February 1, 2003Journal of Biological Chemistry121 citationsOpen Access

Structure of Human FIH-1 Reveals a Unique Active Site Pocket and Interaction Sites for HIF-1 and von Hippel-Lindau

CLCheolju Lee SKSeung Jun Kim DJDae Gwin Jeong

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Abstract

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 β-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. 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 β-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. hypoxia-inducible factor 1 2-oxoglutarate von Hippel-Lindau factor inhibiting HIF-1 oxygen-dependent degradation C-terminal activation domain clavaminic acid synthase 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 (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1672) Google Scholar). 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 (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1672) Google Scholar). HIF-1 is a basic helix-loop-helix/Per-Arnt-Sim homology domain protein composed of two (α and β) subunits. Both the half-life and transactivation function of HIF-1α are regulated by changes in the cellular oxygen level, whereas HIF-1β remains mostly unaffected (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1672) Google Scholar). Two separate domains within HIF-1α 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) (2Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2111) Google Scholar, 3Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3879) Google Scholar, 4Jaakkola P. Mole D.R. Tian Y.M. 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Bullock A.N. Welford R.W. Elkins J.M. Oldham N.J. Bhattacharya S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. 2002; 277: 26351-26355Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 14Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1224) Google Scholar). Hydroxylation of the asparagine residue during normoxia suppresses interaction of CAD with transcription coactivators (15Arany Z. Huang L.E. Eckner R. Bhattacharya S. Jiang C. Goldberg M.A. Bunn H.F. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12969-12973Crossref PubMed Scopus (633) Google Scholar, 16Carrero P. Okamoto K. Coumailleau P. O'Brien S. Tanaka H. Poellinger L. Mol. Cell. Biol. 2000; 20: 402-415Crossref PubMed Scopus (325) Google Scholar, 17Ema M. Hirota K. Mimura J. Abe H. Yodoi J. Sogawa K. Poellinger L. Fujii-Kuriyama Y. EMBO J. 1999; 18: 1905-1914Crossref PubMed Google Scholar, 18Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1274) Google Scholar, 19Sang N. Fang J. Srinivas V. Leshchinsky I. Caro J. Mol. Cell. Biol. 2002; 22: 2984-2992Crossref PubMed Scopus (137) Google Scholar). Both FIH-1 and HIF-1-PH belong to the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily (2Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2111) Google Scholar, 12Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1124) Google Scholar, 13Hewitson K.S. McNeill L.A. Riordan M.V. Tian Y.M. Bullock A.N. Welford R.W. Elkins J.M. Oldham N.J. Bhattacharya S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. 2002; 277: 26351-26355Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 14Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1224) Google Scholar). The family consists of a variety of enzymes catalyzing hydroxylations, desaturations, and oxidative ring closures/rearrangements (20Barlow J.N. Baldwin J.E. Clifton I.J. Gibson E. Hensgens C.M. Hajdu J. Hara T. Hassan A. John P. Lloyd M.D. Roach P.L. Prescott A. Robinson J.K. Zhang Z.H. Schofield C.J. Biochem. Soc. Trans. 1997; 25: 86-90Crossref PubMed Scopus (8) Google Scholar). Crystal structures of the family members have been reported for bacterial proline 3-hydroxylase and other enzymes responsible for biosynthesis of antibiotics (21Roach P.L. Clifton I.J. Hensgens C.M. Shibata N. Schofield C.J. Hajdu J. Baldwin J.E. Nature. 1997; 387: 827-830Crossref PubMed Scopus (390) Google Scholar, 22Valegard K. van Scheltinga A.C. Lloyd M.D. Hara T. Ramaswamy S. Perrakis A. Thompson A. Lee H.J. Baldwin J.E. Schofield C.J. Hajdu J. Andersson I. Nature. 1998; 394: 805-809Crossref PubMed Scopus (315) Google Scholar, 23Zhang Z. Ren J. Stammers D.K. Baldwin J.E. Harlos K. Schofield C.J. Nat. Struct. Biol. 2000; 7: 127-133Crossref PubMed Scopus (252) Google Scholar, 24Clifton I.J. Hsueh L.C. Baldwin J.E. Harlos K. Schofield C.J. Eur. J. Biochem. 2001; 268: 6625-6636Crossref PubMed Scopus (99) Google Scholar). 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 β-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. 