Structure of Factor-inhibiting Hypoxia-inducible Factor (HIF) Reveals Mechanism of Oxidative Modification of HIF-1alpha *

Jonathan M. ElkinsDagger , Kirsty S. HewitsonDagger §, Luke A. McNeillDagger , Jürgen F. SeibelDagger , Imre SchlemmingerDagger , Christopher W. Pugh||, Peter J. Ratcliffe||, and Christopher J. SchofieldDagger §

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha -subunit while hydroxylation of Asn803 in the C-terminal transactivation domain of HIF-1alpha (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 beta -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

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 alpha beta heterodimer, HIF-beta being a constitutive nuclear protein that dimerises with oxygen regulated HIF-alpha 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-alpha residues. In normoxia, 4-hydroxylation of human HIF-1alpha at Pro402 or Pro564 by a set of HIF prolyl hydroxylase isozymes (PHD1-3) (3, 4) mediates HIF-1alpha 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-1alpha Asn803 (10, 11), which blocks interaction with the transcriptional coactivator p300 (12, 13). In hypoxia, lack of hydroxylase activity enables HIF-alpha 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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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 beta -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.

Figures were prepared with Molscript (22), Bobscript (23), Raster3D (24), and Pymol (www.pymol.org).

    RESULTS AND DISCUSSION
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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-2alpha CAD850-862 (equivalent to HIF-1alpha 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. 

                              
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Table I
Summary of FIH:CAD-fragment complex structures
Crystalline FIH-CAD complexes were also obtained with Fe(II), HIF-1alpha 775-786, and 2OG or NOG (data not shown, since no additional CAD residues were resolved over the structures with HIF-1alpha 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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Fig. 1.   The FIH-CAD complex (a-c, structure 1; d, structure 2). a, FIH monomer. The CAD peptide is shown as a ball-and-stick representation in red and the DSBH motif in green. b, FIH dimer. The two molecules of FIH are in dark and light blue, the DSBH motif is in green, and the CAD peptide is in red. c, the 2OG binding site with bound NOG is shown in yellow. The Fe(II) is colored pink, and the 2mFo - DFc electron density map is contoured at 1.5 sigma . d, orientation of CAD Asn803 at the FIH active site. The 2OG and CAD peptide are shown in yellow.

The core of FIH comprises a double-stranded beta -helix (DSBH or jellyroll) motif formed from eight beta -strands, beta 8-beta 11 and beta 14-beta 17 (Fig. 1a) that is characteristic of the 2OG oxygenase superfamily. Residues 220-259 form an insert between strands 4 (beta 11) and 5 (beta 14) of the DSBH. One face of the DSBH is flanked by an additional four beta -strands from the N-terminal region to form an eight-membered antiparallel beta -sheet (Fig. 1a). Interestingly, the N-terminal strand beta 1 bisects the face of the DSBH opposite to the active site (Fig. 1a). The beta 1 strand has a 360° twist located at a PXXP sequence, in between its interactions with beta 14 and beta 2. A similarly positioned beta -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 beta 1, alpha 1, and beta 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-1alpha and HIF-2alpha 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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Fig. 2.   FIH-CAD interactions. a, CAD fragments are shown as stick models in yellow above a van der Waals surface of FIH. FIH residues beneath the surface are colored green. Dotted red lines represent electrostatic bonds. b, alternative view of site 1. Note Asn803 is deeply buried. c, electron density for the bound CAD peptide (structure 1). CAD residues 795-806 (site 1, left) and 812-823 (site 2, right) are shown as ball-and-stick representations in yellow. The difference electron density, contoured at 2.2 sigma  (left) and 1.5 sigma  (right), was calculated after random model perturbation and refinement with CAD omitted to remove model bias.

                              
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Table II
FIH activity with various HIF fragment peptides
Activity was measured by decarboxylation of 2-oxoglutarate. Incubation for 20 min was at the conditions reported previously (10).

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 beta -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 alpha -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 alpha -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-alpha 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 Cbeta -Cgamma 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 beta -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 left-right-arrow Fe(III)-O·] that effects oxidation at the beta -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 beta -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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Fig. 3.   Mechanism for FIH and sequence alignment of FIH with JmjC domain proteins. a, part of the FIH catalytic cycle, showing the role of 2OG and the mechanism of inhibition by NOG. b, partial sequence alignment of FIH with a selection of JmjC domain containing proteins. FIH secondary structure is indicated above the alignment. Selected 2OG binding residues found in FIH are indicated by red triangles under the alignment and the two iron binding residues by green triangles. SWALL accession numbers are indicated on the left of the alignment.

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.

