From the Center for Cellular Switch Protein Structure, Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yuseong-gu, Daejeon 305-806, Korea
Received for publication, October 10, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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The master switch of cellular hypoxia responses,
hypoxia-inducible factor 1 (HIF-1), is hydroxylated by factor
inhibiting HIF-1 (FIH-1) at a conserved asparagine residue under
normoxia, which suppresses transcriptional activity of HIF-1 by
abrogating its interaction with transcription coactivators. Here we
report the crystal structure of human FIH-1 at 2.8-Å resolution.
The structural core of FIH-1 consists of a jellyroll-like
Mammalian cells adapt themselves to low oxygen conditions
(hypoxia) by activating a conserved hypoxic response pathway,
where the transcription factor, hypoxia-inducible factor 1 (HIF-1)1 plays a
major role (1). The protein products of HIF-1-regulated genes are
responsible for angiogenesis, vascular reactivity and remodeling,
glucose and energy metabolism, cell proliferation and survival,
erythropoiesis, and iron metabolism (1). HIF-1 is a basic
helix-loop-helix/Per-Arnt-Sim homology domain protein composed of two
( Two separate domains within HIF-1 Both FIH-1 and HIF-1-PH belong to the 2-oxoglutarate
(2OG)-dependent dioxygenase superfamily (2, 12-14). The
family consists of a variety of enzymes catalyzing hydroxylations,
desaturations, and oxidative ring closures/rearrangements (20). Crystal
structures of the family members have been reported for bacterial
proline 3-hydroxylase and other enzymes responsible for biosynthesis of antibiotics (21-24). Nevertheless, revelation of crystal structures of
FIH-1 and HIF-1-PH is very important in understanding
oxygen-dependent regulation of HIF-1, because these two
enzymes serve as oxygen sensors in the hypoxia response pathway.
Here we report the crystal structure of human FIH-1, one of the two
oxygen sensors. The structural core of FIH-1 consists of a Cloning, Expression, and Purification of FIH-1--
The gene for
FIH-1 was amplified by PCR from human colon cDNA library
(Clontech) using 5'-gga att cca tat ggc ggc gac agc ggc gga gg-3' as a forward primer and 5'-cta tgg atc ctg gca gga ggc
ctt gac ccc-3' as a reverse primer and cloned into
NdeI/BamHI restriction sites of pET-28a vector
(Novagen). The N-terminal His6-tagged FIH-1 fusion protein
containing the full-length of FIH-1 (residues 1-349) was overexpressed
from Escherichia coli BL21(DE3) and purified by nickel
affinity chromatography (Qiagen). After removal of the N-terminal tag
by cleavage with thrombin, FIH-1 was further purified by anion exchange
chromatography using Q-Sepharose. Protein purity was confirmed by
SDS-PAGE, and concentration was determined using Crystallization--
FIH-1 (apo form) was crystallized at
25 °C by the hanging-drop vapor diffusion method. Tetragonal
crystals were obtained in drops containing 1.8 µl of protein
solution (20 mg/ml) and 1.8 µl of reservoir solution (0.4 M lithium sulfate, 20% PEG 4000, 0.1 M Tris,
pH 8.5). The crystals belonged to the P41212
space group with unit cell dimensions of a = b = 86.89 Å and c = 143.42 Å.
Data Collection, Structure Determination, and
Refinement--
Crystals grown from selenomethionyl-derivatized
protein was used for the MAD data collection at the Pohang Accelerator
Laboratory beamline 6B. Data collected at three wavelengths (peak,
edge, and remote) were processed and scaled with the program DENZO and SCALEPACK (25). Seven selenium sites out of eight expected sites were
located by the program SOLVE (26), and heavy atom parameters were
refined by the program SHARP (27). The phases were subsequently improved by solvent-flattening using the program DM (28). The resulting
experimental map was of high quality and allowed us to build the
majority of the residues. Data collected at the edge wavelength were
used in the refinement. The model was built in the program O (29) and
refined with the program CNS (30) in the resolution range of 99 to 2.8 Å. The randomly selected 5% of the data were set aside for the
Rfree calculation. Refinement included an
overall anisotropic B factor and bulk solvent correction. The
Rcryst and the Rfree are
22.8 and 27.5%, respectively (Table I).