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 intoNdeI/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 ε280 = 1.69 (mg/ml)−1 cm−1 in 6 mguanidine. 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 (0.4m 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. 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 (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38556) Google Scholar). Seven selenium sites out of eight expected sites were located by the program SOLVE (26Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar), and heavy atom parameters were refined by the program SHARP (27de La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The phases were subsequently improved by solvent-flattening using the program DM (28Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19761) Google Scholar). 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 (29Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar) and refined with the program CNS (30Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16963) Google Scholar) in the resolution range of 99 to 2.8 Å. The randomly selected 5% of the data were set aside for theR free calculation. Refinement included an overall anisotropic B factor and bulk solvent correction. TheR cryst and the R free are 22.8 and 27.5%, respectively (Table I). The stereochemical analysis using the program PROCHECK (31Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) 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.Table ICrystallographic dataSe-MetPeak (λ1)Edge (λ2)Remote (λ3)A. Data collection statisticsWavelength (Å)0.97920.97940.9716Space groupP41212Cell dimension (Å)86.89 × 86.89 × 143.42Highest resolution (Å)2.82.82.8Unique reflections (total)13,768 (91,630)13,563 (98,205)13,783 (93,608)Completeness (%)aThe values in parentheses (completeness andR merge) are for the highest resolution bin.97.0 (91.5)95.9 (89.7)97.3 (93.7)R mergebR merge = Σi ‖ I i − 〈I〉 ‖/ Σ ‖ 〈I〉 ‖, where I is the intensity for the ith measurement of an with the Refinement range reflections cryst = Σ ‖ c ‖/ Σ and c are the and structure factor R free was from 5% of data that were used in the The values in parentheses (completeness andR merge) are for the highest resolution R merge = Σi ‖ I i − 〈I〉 ‖/ Σ ‖ 〈I〉 ‖, where I is the intensity for the ith measurement of an with the R cryst = Σ ‖ c ‖/ Σ and c are the and structure factor The R free was from 5% of data that were used in the refinement. in a were by using the P.J. J. Appl. Crystallogr. 1991; Google Scholar), D.J. Methods Enzymol. 1997; 277: PubMed Scopus Google Scholar), J. Mol. 1997; PubMed Scopus Google Scholar), M. Methods Enzymol. 1997; 277: PubMed Scopus Google Scholar), and A. 1991; PubMed Scopus Google Scholar). The overall of FIH-1 is composed of a β-barrel by eight with an dimension of × × Å The structure of FIH-1 reveals two I from to residue and is composed of the β-barrel and and and (residues two and from domain I. which is in structures of the dioxygenase with the region of the molecule 1 is further by with helix of FIH-1 revealed by the crystal structure was confirmed by chromatography and which is composed of that to of the of the FIH-1 low the overall structure of FIH-1 is to of other 2OG-dependent as the of the residues K.S. McNeill L.A. Riordan M.V. Tian Y.M. Bullock A.N. Welford R.W. Elkins J.M. Oldham N.J. Bhattacharya S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. 2002; 277: 26351-26355Abstract Full Text Full Text PDF PubMed Scopus (600) Google D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1224) Google Scholar). In a for structures by using the members of the 2OG-dependent including clavaminic acid synthase and synthase were identified with ranging from to are in the β-barrel we the structure of FIH-1 with that of were with a of Å are in the region of the β-barrel the of are and be to other in the structure of FIH-1 the long insertions in N- and C-terminal regions are also in several and in the middle of the have a of FIH-1 for and K.S. McNeill L.A. Riordan M.V. Tian Y.M. Bullock A.N. Welford R.W. Elkins J.M. Oldham N.J. Bhattacharya S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. 