Binding of Natively Unfolded HIF-1α ODD Domain to p53

Molecular Cell, Vol. 17, 11–21, January 7, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2004.11.019

Binding of Natively UnfoldedHIF-1� ODD Domain to p53

accumulation itself involves three steps: hypoxia-inducedposttranslational stabilization, nuclear translocation,and hypoxia-induced transcriptional activation. HIF-1�

Nuria Sanchez-Puig, Dmitry B. Veprintsev,and Alan R. Fersht*Centre for Protein Engineering

is stable under hypoxic conditions but in the presenceMedical Research Councilof oxygen is targeted for proteosomal degradation byHills Roadthe VCB ubiquitination complex (von Hippel-Lindau pro-CB2 2QH, Cambridgetein [VHL], elongin B, and elongin C) (Cockman et al.,United Kingdom2000). The formation of this complex occurs only undernormoxic conditions and as a result of the hydroxylationof two conserved prolines (prolines 402 and 564) in theSummaryHIF-1� ODD domain (Masson et al., 2001; Min et al.,2002). Under hypoxic conditions, HIF-1� accumulatesHypoxia-inducible factor-1 (HIF-1) is a heterodimericand is subsequently exported to the nucleus where ittranscription factor that plays a crucial role in mediat-heterodimerizes with Arnt. The heterodimer then inter-ing oxygen response in the cell. Using biophysicalacts with p300/CBP (Arany et al., 1996; Kallio et al.,techniques, we characterized two fragments of the1998), binds to a cognate hypoxia-response elementHIF-1� subunit, one the full-length ODD domain (resi-(HRE) (Semenza et al., 1996), and thereby transactivatesdues 403–603) and the second comprising the N-TADHRE-containing promoters and enhancers. The interac-(N-transactivation domain) and inhibitory domain (res-tion between p300/CBP and HIF-1� is also regulatedidues 530–698). Both were unstructured in solutionby posttranslational hydroxylation. Asparagine 803 isunder physiological conditions and so belong to thehydroxylated by FIH-1 (Factor Inhibiting HIF-1�) duringfamily of natively unfolded proteins. The HIF-1� ODDnormoxia, thereby preventing its interaction with p300/domain binds weakly to the isolated p53 core domainCBP and thus silencing transactivation (Dames et al.,but tightly to full-length p53 to give a complex of one2002; Freedman et al., 2002; Lando et al., 2002; MahonHIF-1� ODD domain with a p53 dimer. By being un-et al., 2001). In addition, FIH-1 also binds to VHL andstructured, the HIF-1� ODD domain can thread bothacts as a transcriptional corepressor, inhibiting HIF-1�its binding sites through the p53 multimer and bindtransactivation by recruiting histone deacetylases. More-tightly by the “chelate effect.” These results supportover, acetylation of Lys532 by ARD1 in the HIF-1� ODDthe idea that hypoxic p53-mediated apoptosis doesdomain increases the interaction with VCB and, there-involve the direct binding of HIF-1� to p53.fore, induces VCB-mediated ubiquitination (Jeong et al.,2002). Other reports indicate that p53 inhibits hypoxia-Introductioninducible levels of HIF-1� by facilitating its ubiquitinationand subsequent degradation (Blagosklonny et al., 1998;Hypoxia-inducible factor-1 (HIF-1) is a transcriptionalRavi et al., 2000), but it is still not clear if the interactionactivator that functions as a global regulator of oxygenis direct or mediated by Mdm2 (An et al., 1998; Chen ethomeostasis, facilitating oxygen delivery and adapta-al., 2003; Hansson et al., 2002).tion to oxygen deprivation. HIF-1 is a heterodimeric pro-

