From the Oxford Centre for Molecular Sciences and the
Dyson Perrins Laboratory, University of Oxford, South Parks Road,
Oxford OX1 3QY, United Kingdom and the
Cellular Physiology
Group, Henry Wellcome Building of Genomic Medicine, Roosevelt
Drive, Oxford OX3 7BN, United Kingdom
Received for publication, November 14, 2002
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ABSTRACT |
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The activity of the transcription factor
hypoxia-inducible factor (HIF) is regulated by
oxygen-dependent hydroxylation. Under normoxic conditions,
hydroxylation of proline residues triggers destruction of its
In hypoxic cells, activation of the
HIF1 transcriptional cascade
directs a series of adaptive responses that enhance oxygen delivery or
limit oxygen demand (1). Activation of HIF in cancer and
ischemic/hypoxic vascular diseases has indicated a central role in
human pathology (1). The transcriptional complex is composed of an
Protein Expression, Purification, and
Crystallization--
FIH, CAD775-826, and
CAD786-826 were prepared as described (10).
Selenomethionine (SeMet) substituted FIH was produced using a metabolic
inhibition protocol and LeMaster media supplemented with 50 mg/liter
L-selenomethionine. SeMet incorporation was >95% by
electrospray ionization-mass spectrometry. Aerobic crystallization of SeMet FIH (at 11 mg/ml) was accomplished by hanging-drop vapor diffusion at 17 °C. The mother liquor consisted of 1.2 M ammonium sulfate, 4% PEG 400 and 0.1 M Hepes pH 7.5. Crystallization of FIH-Fe-CAD fragment
complexes was accomplished under an anaerobic atmosphere of argon in a
Belle Technology glove box (0.3-0.4 ppm O2) using the same
mother liquor and a solution containing FIH (at 11 mg/ml),
Fe2+ (1 mM), 2OG/NOG (2 mM), and
CAD fragment (1 mM). Crystallization of FIH-Zn-CAD fragment
was accomplished aerobically under similar conditions. Peptides were
either synthesized by solid phase peptide synthesis or purchased from
Biopeptide Co. (San Diego, CA).
Crystallographic Data Collection and Structure
Solution--
Crystals were cryocooled by plunging into liquid
nitrogen and x-ray data were collected at 100 K using a nitrogen
stream. Cryoprotection was accomplished by sequential transfer into a solution containing 1.2 M ammonium sulfate, 3% PEG 400, 0.1 M Hepes, pH 7.5, and 10% followed by 24% glycerol. A
three-wavelength multiple anomalous dispersion data set was collected
to 2.9-Å resolution on beamline 14.2 of the Synchrotron Radiation
Source, Daresbury, UK. Data from crystals of FIH-CAD complexes were
collected on beamlines 14.2, 9.6, or 9.5 using ADSC Quantum 4 (14.2 and 9.6) or MarCCD detectors (9.5). All data were processed with MOSFLM and
the CCP4 suite (15). The crystals belonged to space group P41212. The crystallographic
asymmetric unit contains one FIH molecule. Six selenium positions were
located and phases calculated using SOLVE (16). Density modification,
which increased the figure of merit from 0.56 to 0.66, was performed
using RESOLVE (17).
Refinement--
An initial model was built using O (18) and
refined against the SeMet data (remote wavelength) using CNS (19). One
cycle of simulated annealing followed by grouped B-factor
refinement brought the Rfree to 36.2%.
Following further rebuilding and refinement, which brought the
Rfree to 32.3%, the model was transferred to the 2.15-Å data set. Rebuilding and refinement using REFMAC5 (20), including addition of iron, substrate and solvent molecules, and refinement of TLS parameters brought the conventional
R-factor to 17.8% and the Rfree to
21.3%. The following residues are missing in the current model: 1-15
and 304-306 of FIH and 786-794, 807-811, and 824-826 of the CAD
fragment. According to PROCHECK (21) there are no Ramachandran
disallowed residues, and 90.7% of residues have most favorable
backbone conformations. For the CAD peptide, 77.8% of residues are in
the most favorable region with the remaining 22.2% in additionally
allowed regions.
Other structures were solved by molecular replacement using the
coordinates from the 2.15-Å data and refinement using REFMAC5. In all
structures electron density for the iron and 2OG/NOG was visible
throughout refinement. Significant positive difference electron density
was observed between the iron and the CAD Asn803
Figures were prepared with Molscript (22), Bobscript (23), Raster3D
(24), and Pymol (www.pymol.org).
To obtain an FIH-CAD complex without oxidation of the CAD or the
Fe(II), anaerobic conditions were employed to crystallize
FIH in the presence of Fe(II), 2OG, and various CAD
polypeptides from 7-52 residues. Crystals were also obtained
anaerobically for FIH complexed with Fe(II) and the FIH
inhibitor NOG and aerobically for FIH complexed with Zn(II)
and NOG. The structures were solved by molecular replacement using a
model obtained by multiple anomalous dispersion on
selenomethionine-substituted apo-FIH.