    ACKNOWLEDGEMENTS

We thank the staff at beamlines 14.2, 9.6, and 9.5 of the Synchrotron Radiation Source, Daresbury, UK for expert assistance.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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 beta -helix; CBP, CREB-binding protein (where CREB is cAMP-response element-binding protein).

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Semenza, G. L. (2000) Genes Dev. 14, 1983-1991[Free Full Text]
2. Semenza, G. L. (1999) Annu. Rev. Cell Dev. Biol. 15, 551-578[CrossRef][Medline] [Order article via Infotrieve]
3. Epstein, A. C. R., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O'Rourke, J., Mole, D. R., Mukherji, M., Metzen, E., Wilson, M. A., Dhanda, A., Tian, Y.-M., Masson, N., Hamilton, D. L., Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P. H., Pugh, C. W., Schofield, C. J., and Ratcliffe, P. J. (2001) Cell 107, 43-54[Medline] [Order article via Infotrieve]
4. Bruick, R. K., and McKnight, S. L. (2001) Science 294, 1337-1340[Abstract/Free Full Text]
5. Jaakkola, P., Mole, D. R., Tian, Y.-M., Wilson, M. I., Gielbert, J., Gaskell, S. J., von Kriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Science 292, 468-472[Abstract/Free Full Text]
6. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J. M., Lane, W. S., and Kaelin, W. G., Jr. (2001) Science 292, 464-468[Abstract/Free Full Text]
7. Hon, W.-C., Wilson, M. I., Harlos, K., Claridge, T. D. W., Schofield, C. J., Pugh, C. W., Maxwell, P. H., Ratcliffe, P. J., Stuart, D. I., and Jones, E. Y. (2002) Nature 417, 975-978[CrossRef][Medline] [Order article via Infotrieve]
8. Min, J.-H., Yang, H., Ivan, M., Gertler, F., Kaelin Jr, W. G., and Pavletich, N. (2002) Science 296, 1886-1889[Abstract/Free Full Text]
9. Mahon, P. C., Hirota, K., and Semenza, G. L. (2001) Genes Dev. 15, 2675-2686[Abstract/Free Full Text]
10. Hewitson, 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., and Schofield, C. J. (2002) J. Biol. Chem. 277, 26351-26355[Abstract/Free Full Text]
11. Lando, D., Peet, D. J., Gorman, J. J., Whelan, D. A., Whitelaw, M. L., and Bruick, R. K. (2002) Genes Dev. 16, 1466-1471[Abstract/Free Full Text]
12. Dames, S. A., Martinez-Yamout, M., De, Guzman, R. N., Dyson, H. J., and Wright, P. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5271-5276[Abstract/Free Full Text]
13. Freedman, S. J., Sun, Z.-Y. J., Poy, F., Kung, A. L., Livingston, D. M., Wagner, G., and Eck, M. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5367-5372[Abstract/Free Full Text]
14. Lando, D., Peet, D. J., Whelan, D. A., Gorman, J. J., and Whitelaw, M. L. (2002) Science 295, 858-861[Abstract/Free Full Text]
15. Collaborative Computational Project Number 4. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
16. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
17. Terwilliger, T. C. (2000) Acta Crystallogr. Sect. D Biol. Crystallogr. 56, 965-972[CrossRef][Medline] [Order article via Infotrieve]
18. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
19. Brunger, 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., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
20. Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S., and Dodson, E. J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 247-255[CrossRef][Medline] [Order article via Infotrieve]
21. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
22. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
23. Esnouf, R. M. (1997) J. Mol. Graph. Model. 15, 132-134[CrossRef][Medline] [Order article via Infotrieve]
24. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524
25. Jones, S., and Thornton, J. M. (1995) Prog. Biophys. Mol. Biol. 63, 31-65[CrossRef][Medline] [Order article via Infotrieve]
26. Fusetti, F., Schröter, K. H., Steiner, R. A., van Noort, P. I., Pijning, T., Rozeboom, H. J., Kalk, K. H., Egmond, M. R., and Dijkstra, B. W. (2002) Structure (Lond.) 10, 259-268[CrossRef][Medline] [Order article via Infotrieve]
27. Zhou, J., Kelly, W. L., Bachmann, B. O., Gunsior, M., Townsend, C. A., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 7388-7398[CrossRef][Medline] [Order article via Infotrieve]
28. Zhang, Z. H., Ren, J. S., Harlos, K., McKinnon, C. H., Clifton, I. J., and Schofield, C. J. (2002) FEBS Lett. 517, 7-12[CrossRef][Medline] [Order article via Infotrieve]
29. Clissold, P. M., and Ponting, C. P. (2001) Trends Biochem. Sci. 26, 7-9[CrossRef][Medline] [Order article via Infotrieve]


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