The stereochemical analysis using the program PROCHECK (31) showed that
79.2% of the refined residues are in most favored regions and none
belongs to the regions of disallowed conformations. The final model
contains residues 12-349 of FIH-1 and one sulfate ion.
Figs. were prepared by using the programs MOLSCRIPT (32), RASTER3D
(33), BOBSCRIPT (34), RIBBONS (35), and GRASP (36).
Overall Structure--
The overall fold of FIH-1 is
composed of a central
Despite low sequence homology, the overall structure of FIH-1 is
similar to those of other 2OG-dependent oxygenases as
proposed based on the conservation of the signature motif residues (13, 14). In a search for homologous structures by using the Dali server,
seven members of the 2OG-dependent oxygenases including clavaminic acid synthase (CAS), isopenicillin synthase, and
anthocyanidin synthase were identified with similar z-values ranging
from 9.0 to 7.0. Structural similarities are mainly found in the
central Active Site Conformation--
Previous investigations have
demonstrated a strict requirement of FIH-1 for ferrous ion and 2OG (13,
14). At the center of the
Whereas conformations of the facial triad residues are highly conserved
between FIH-1 and other 2OG-dependent dioxygenases, the
putative binding site for 2OG in FIH-1 is substantially different from
that in other 2OG-dependent dioxygenases. In CAS,
5-carboxylate of 2OG is bound to the charged side chain of Arg-293 that
is highly conserved in 2OG-dependent dioxygenases (2, 37).
Arg-281 and Arg-297 also are positioned near the 5-carboxylate,
contributing to the binding of 2OG. However, in FIH-1, those arginines
are replaced with hydrophobic or non-ionic residues such as Thr-290, Ile-281, and Asn-294 (Fig. 2). Instead, side chain of Lys-214 protruding from different secondary structural element (strand The Putative Binding Sites for HIF- Implications for the Regulation of HIF-1 Activity--
The FIH-1
structure suggests a mechanism for the physiological regulation of the
HIF-1 activity by revealing three-dimensional locations of regions in
FIH-1 that are implicated in the interactions with HIF-1
Appropriate hypoxia responses are important in overcoming oxygen and
nutrient deficiency during hypoxic states in ischemic diseases such as
stroke and cardiovascular diseases. The crystal structure of FIH-1
reveals critical information about the active site of the enzyme such
as the wide-opened entrance for the catalytic site and a distinctive
2OG-binding site. These characteristic features would be an aid in
designing specific drugs that can inhibit the enzyme activity and
promote the hypoxia-inducible responses in ischemic diseases. The
prominent groove near the active site, which is likely the HIF-1-barrel containing the conserved ferrous-binding triad residues,
confirming that FIH-1 is a member of the
2-oxoglutarate-dependent dioxygenase family. Except for the
core structure and triad residues, FIH-1 has many structural deviations
from other family members including N- and C-terminal insertions
and various deletions in the middle of the structure. The
ferrous-binding triad region is highly exposed to the solvent, which is
connected to a prominent groove that may bind to a helix near the
hydroxylation site of HIF-1. The structure, which is in a dimeric
state, also reveals the putative von Hippel-Lindau-binding site that is
distinctive to the putative HIF-1-binding site, supporting the
formation of the ternary complex by FIH-1, HIF-1, and von
Hippel-Lindau. The unique environment of the active site and
cofactor-binding region revealed in the structure should allow design
of selective drugs that can be used in ischemic diseases to promote
hypoxia responses.
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ABSTRACT
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and
) subunits. Both the half-life and transactivation function
of HIF-1
are regulated by changes in the cellular oxygen level,
whereas HIF-1
remains mostly unaffected (1).
are responsible for the mechanisms
by which cellular oxygen regulates HIF-1 activity. The first is the
oxygen-dependent degradation (ODD) domain, which is
hydroxylated by a specific proline hydroxylase (HIF-1-PH) (2-6), and
then recognized by the von Hippel-Lindau (VHL) ubiquitin-protein ligase
complex for targeting to the proteasome (7-11). A second hypoxic
switch has recently been identified to operate in the C-terminal
activation domain (CAD), where a conserved asparagine residue is
hydroxylated by factor inhibiting HIF-1 (FIH-1) (12-14). Hydroxylation
of the asparagine residue during normoxia suppresses interaction of CAD
with transcription coactivators (15-19).