The tumor suppressor p53 is a homotetramer stabi-tein of the bHLH-PAS (basic Helix-Loop-Helix, Per, Arnt,lized by numerous stimuli such as DNA damage, abnor-and Sim protein) superfamily, which regulates hypoxia-mal proliferation signals, hypoxia, and osmotic stress.inducible genes (Wang et al., 1995). The products ofp53 is a transcription factor that activates a variety of

these genes are involved in angiogenesis, vascular re-genes involved in DNA damage repair, cell cycle arrest,

modeling, energy metabolism, and cell proliferation andand apoptosis. Diverse evidence suggests that hypoxia

survival (Semenza, 2001). HIF-1 is composed of two causes p53 accumulation, although the molecularsubunits, the HIF-1� and HIF-1� subunits. The HIF-1� mechanisms leading to either cell cycle arrest or apopto-subunit is a protein unique to HIF-1 and contains 826 sis during hypoxia are not yet clear (Blagosklonny et al.,amino acids, whereas the HIF-1� subunit (Arnt) has been 1998; Carmeliet et al., 1998; Goda et al., 2003; Graebershown to encode the 774 and 789 amino acid isoforms et al., 1994, 1996; Green and Giaccia, 1998). Studiesof the aryl hydrocarbon receptor (AHR) nuclear translo- show that p53 accumulation frequently occurs withincation protein. The HIF-1� functional motifs comprise hypoxic regions in tumors (Zhong et al., 1999) and thatan N-terminal part that contains a bHLH domain whose this strongly correlates with cells undergoing apoptosisfunction is to mediate DNA binding and protein dimeriza- (Graeber et al., 1994, 1996), while cells lacking p53 aretion (Jiang et al., 1996) and a PAS domain that acts as more resistant to hypoxia-induced apoptosis. Contrarya second dimerization interface. It also has a C-terminal reports, however, have shown that hypoxia is not suffi-part that contains the oxygen-dependent degradation cient to induce p53 accumulation (Wenger et al., 1998),(ODD) domain (Masson et al., 2001) and two transactiva- or accumulation only occurs when accompanied bytion domains (Jiang et al., 1997), one of which overlaps acidosis and nutrient deprivation (Pan et al., 2004). Addi-with the ODD domain. The main regulatory step of HIF1 tional evidence suggests that direct association be-involves the accumulation of its HIF-1� subunit. The tween p53 and HIF-1� mediates p53 hypoxic accumula-

tion (An et al., 1998; Hansson et al., 2002; Ravi et al.,2000). p53 accumulates in wild-type embryonic stem*Correspondence: [email protected]

Molecular Cell12

cells when challenged with anoxia/hypoglycaemia, butnot in HIF-1�-deficient cells (Carmeliet et al., 1998). Onthe contrary, Chen and coworkers found that the interac-tion between HIF-1� and p53 is mediated by Mdm2,which acts as a bridge between the two and suppressesMdm2-mediated p53 ubiquitination (Chen et al., 2003).

In this study, we used biophysical techniques to char-acterize in vitro two fragments of HIF-1�. The fragmentscomprised residues 530–698 (N-TAD and inhibitory do-main) and residues 403–603 (full-length ODD domain),respectively. We found that under physiological condi-tions both fragments exhibited the properties of nativelyunfolded proteins and behaved as random coils. In orderto gain insight into the relationship between HIF-1� andp53, we used analytical ultracentrifugation and fluores-cence anisotropy to study their interaction. We demon-strated that the HIF-1� ODD domain contained two bind-ing sites for p53 core. Interaction of the isolated p53core domain with the HIF-1� ODD domain was veryweak at physiological ionic strength. In contrast, underphysiological conditions, the binding was tighter withp53 tetrameric full-length protein as a result of a cooper-ative effect and was located to its DNA binding region.