Crystalline FIH-CAD complexes were obtained with
CAD786-826 and CAD775-826. Crystallization
attempts with CAD787-806, HIF-2-subunit while hydroxylation of Asn803 in the
C-terminal transactivation domain of HIF-1
(CAD) prevents its
interaction with p300. Here we report crystal structures of the
asparagine hydroxylase (factor-inhibiting HIF, FIH) complexed with
Fe(II), 2-oxoglutarate cosubstrate, and CAD fragments,
which reveal the structural basis of HIF modification. CAD binding to
FIH occurs via an induced fit process at two distinct interaction
sites. At the hydroxylation site CAD adopts a loop conformation,
contrasting with a helical conformation for the same residues when
bound to p300. Asn803 of CAD is buried and precisely
orientated in the active site such that hydroxylation occurs at its
-carbon. Together with structures with the inhibitors
Zn(II) and N-oxaloylglycine, analysis of the
FIH-CAD complexes will assist design of hydroxylase inhibitors
with proangiogenic properties. Conserved structural motifs within FIH
imply it is one of an extended family of Fe(II) oxygenases
involved in gene regulation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
heterodimer, HIF-
being a constitutive nuclear protein that
dimerises with oxygen regulated HIF-
subunits (2). The activity of
the HIF system is regulated by a series of Fe(II) and
2OG-dependent dioxygenases that catalyze hydroxylation of specific HIF-
residues. In normoxia, 4-hydroxylation of human HIF-1
at Pro402 or Pro564 by a set of HIF
prolyl hydroxylase isozymes (PHD1-3) (3, 4) mediates HIF-1
recognition by the von Hippel-Lindau ubiquitin ligase complex
leading to its proteasomal destruction (5-8). In a complementary
mechanism FIH (9) catalyzes hydroxylation of HIF-1
Asn803 (10, 11), which blocks interaction with the
transcriptional coactivator p300 (12, 13). In hypoxia, lack of
hydroxylase activity enables HIF-
to escape destruction and become
transcriptionally active. Inhibition of HIF hydroxylases by
Fe(II) chelators and 2OG analogues activates the HIF
transcriptional cascade even in normoxia (3, 5, 14). The HIF
hydroxylases therefore provide a focus for understanding cellular
responses to hypoxia and a target for therapeutic manipulation. Here we report crystal structures for the HIF asparagine hydroxylase (FIH) alone and complexed with CAD polypeptides, cosubstrates, and inhibitors.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-carbon. Since B-factor differences between FIH and CAD
imply that the CAD is not at 100% occupancy, this may represent an
alternative binding mode for the 20G 1-carboxylate in the absence of
substrate, although it could also be due to a ligating water molecule,
again in the absence of substrate.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
CAD850-862
(equivalent to HIF-1
CAD802-814), and
CAD800-806 did not result in FIH-CAD complexes. Thus, a
CAD peptide of greater than 20 residues was required (see below). Structures 1-3 (Table I) are of the
FIH-CAD complexes, while structure 4 is of FIH in the absence of
CAD. Unless otherwise indicated, the following discussion refers to
structure 1.
Summary of FIH:CAD-fragment complex structures
775-786, and 2OG or NOG (data not shown, since no additional
CAD residues were resolved over the structures with HIF-1
786-826).
r.m.s.d., root mean square deviation.
Analysis of crystallographic symmetry revealed a dimeric form of FIH,
consistent with native gel-electrophoresis analysis (data not shown).
The dimer interface involves the two C-terminal helices of each
molecule in an interlocking arrangement predominantly involving
hydrophobic interactions (Fig. 1,
a and b). This unusual interface buries a surface
area of 3210 Å2, which is large by comparison with other
dimeric proteins of this size (25).
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The core of FIH comprises a double-stranded -helix (DSBH or
jellyroll) motif formed from eight
-strands,
8-
11 and
14-
17 (Fig. 1a) that is characteristic of the 2OG
oxygenase superfamily. Residues 220-259 form an insert between strands
4 (
11) and 5 (
14) of the DSBH. One face of the DSBH is flanked by
an additional four
-strands from the N-terminal region to form an
eight-membered antiparallel
-sheet (Fig. 1a).
Interestingly, the N-terminal strand
1 bisects the face of the DSBH
opposite to the active site (Fig. 1a). The
1 strand has a
360° twist located at a PXXP sequence, in between its
interactions with
14 and
2. A similarly positioned
-strand is
found in most 2OG dependent oxygenases, although not always from the
same region of the protein. The sheet-helix-sheet motif formed by
1,
1, and
2 is conserved in all enzymes of this class except proline
3-hydroxylase. The DSBH topology of FIH defines it as a member of a
superfamily of DSBH proteins, including non iron-containing enzymes
such as the Cu(II) utilizing quercetin 2,3-dioxygenase (26)
and Mn(II) utilizing type II phosphomannose isomerase.
The structures unexpectedly reveal the existence of two distinct
FIH-CAD interaction sites, one involving the hydroxylation site itself
(CAD795-806, site 1) and a second lying to the C-terminal
side of this site (CAD813-822, site 2) (Fig. 2, a and b). The
binding sites involve contact surface areas of 1640 Å2 and
1080 Å2, respectively, and CAD residues in these regions
are highly conserved in all known HIF-1 and HIF-2
sequences.
Kinetic analyses were employed to investigate the relative importance
of sites 1 and 2. CAD fragments shorter than 20 residues are not
efficient in vitro substrates (Table
II). Those containing site 1 only are hydroxylated by FIH2
but less efficiently than those containing both sites, consistent with
the crystallographic data. Electron density for site 1 is of good
quality, with only the side chain of Tyr798 poorly defined,
while that for site 2 is at a lower level and quality, probably
reflecting weaker binding at this site (Fig. 2c).
CAD804-806, and presumably also CAD807-811
for which density was not observed, do not form direct interactions with FIH.
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At site 1, HIF-CAD795-803 residues are bound in a groove
(Fig. 2b) and adopt a largely extended conformation linked to FIH by ten hydrogen bonds. Asn803 of CAD is completely
buried at the active site and lies directly adjacent to the
Fe(II). CAD Asn803 and Ala804 form
a tight turn, stabilized by a hydrogen bond between the backbone
carbonyl of Val802 and NH of Ala804, which
projects the side chain of Asn803 toward the Fe(II). This
side chain is precisely orientated by three hydrogen bonds to enable
hydroxylation at the pro-S position of the -carbon (Fig.
1d), consistent with NMR assignment of hydroxylation at this
site.2 The primary amide of CAD Asn803 is
sandwiched between FIH residue Tyr102 and the
Fe(II). It forms hydrogen bonds with the side chains of FIH
residues Gln239 and Arg238 (Fig.
1d), residues located on the insert to the DSBH motif, rationalizing the unusual selectivity of FIH for asparagine over aspartate (10). Interestingly, the substrate and Fe(II)
binding sites are directly linked, since the backbone nitrogen of CAD
Asn803 also forms a hydrogen bond (~3 Å) with the
carboxylate oxygen of Asp201 that is not complexed to the
iron. Six additional hydrogen bonds stabilize the binding of FIH to
CAD795-801. In contrast with site 1, site 2 is bound on
the FIH surface and involves only two hydrogen bonds (Fig.
2a). CAD816-823 of site 2 form an
-helix, in
exact agreement with the structure of this region in complex with
CBP/p300 (12, 13). As in that complex, the highly conserved
Leu818, Leu819, and Leu822 sit in a
hydrophobic pocket on the surface of FIH (Fig. 2a); it is
not possible for CAD to bind simultaneously to CBP/p300 and FIH.
The extended loop conformation adopted by the CAD residues at site 1 contrasts with the -helical conformation adopted by the same
residues when complexed with the 1st transcriptional adaptor
zinc-binding domain (TAZ1) of CBP/p300 (12, 13). The disordered
structure observed for the CAD, and other HIF-
residues (7), when
free in solution may thus reflect a requirement to adopt more than one
conformation for complex formation with different proteins. The changes
in the conformation of CAD on binding are complemented by changes in
FIH revealing an induced fit binding process. Most strikingly
Trp296 of FIH undergoes a 50° rotation about
C
-C
to accommodate CAD
Val802, while both Tyr102 and
Tyr103 become more ordered. This ordering of FIH on
substrate binding may be reflected in the significant improvement in
resolution for the structures obtained with CAD fragments bound over
those without.
Analysis of the active site reveals that the Fe(II) is
bound in an almost octahedral manner by the side chains of
His199, Asp201, and His279, and the
2-oxo and 1-carboxylate groups of 2OG (Fig. 1c). While bidentate chelation of 2OG with Fe(II) in this structure
was anticipated, the binding interactions for the 2OG 5-carboxylate,
which forms hydrogen bonds with the side chains of Lys214,
Thr196, and Tyr145, are unprecedented. In many
2OG oxygenases Arg and Ser/Thr residues fulfil an analogous role. FIH
is further unusual in that Lys214 is on the fourth DSBH
-strand, whereas previously assigned basic 2OG-5-carboxylate binding
residues are at the beginning of the eighth DSBH strand.
In the FIH-CAD complexes (structures 1-3 in Table I), there is a
vacant position opposite His279 (Fig. 1, c and
d) revealing that the enzyme is primed for dioxygen binding
(27), although there could be rearrangement of the ligands during the
catalytic cycle (28). As with other 2OG oxygenases it is assumed that
following binding of dioxygen, decarboxylation of 2OG yields an
iron-oxo species [Fe(IV) = O Fe(III)-O·] that effects oxidation at the
-carbon of CAD Asn803 (Fig.
3a). Interestingly,
accommodation of a dioxygen ligand (or an alternate 2OG conformation)
opposite His279 may require disruption of the unusual
hydrogen bond between Asp201 and CAD Asn803
(the Fe(II) and Asn803
-carbon are only
~4.9 Å apart) that is observed in the anaerobic enzyme substrate
complex (Fig. 1d). This hydrogen bond may have energetic
consequences for the binding of dioxygen that are relevant to the
oxygen sensing function of the enzyme.
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To understand the mechanisms of action of hydroxylase inhibitors, structures were also obtained for FIH complexed with NOG and with Zn(II). FIH was demonstrated to bind Zn(II) in an identical manner to Fe(II) (structure 3), consistent with the metal-mediated mimic of hypoxia being due to displacement of Fe(II) from the active site of HIF hydroxylases. FIH-CAD structures with NOG reveal that like 2OG it is ligated to Fe(II) in a bidentate manner (Fig. 1c) and imply that it is an inhibitor due to decreased susceptibility to attack by an iron bound (su)peroxide. Kinetic analyses of a series of inhibitors based upon N-oxaloyl amino acids demonstrated that the R-enantiomer (IC50 <0.4 mM) of N-oxaloylalanine was significantly more potent than the S-enantiomer (IC50 2.5 mM). Analysis of the 2OG binding pocket in FIH suggests that the binding of the S-enantiomer is disfavored by interactions with Thr196 and Ile281 in the 2OG binding pocket. Since a reversed selectivity (i.e. the S-enantiomer was more potent) was observed both for procollagen prolyl hydroxylase and the PHD isozymes) (5), it should be possible to develop selective inhibitors for individual types of HIF hydroxylase based on such structural constraints. The unusual and precise structural determinants of both CAD and 2OG binding to FIH may aid inhibitor design via linkage of the 2OG and CAD binding sites and development of heterocyclic compounds that mimic the tight turn adopted by the CAD802-804 when complexed to FIH.
Recognition of post-translational hydroxylation as a major mode of
regulation of the HIF pathway raises an important question as to its
general role in biological signaling. Sequence analyses based on the
FIH structure indicate that the 2OG oxygenase superfamily extends
further than has previously been foreseen (Fig. 3b). Of particular interest are similarities with the JmjC homology region of
the jumonji transcription factors (10, 29). These proteins are
predicted to have a DSBH core and have been implicated in diverse
biological processes such as cell growth and heart development. Conserved HX(D/E) residues had been identified in some JmjC
domains but not assigned as an iron binding motif (29). In the light of
the FIH structure it is clear that many JmjC proteins have conserved
residues including both this motif and the newly defined 2OG
5-carboxylate binding site involving FIH residues Lys214
and Thr196 on the fourth strand of the DSBH (Fig.
3b). The structure therefore implies that FIH is a one of a
large family of iron- and 2OG-dependent oxygenases that are
involved in the regulation of gene expression.
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ACKNOWLEDGEMENTS |
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We thank the staff at beamlines 14.2, 9.6, and 9.5 of the Synchrotron Radiation Source, Daresbury, UK for expert assistance.
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FOOTNOTES |
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* This work was supported by The Wellcome Trust, Medical Research Council, Biochemical and Biophysical Research Council, Engineering and Physical Sciences Research Council, and European Union.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 1H2K, 1H2L, 1H2M, and 1H2N) 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: Oxford Centre for Molecular Sciences, Dyson Perrins Laboratory, South Parks Rd., Oxford OX1 3QY, UK. Tel.: 44-1865-275625; Fax: 44-1865-275674; E-mail: christopher.schofield@chem.ox.ac.uk or kirsty.hewitson{at}chem.ox.ac.uk.
¶ Supported by a German Deutsches Akademischer Austauschdienst fellowship.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.C200644200
2 L. A. McNeill, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
HIF, hypoxia-inducible factor;
CAD, C-terminal transactivation domain;
FIH, factor inhibiting HIF;
2OG, 2-oxoglutarate;
NOG, N-oxaloylglycine;
SeMet, selenomethionine;
PEG, polyethylene
glycol;
DSBH, double-stranded -helix;
CBP, CREB-binding protein
(where CREB is cAMP-response element-binding protein).
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