-barrel
like other members of the 2OG-dependent dioxygenase family.
In comparison to other family members, the structure of FIH-1 shows
several distinctive features such as a unique cofactor-binding site in
the wide-opened active site pocket, a dimerization domain at the C
terminus, and a long and wide groove at the center of the molecule
ranging from the active site of the enzyme toward the dimerization
domain. The structure-based interpretation of previous biochemical
analyses suggests a mechanism of hypoxia regulation by utilizing a
multicomponent complex made of FIH-1, HIF-1, and VHL.
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280 = 1.69 (mg/ml)
1 cm
1 in 6 M guanidine.
Crystallographic data
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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-barrel surrounded by eight
-helices with
an approximate dimension of 60 × 45 × 35 Å (Fig.
1a). The structure of FIH-1
reveals two discrete domains. Domain I runs from N terminus to residue
300 and is mainly composed of the central
-barrel (sheet 1,
2-
7,
9,
12, and
14; sheet 2,
1,
8,
10,
11,
and
13) and six
-helices (
1-
6) surrounding them. Domain II
(residues 301-349) comprising two consecutive
-helices (
7 and
8) stretches away from domain I. Domain II, which is completely
missing in all known structures of the dioxygenase family, forms strong
interactions with the same region of the 2-fold symmetry related
molecule (Fig. 1b). It is further stabilized by interactions
with helix
6. Dimeric association of FIH-1 revealed by the crystal
structure was confirmed by gel filtration chromatography and dynamic
light scattering experiments (data not shown). Dimeric interface, which
is predominantly composed of hydrophobic interactions, buries 1,516 Å2 that corresponds to 8.7% of the total surface of
the FIH-1 monomer.
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Fig. 1.
Monomeric and dimeric structures of
FIH-1. a, monomeric structure. The schematic ribbon
diagram of the FIH-1 monomer is shown with secondary structural
elements labeled. -Helices and
-strands are colored
red and violet, respectively. Boundaries of the
secondary structural elements are
1 (residues 39-41),
2
(44-45),
3 (63-64),
4 (89-95),
5 (119-124),
6
(143-149),
7 (182-190),
8 (195-200),
9 (204-211),
10
(214-219),
11 (260-265),
12 (270-273),
13 (278-283),
14
(290-297),
1 (50-57),
2 (87-84),
3 (125-138),
4
(156-164),
5 (167-176),
6 (224-227),
7 (312-330), and
8
(336-344). b, dimeric structure. The FIH-1 dimer found
in the crystal is shown as a ribbon representation. Two monomers in the
FIH-1 dimer are in red (Mol A) and yellow (Mol
B), respectively. The region involved in the dimeric association is
indicated with a circle. Helices
6-
8 playing a major
role in the dimerization are labeled in the figure.
-barrel region. When we aligned the structure of FIH-1 with
that of CAS, 103 C
atoms were superimposed with a root mean square deviation of 2.50 Å (Fig. 2). Although
there are good alignments in the region of the central
-barrel
region, the arrangements of
-helices are quite different and cannot
be aligned to each other well. Other differences in the structure of
FIH-1 include the long insertions in N- and C-terminal regions (Fig.
2). Substantial differences are also found in several loops and
-helices in the middle of the protein.
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Fig. 2.
Structure-based sequence alignment of FIH-1
with CAS. Sequences of FIH-1 and CAS were aligned by C -carbon
superposition of the two structures. In the structural superposition,
the initial transformation operator between the two structures was
obtained from the result of the Dali search. The initial alignment was
further refined with the program O by using the default constraints
(less than 3.80 Å deviation for more than three consecutive
C
-carbon atoms). As the result, 103 residues were superposed with an
overall root mean square deviation of 2.50 Å. The superposed residues
are shaded yellow in the figure. The secondary structural
elements of FIH-1 are shown in blue above the
alignment and those of CAS are in red below the alignment.
The facial triad residues (His-199, Asp-201, and His-279 of FIH-1) are
indicated by inverted black triangles. A
black circle below the alignment indicates the
arginine residue (Arg-293 of CAS) involved in coordinating the
5-carboxylate of 2OG in CAS. A lysine residue (Lys-214 of FIH-1)
implicated in the 2OG binding of FIH-1 (see text) is indicated as a
black circle above the alignment.
