosphatase by Mutagenesis and Expression in Human Prostate Cancer Cells J. Biol. Chem., January 19, 2001; 276(4): 2544 - 2550. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |