From the Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Heparan sulfate
N-deacetylase/N-sulfotransferase (HSNST)
catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-Å resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate (PAP). NST1
is approximately spherical with an open cleft, and consists of a single
Heparan sulfate chains are ubiquitous as proteoglycans on cell
surfaces and in the extracellular matrix. They have been increasingly implicated in various biological processes including cell growth, cell
differentiation, blood coagulation, and viral and bacterial infections
(1, 2). Reduced biosynthesis of heparan sulfates, for instance, results
in defective WINGLESS/WNT signaling in Drosophila (3, 4).
Mice lacking the heparan sulfate 2-O-sulfotransferase gene
die neonatally from defective kidney development (5). The recent
crystal structures of the fibroblast growth factor-heparin and
antithrombin-heparin complexes have shown specific protein-sulfate interactions (6, 7). Modification by sulfation thus, can alter
functional specificity and diversity of heparans and heparins.
Heparan sulfation is catalyzed by a group of the Golgi-membrane enzymes
called heparan sulfate sulfotransferases. The superfamily includes also
a large number of cytosolic sulfotransferases that sulfate low
molecular weight substrates such as steroids, bioamines, pharmaceutical
drugs, and environmental chemicals. The membrane and cytosolic
sulfotransferases share little overall sequence similarity, whereas all
sulfotransferases use 3'-phosphoadenosine 5'-phosphosulfate
(PAPS)1 as the ubiquitous
sulfate donor. The crystal structure of the estrogen sulfotransferase
(EST)-PAP-estradiol (E2) complex has revealed the structural motifs for
the 5'- and 3'-phosphate binding of PAP (8, 9). It remains to be
structurally determined whether these motifs are also conserved in
heparan sulfate sulfotransferases. Multiple sequence alignments have
suggested that these motifs may be conserved (9). The reaction
mechanisms and specific substrate binding that lead to the diverse
heparan sulfations are poorly understood.
The bi-functional enzyme HSNST sequentially deacetylates and sulfates
the amino group of the disaccharide glucuronic
acid-N-acetylglucosamine (GlcA-GlcNAc) unit of heparan
sulfate (10, 11). Amino acid sequence alignment of the human HSNST1
with EST identified the N-sulfotransferase domain (NST1)
(12). Human NST1 is similar to the corresponding domain of mouse HSNST
reported by Berninsone and Hirschberg (13). Subsequently, site-directed
mutagenesis has shown that Lys-614 is a critical residue for NST1
catalysis (12). Using NST1 crystals grown as previously reported (12), we now describe herein the crystallographic structure NST1. This structure displays the conserved nature of the structure of the PAP-binding site and identifies possible catalytic residues. In addition, a substrate binding site is suggested from the structure.
Protein Expression, Purification, Crystallization, and Enzyme
Assay--
Selenomethionyl NST1, using a pGEX-4T3-NST1 plasmid, was
expressed in the methionine auxotrophic Escherichia coli
strain B834 (DE3) with a defined minimal essential medium (without
methionine) containing 50 mg of selenomethionine per 1 liter of
culture. The NST1 was then purified, and crystals
(P21212 or P21) were grown under
the same conditions as described previously (14). Heparan sulfate
sulfotransferase activity of NST1 was also measured according to the
previously described procedure (12).
Crystallographic Data Collection and Processing--
Two MAD
data sets of selenomethionyl NST1 were collected at Structure Determination and Refinement--
All data were
processed using SCALEPACK and DENZO (14). Because of heavy ice rings in
data set 1 and data set 2 being weak, F(A)s were calculated with CCP4
(15) using data between 20 and 4 Å of data set 1. Positions for four
of the six selenium atoms were determined using SHELX96 (16). Data set
1 was reprocessed to eliminate all reflection near the ice rings
between 3.95 and 3.1 Å. Subsequently, the reprocessed data set 1 was
merged with data set 2 to obtain a complete data set to 2.85-Å
resolution. SHARP (17) was then used for refinement of the selenium
sites. Solvent flattening and histogram matching were carried out using DM and Solomon from CCP4 (15). In the model building process using O
(18), SigmaA maps were generated by combining the phases from
polyalanine fragments with the MAD phases (15). After multiple cycles
of positional, torsion angle, and b factor refinements using X-PLOR
(19), the R-factor and Rfree were
23.8 and 31.9%, respectively. Because some of the loop regions still
lacked interpretable density, molecular replacement was employed to
determine phases for the data from the P21 crystal.
Multiple cycles of manual rebuilding and refinement using the
P21 data at 2.3 Å reduced the R and
Rfree factors to 20.9 and 25.8%, respectively.
The stereochemistry of the refined model was verified using PROCHECK
(15). Data collection and refinement statistics are summarized in Table
I. The coordinates have been deposited in the Protein Data Bank with
code 1NST.
The overall structure of NST1 is roughly spherical with an open
cleft (Fig. 1A). This
structure is composed of a five-stranded parallel /
fold with a central five-stranded parallel
-sheet and a
three-stranded anti-parallel
-sheet bearing an interstrand disulfide
bond. The structural regions
1,
6,
1,
7, 5'-phosphosulfate binding loop (between
1 and
1), and a random coil (between
8 and
13) constitute the PAP binding site of NST1. The
6 and random coil (between
2 and
2), which form an open cleft near the
5'-phosphate of the PAP molecule, may provide interactions for
substrate binding. The conserved residue Lys-614 is in position to form
a hydrogen bond with the bridge oxygen of the 5'-phosphate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
Results and Discussions
Conclusion
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
Results and Discussions
Conclusion
REFERENCES
180 °C from
two separate single crystals (both P21212) on a
MAR detector at beamline X9B of the NSLS, Brookhaven National
Laboratory. Three wavelengths were selected from the fluorescence
spectra: f1 (0.97163 Å: remote), f2 (0.97907 Å: peak), and f3
(0.97940 Å: edge) (Table I). Native data
of an NST1 crystal (P21) were collected at
180 °C on
an R-axis IV with an RU300 rotating anode generator.
Data collection and refinement statistics
Results and Discussions
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
Results and Discussions
Conclusion
REFERENCES
-sheet (
1,
2,
3,
4, and
5) with
helices on both sides of the
-sheet (Fig. 1B). This fold is similar to EST (the 1.9-Å
r.m.s. deviation for 97 C
s in
1,
3,
4,
5,
1,
6,
11,
12, and
13) as well as to the nucleotide binding motif
observed in nucleotide kinases (20). The loop between
1 and
1
adopts the same PSB-loop configuration as the 5'-phosphate binding site of PAPS. A cavity formed between the PSB-loop, and
6 defines the PAP
binding site. Three
strands (
6,
7, and
8) near the C
terminus form an anti-parallel
-sheet with a single disulfide bond
between
7 and
8. An open cleft that runs perpendicular to the PAP
binding cavity is large enough to contain a hexasaccharide chain. The
6 and random coil between
2 and
2 constitute the cleft near
the 5'-phosphate of PAP and thus may constitute part of the substrate
binding site.
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Fig. 1.
Overall structure and topology of NST1.
A, ribbon representation of the NST1 structure in complex
with PAP. Helices are in yellow, -strands in
green, random coil in blue, disulfide bond in
light blue, and PAP molecule in red.
B, topological representation of the polypeptide fold of
NST1. The residue numbers define the secondary structural elements. The
N-terminal 18 residues (587-600) and 4 residues from 665 to 669 are
disordered in the NST1 crystal. These figures were prepared with SETOR
(23).
The secondary structural elements that comprise the PAP binding site in
NST1 and residues forming specific interactions to the PAP molecule are
depicted (Fig. 2), respectively. The PSB-loop (residues 612-617) and
1 of NST1 constitute the 5'PSB motif and provide the major binding
sites for the 5'-phosphate of the PAP molecule. Backbone amide
nitrogens from PSB-loop residues 614-618 are all within hydrogen
bonding distance of the 5'-phosphate. The side-chain N
of Lys-614
and the O
s of both Thr-617 and Thr-618 are also hydrogen-bonded to
the 5'-phosphate.
6 and
4 are the key elements of the 3'PB motif,
and the O
of Ser-712 from this helix forms a hydrogen bond to the
3'-phosphate of the PAP molecule. The PAP molecule in NST1 is bound in
the same orientation (relative to the PSB-loop) as seen in EST. The PAP
binding site, found in the EST structure determined previously, is
conserved. The r.m.s. deviation (with EST) for 47 C
s in
1,
6,
1,
4, and PSB-loop is 1.16 Å.
The anti-parallel -sheet (
6,
7, and
8) and the following
random coil provide the remaining interactions for the PAP binding site
(Fig. 2). These interactions reveal
diversity in the binding site of NST1. The side-chains of Lys-833 and
Tyr-837 from this random coil are within hydrogen bonding distance to
two oxygen atoms of the 5'-phosphate and the oxygen atom of the
3'-phosphate, respectively. Besides these side-chain interactions, the
backbone nitrogens of Gly-834 and Arg-835 are also within hydrogen
bonding distance of a 3'-phosphate oxygen of the PAP molecule. The
adenine ring from the PAP molecule is in position to form a parallel
ring stacking interaction with Phe-816 of
7. Moreover, the backbone oxygen of Trp-817 is within hydrogen bonding distance to the N-6 of the
adenine. The interactions of these residues with the PAP molecule are
unique features in NST1 that are not present in the crystal structure
of the EST-PAP complex (8).
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Lys-614 of NST1 is known to be conserved in other heparan sulfate
sulfotransferases as well as in all cytosolic sulfotransferase (9, 12).
Although this residue plays a critical role in NST1 activity (12), the
structural basis of its role in catalysis has remained unresolved. The
crystal structure of the EST-PAP-vanadate complex (Fig.
3) has recently been solved and has
provided a possible transition state template for the sulfuryl transfer
reaction (21). Superimposition of NST1 on this structure indicates that
the side-chains of Lys-614 (in NST1) and Lys-48 (in EST) exhibit a
similar orientation and conformation (Fig. 3). N of Lys-614 is found
to be directly coordinated to an oxygen of the PAP molecule in NST1.
This oxygen is also coordinated to Lys-48 (N
) in EST and is
implicated as the bridge oxygen of the leaving phosphate group of PAP
(21). Moreover, the mutation of Lys-614 to Arg gives a variant with a
significant level of NST1 activity (63 ± 7.0 and 9.4 ± 2.4 nmol of sulfate/min/mg of protein in the wild-type and K614R mutant, respectively), whereas the K614A mutation abolishes activity completely (12). These structural and mutational data suggest that Lys-614 may act
as a possible proton donor in catalysis, similar to Lys-48 in EST (21).
Lys-833 of NST1 is also coordinated with the bridge oxygen (Fig. 3).
Lys-833 is, in fact, conserved not only in Caenorhabditis elegans HSNST but also in human heparan sulfate
3-O-sulfotransferase (see the sequence alignments in Shworak
et al. (22)). Thus, Lys-833 and its counterparts may play a
significant role in catalysis.
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In sharp contrast to the hydrophobic pocket of estrogen binding site in
EST, the putative substrate binding site of NST1 appears to be a large
open cleft with a hydrophilic surface, with a random coil (residues
640-647, approximately 12 Å in length) and 6 forming the center of
the cleft near the 5'-phosphate of the PAP molecule. This amphipathic
random coil positions negatively charged side-chains (Glu-641, Glu-642,
Gln-644, and Asn-647) toward the center, whereas the hydrophobic
side-chains (Ile-643, Phe-645, and Phe-656) are buried in the
hydrophobic core of NST1. The side-chains of residues (Trp-713,
His-716, Gln-717, and His-720) in
6 constitute the opposing face of
the cleft. The center of this cleft (approximate dimensions: 12 Å in
length, 8 Å in width, and 8 Å in depth) is large enough to
accommodate a trisaccharide unit of polysaccharide chain. Further
studies, such as the determination of the complex structure of NST1
complexed with polysaccharide, are needed to conclude whether this
center portion of the cleft is, in fact, the substrate binding site.
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Conclusion |
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The striking similarities between the PAP binding orientation in
NST1 and EST provide structural evidence that the Golgi membrane and
cytosolic enzymes belong to the same family of enzymes. The similar
topology and function of Lys-614 to Lys-48 of EST suggest a common
reaction mechanism in all sulfotransferases. Lys-833 may be an
additional catalytic residue not present in the cytosolic enzymes. The
NST1 structure provides an excellent model for investigating the
substrate specificity of heparan sulfate sulfotransferases so that we
may better understand sulfation at specific positions of glucuronic
acid-N-acetylglucosamine.
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ACKNOWLEDGEMENTS |
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We thank Rick Moore for excellent technical assistance, Dr. M. Duffel for supply of PAP, Dr. Z. Dauter for assistance with data collection at NSLS, and Drs. T. Hall and I. Tanaka for helping with MAD phasing. Our sincere appreciation is also acknowledged to Drs. Lee Pedersen, W. Beard, and T. Hall for comments on this manuscript.
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FOOTNOTES |
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* 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 (code 1NST) has been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
JSPS Research Fellow at National Institutes of Health.
§ To whom correspondence should be addressed: Head, Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709. Tel.: 919-541-2404; Fax: 919-541-0696; E-mail: negishi{at}niehs.nih.gov.
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ABBREVIATIONS |
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The abbreviations used are: PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PAP, 3'-phosphoadenosine 5'-phosphate; PSB-loop, 5'-phosphosulfate binding loop; 5'PSB, 5'-phosphosulfate binding motif; 3'PB, 3'-phosphate binding motif; NST1, the sulfotransferase domain of heparan sulfate N-deacetylase/N-sulfotransferase; EST, estrogen sulfotransferase.
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