-barrel structure of FIH-1, there is a big
cavity that is lined with the residues implicated in coordinating a
ferrous ion (His-199, Asp-201, and His-279), termed the
2-His-1-carboxylate facial triad (Fig. 3,
a and b; Fig.
4a). In one side of the facial triad, there is an opening that is spacious enough to accommodate the
cofactor, 2OG. Side chains of the facial triad residues are aligned as
if they were coordinating a ferrous ion, despite that the FIH-1
structure is in apo state (Fig. 3a). Positions of the triad
residues in FIH-1 match well with those of CAS (Fig. 3a). Consistent with the crystal structure, mutation of either His-199 or
Asp-201 to Ala impaired the FIH-1 activity (14). The facial triad
residues are highly exposed to the solvent due to the wide opening of
the pocket entrance (~15 Å wide, Fig 4a). In CAS the entrance of pocket is blocked by three extended loops (residues 111-117, 131-135, and 202-205 in CAS), allowing only small molecule substrates to enter the active site. The wide-opened pocket entrance of
FIH-1 indicates that the substrate may have a helical conformation rather than an extended strand (see below).
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Fig. 3.
The active site conformation.
a, the active site residues in FIH-1 are presented as
superimposed with the corresponding residues of CAS in complex with 2OG
and ferrous ion. The two structures were superposed as in Fig. 2. In
the figure, facial triad residues (His-199, Asp-201, and His-279 of
FIH-1; His-144, Glu-146, and His-279 of CAS) and the residue implicated
in the 2OG binding (Lys-214 of FIH-1; Arg-293 of CAS) are presented.
Side chains of FIH-1 and CAS are drawn in blue and
yellow sticks, and labeled black and
red, respectively. One of the facial triad residues
(His-279) is common in both proteins (labeled magenta). Main
chains near the presented residues are drawn as tubes of C -carbon
trace (FIH-1, purple; CAS, gray).
b, the 2Fo
Fc
electron density map around the facial triad residues (His-199,
Asp-201, and His-279) is presented in stereo. The map is contoured at
1.0
level.
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Fig. 4.
The putative binding sites for
HIF-1 and VHL. a, the
electrostatic potential surface of FIH-1 is presented with the docked
peptide representing the region near the hydroxylation site of HIF-1
CAD. Positive and negative potentials are colored blue and
red, respectively. In the figure, an ideal
-helical
polyalanine model (13 residues) was manually docked on the prominent
groove near the active site of FIH-1. The facial triad residues
(His-199, Asp-201, and His-279) in the active site also are shown in
the figure. b, the putative HIF-1
CAD- and
VHL-binding sites are represented on a ribbon diagram of FIH-1 with the
same orientation as a. Two parts of the N-terminal 126 residues were colored differently (residues 12-88, pink;
residues 89-126, orange). The rest of the molecule is
colored gray. The
-helical polyalanine model docked in
the putative substrate-binding groove and the facial triad residues are
shown as in a.
10 of
sheet 1) appears to be involved in the binding of 2OG. Although the
C
position of Lys-214 is different from that of Arg-293 of CAS, the
charged moiety of Lys-214 is located at the similar position as that of
CAS (Fig. 3a). Lys-214 is completely conserved in the
subfamily members of FIH-1 from various species (14), supporting its
possible role in the FIH-1 activity by coordinating 2OG.
CAD and VHL--
The most
prominent feature in the FIH-1 structure is a distinctive groove that
extends from the active site toward the interconnecting loop between
domains I and II (loop D1-D2) (Fig. 4, a and b). The groove, being ~15 Å wide and ~40 Å long, is lined mainly by residues from helix
7, loop D1-D2, and strands
7 and
14. In CAS, the region corresponding to the FIH-1 groove is occupied by
several loop structures (residues 111-117, residues 148-153, residues
202-205, and residues 317-324 in CAS). The groove surface consists of
many hydrophobic residues and is wide enough to accommodate a single
-helix (Fig. 4, a and b). Recently, NMR
structures for the complex of the CH1 domain of P300 with the HIF-1
CAD were reported (38, 39). In the complex structures, residues
preceding the hydroxylation site, Asn-803 has an
-helical
conformation. Thus, we propose that the long groove found in the FIH-1
structure serves as the binding site for HIF-1
CAD. Previous
analyses (12) suggested that the HIF-binding site is located at the
C-terminal region of FIH-1, consistent with the location of the
putative helix-binding groove revealed from the structure. The
VHL-binding site is located N-terminal (until residue 126) to the
HIF-binding site (12). In the FIH-1 structure, the N-terminal 126 residues are divided into two structural segments that overlap the core of the protein from opposite sides (Fig. 4b). The first
segment comprising the N-terminal 89 residues lies at the opposite side of the active site, whereas the second segment (residues 90-126) is
positioned at the entrance of active site. The second segment is close
to the putative binding site for HIF-1
CAD (Fig. 4b), indicating that there may be a steric hindrance if VHL were to bind to
the second segment. Thus, VHL is likely to bind FIH-1 by utilizing the
first segment, and FIH-1 may act as a bridge in the interaction between
VHL and HIF1
CAD (Fig. 5). Consistent with this structural implication, VHL interacts with HIF-1
CAD in
the presence but not in the absence of FIH-1 (12).
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Fig. 5.
A hypothetical model of the complex formation
by the FIH-1 dimer, VHL, and HIF-1 . The
model based on the structural and functional implications of FIH-1 in
the hypoxia regulation is presented in the figure. The model was
constructed to represent that FIH-1 acts as a bridge for the
association between VHL and HIF-1
CAD, whereas VHL interacts also
with the HIF-1
ODD domain and histone deacetylases (see text). In
normoxia, VHL binds to the HIF-1
ODD domain through hydroxyproline
in the domain, which brings the VHL-associated FIH-1 around HIF-1
CAD. FIH-1 then hydroxylates Asn-803 of HIF-1
CAD, which prevents
transcription coactivators from binding to CAD. The dimerization of
FIH-1 leads to a large complex consisting of two molecules of FIH-1,
two molecules of HIF-1
, two molecules of VHL, and several pairs of
associated histone deacetylases. In hypoxia, the complex is not likely
to form due to the lack of the proline hydroxylation in the HIF-1
ODD domain. The N-terminal region of HIF-1
including the DNA binding
domain and Per-Arnt-Sim homology domain is not shown in the figure
(indicated as broken lines).
CAD and
VHL. The distinctive binding sites for HIF-1
CAD and VHL on the
surface of FIH-1 support the possibility of the ternary complex and
indicate that the cooperative binding of the three proteins may be
required in the hydroxylation of HIF-1
CAD by FIH-1 (Fig. 5).
Previous analyses reported that FIH-1 was active under hypoxic
conditions despite its expected role as an oxygen sensor, whereas the
activity of HIF-1-PH was suppressed in hypoxia (2, 12, 14). This
implies that the FIH-1 active site itself may not act as a direct
oxygen sensor. Instead, the oxygen dependence of the hydroxylation by
FIH-1 may be due to its substrate recognition mechanism that requires a complex formation with VHL and HIF-1
. In normoxia, VHL binds to the
HIF-1
ODD domain through the hydroxyproline and then recruits FIH-1
around HIF-1
CAD for the hydroxylation of Asn-803, whereas in
hypoxia, FIH-1 cannot bind the substrate due to the absence of the
HIF-1
-bound VHL. The dimerization of FIH-1 may lead to the formation
of a large complex that includes two molecules of HIF-1
and VHL as
well as various histone deacetylases (Fig. 5). The
multimerization-dependent regulation of FIH-1 activity may have an advantage for the HIF-1
regulation by providing sequential checks for the cellular oxygen level by using different mechanisms.
CAD-binding site, may also be exploited for the design of inhibitors.
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ACKNOWLEDGEMENTS |
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We thank Drs. S.-S. Cha, K.-H. Kim, and H.-S. Lee at the Pohang Accelerator Laboratory for help in data collection.
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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 the structure factors (code 1IZ3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Both authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 82-42-860-4149; Fax: 82-42-860-4598; E-mail: ryuse@kribb.re.kr.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210385200
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ABBREVIATIONS |
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The abbreviations used are: HIF-1, hypoxia-inducible factor 1; 2OG, 2-oxoglutarate; VHL, von Hippel-Lindau; FIH-1, factor inhibiting HIF-1; ODD, oxygen-dependent degradation; CAD, C-terminal activation domain; CAS, clavaminic acid synthase.
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