Results

Hydrodynamic Behavior of HIF-1� 530–698and HIF-1� 403–603The hydrodynamic dimension of a protein is characteris-tic of its conformational state. Size-exclusion chroma-tography has proven very useful to study the degree ofcompactness of a protein. HIF-1� 530–698 and HIF-1�

403–603 eluted from the gel filtration column as well-defined peaks corresponding to Mr 95,500 and 134,700,respectively (Figure 1A), values five and six times largerthan the calculated theoretical values of 18.8 and 22.3kDa. Further, experimental molecular weights obtainedby mass spectroscopy were in agreement with the cal-culated theoretical values. Such a high Mr may resultfrom the formation of soluble aggregates or from an

Figure 1. Hydrodynamic Characterization of HIF-1� 530–698 andextended rather than globular conformation. Analysis ofHIF-1� 403–603the sedimentation equilibrium centrifugation experi-(A) Size-exclusion chromatography elution profile of HIF-1� 530–698ment, however, showed that both proteins were mono-(circles) and HIF-1� 403–603 (triangles) monitored at 220 nm. Aster-meric with a Mr of 18,000 � 120 and 22,000 � 500 (Figureisks denote the positions of the molecular weight standards, from

1B; as an example for HIF-1� 530–698). In addition, left to right: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa),HIF-1� 530–698 and HIF-1� 403–603 both migrated as albumin (67 kDa), ovoalbumin (43 kDa), chymotrypsinogen (25 kDa).a 32 kDa protein in an SDS-PAGE gel even after heat (Inset) Purified recombinant HIF-1� 530–698 and HIF-1� 403–603.

10% SDS-PAGE stained with Coomassie Brilliant Blue R-250. Lanedenaturation (Figure 1A, inset). The Stokes radius of1, molecular weight markers (kDa); lane 2, HIF-1� 530–698; lane 3,each protein was estimated from its Mr using EquationsHIF-1� 403–603.2 and 3 (Uversky, 1993). The value of 38.3 � 0.04 A(B) Equilibrium sedimentation experiments of HIF-1� 530–698 at

obtained for HIF-1� 530–698 (Mr 95,500) compared 10�C and 30,000 rpm. Residuals correspond to the fitting of the datamuch more favorably with the theoretical Stokes radius to a single exponential model to deduce the molecular weight.predicted for an unfolded protein of 18.8 kDa (38.3 �

0.2 A) than that predicted for a native globular mono-meric protein (21 � 0.06 A). Similar analysis for HIF-1� Secondary Structure Analysis of HIF-1� 530–698403–603 showed its hydrodynamic radius corresponded and HIF-1� 403–603to 44.3 � 0.06 A, comparable to that of 41.8 � 0.05 A Circular dichroism is a sensitive spectroscopic methodexpected for an unfolded protein of the same Mr. These for analyzing the secondary structure content of a pro-results imply that both HIF-1� 530–698 and HIF-1� 403– tein, particularly for detecting the presence of � helices.603 have the dimensions of denatured proteins and have The far-UV CD spectra for HIF-1� 530–698 and HIF-1a

403–603 (Figures 2A and 2C) lacked the typical signa-extended conformations.