2002; 277: 26351-26355Abstract Full Text Full Text PDF PubMed Scopus (600) Google D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1224) Google Scholar). the center of the β-barrel structure of FIH-1, is a that is with the residues in a and the triad and In one of the is an that is to the of the triad residues are as were a that the FIH-1 structure is in of the triad residues in FIH-1 with of with the crystal structure, of to the FIH-1 activity D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1224) Google Scholar). The triad residues are highly exposed to the solvent to the wide of the Å 4 In the of is by three (residues and in molecule to the active The wide-opened of FIH-1 that the may have a an putative sites for HIF-1 and VHL. the of FIH-1 is with the the region near the hydroxylation site of HIF-1α and are In the an model was the prominent groove near the active site of The triad residues and in the active site also are in the the putative HIF-1α and sites are a of FIH-1 with the as Two of the N-terminal residues were (residues residues The of the molecule is The model in the putative groove and the triad residues are as in of the triad residues are highly conserved FIH-1 and other 2OG-dependent the putative site for in FIH-1 is from that in other 2OG-dependent In of is to the of that is highly conserved in 2OG-dependent (2Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2111) Google Scholar, L. Biol. 2001; 2: Google Scholar). and also are near the to the of in FIH-1, are with residues such as and of from structural of to be in the of the of is from that of of the of is located at the as that of is conserved in the members of FIH-1 from various D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1224) Google Scholar), supporting its role in the FIH-1 activity by The most prominent in the FIH-1 structure is a distinctive groove that from the active site toward the domains I and a and The Å wide and Å is by residues from helix and and In the region to the FIH-1 groove is by several structures (residues residues residues and residues in The groove consists of many residues and is wide to a a and structures for the complex of the domain of with the HIF-1α CAD were reported M. H.J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, S.J. F. Livingston D.M. G. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). In the complex residues the hydroxylation site, has an we that the long groove in the FIH-1 structure as the site for HIF-1α analyses (12Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1124) Google Scholar) that the site is located at the C-terminal region of FIH-1, with the of the putative groove revealed from the structure. The site is located N-terminal residue to the site (12Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1124) Google Scholar). In the FIH-1 structure, the N-terminal residues are two structural that the core of the protein from 4 The first the N-terminal residues at the of the active site, whereas the second (residues is at the of active The second is to the putative site for HIF-1α CAD 4 that may be a were to bind to the second is to bind FIH-1 by utilizing the first and FIH-1 may as a in the interaction and CAD with structural with HIF-1α CAD in the in the of FIH-1 (12Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1124) Google Scholar). The FIH-1 structure suggests a mechanism for the regulation of the HIF-1 activity by of regions in FIH-1 that are in the with HIF-1α CAD and VHL. The distinctive sites for HIF-1α CAD and the of FIH-1 the of the ternary complex and that the of the three may be in the hydroxylation of HIF-1α CAD by FIH-1 analyses reported that FIH-1 was active under hypoxic conditions its expected role as an oxygen whereas the activity of HIF-1-PH was in hypoxia (2Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2111) Google Scholar, 12Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1124) Google Scholar, 14Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1224) Google Scholar). that the FIH-1 active site may as a oxygen the oxygen of the hydroxylation by FIH-1 may be to its mechanism that a complex formation with and In normoxia, to the HIF-1α domain the and then FIH-1 HIF-1α CAD for the hydroxylation of whereas in FIH-1 bind the to the of the VHL. The dimerization of FIH-1 may to the formation of a complex that two of HIF-1α and as as various The regulation of FIH-1 activity may have an for the HIF-1α regulation by for the cellular oxygen by using hypoxia are important in oxygen and during hypoxic in ischemic diseases such as and The crystal structure of FIH-1 reveals the active site of the enzyme such as the wide-opened for the site and a distinctive features be an in specific drugs that can the enzyme activity and promote the hypoxia-inducible in ischemic The prominent groove near the active site, which is the HIF-1α site, may also be for the design of and Lee at the Pohang Accelerator Laboratory for in data

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