Binding of Natively Unfolded HIF-1� ODD Domain to p5313

tures of secondary structure (negative bands at 222 and208 nm for � helices and a negative band at 217 nm for� sheets), exhibiting instead only a small signal at 200nm. The values of the CD signal for HIF-1� 530–698 at200, 217, and 222 nm were �1700� cm2 dmol�1, �440�cm2 dmol�1, and �456� cm2 dmol�1, respectively. TheCD spectrum of HIF-1� 530–698 did not change mark-edly with increasing temperature and lacked any coop-erative transition (Figure 2B). The linear shape of thespectrum with a slight negative slope was typical ofrandom coil polypeptides. These results indicate thatHIF-1� 530–698 does not have significant regular sec-ondary structure and that it lacks a compact globularstructure. In comparison, CD studies of HIF-1� 403–603(Figure 2C) showed that the minimum at 200 nm(�17,000� cm2 dmol�1) is more pronounced than that forrandom coils, suggesting that the protein contained asmall amount of poly(L-proline) helix type II (PPII). Thecharacteristics of a PPII are a small positive CD signalat 228 nm with 4,630� cm2 dmol�1 and a large negativeminimum at 205 nm with �50,000� cm2 dmol�1. HIF-1�403–603 did not exhibit the maximum at 228 nm, whichmay be due to the fact that it contains a mixture of mainlyrandom coil conformation and only a small amount ofPPII. The CD spectra of HIF-1� 403–603 at differenttemperatures showed a red shift of approximately 2 nm,and the minimum became less negative. Also, a well-defined isodichoric point at 212 nm was present, a fea-ture described for temperature-dependent CD spectraof peptides that contain PPII structures and attributedto the destruction of the PPII favoring the unstructuredstate (Makarov et al., 1992). In addition, the 15N-HSQCspectrum of HIF-1� 530–698 with its very narrow linewidth (data not shown) clearly corresponded to that ofan unfolded protein, thereby corroborating the previousresults. The chemical shifts in the spectrum were clus-tered and displayed very little dispersion; the protonchemical shifts dispersion of the backbone resonanceswas only 0.5 ppm. The 15N chemical shifts showed asimilar amino acid type-specific clustering as previouslydescribed for urea-denatured proteins (Peti et al., 2001).

Effect of the Environment on the SecondaryStructure of HIF-1� 530–698Changes in the environment can influence the conforma-tion of natively unfolded proteins (reviewed in Uversky,2002b). An extensive analysis on the influence of theenvironment conditions was done for HIF-1� 530–698.

Trifluorethanol (TFE) increases the population of �helices and � sheet content in proteins by reinforcinghydrogen bonds within the polypeptide backbone. CDspectra recorded at increasing concentrations of TFE(Figure 3A) showed an increasing gain in helical contentas indicated by the appearance of two minima at 208

Figure 2. Secondary Structure Analysis of HIF-1� 530–698 and and 222 nm and a maximum at 190 nm, specifically atHIF-1� 403–603 by CD 30% and 40% TFE. The values for these two minima(A) Far-UV CD spectra of HIF-1� 530–698 at different temperatures. were lower than those expected for fully formed helices(B) Temperature dependence of the molar ellipticity of HIF-1� 530– �26,000� cm2 dmol�1). However, treatment with TFE re-698 followed at 222 nm. vealed the � helix-forming propensity of HIF-1� 530–(C) Far-UV CD spectra of HIF-1� 403–603 at different temperatures.

698. Concentrations of TFE higher than 40% could notAll spectra were recorded in 25 mM NaPi (pH 7.2), 150 mM KCl, andbe recorded because under these conditions the protein1 mM DTE.precipitated; the induction of structure formation wasreversible upon removal of TFE by dialysis.

Molecular Cell14

Figure 3. Effect of the Solution Environment on the CD Spectra of HIF-1� 530–698

(A) TFE. (B) SDS. (C) pH in the absence of salt. (D) pH in the presence of salt. All spectra were recorded in 25 mM NaPi (pH 7.2), 150 mM KCl,1 mM DTE, except for those studying the effect of pH without salt.

The effect of anionic detergent SDS (sodium dodecyl alter the HIF1-� 530–698 CD spectrum, and it aggre-gated in solutions containing as low as 50 mM ammo-sulfate) is shown in Figure 3B. At submicellar concentra-

tion (2 mM) SDS tends to induce � strands, while at nium sulfate (data not shown).micellar concentrations (50 mM) SDS mimics the mem-brane environment and often induces helices. The struc- p53 Core Binds to HIF-1� 530–698 and HIF-1�

403–603 in an Ionic Strength-Dependent Mannerture of HIF-1� 530–698 at both concentrations remainedalmost the same: fully unfolded, with just a small red Two peptides (residues 419–436 and 551–579) con-

tained within the ODD domain of HIF-1� have been de-shift in the minimum at 200 nm.The effect of pH in the presence and absence of salt scribed to bind p53 core and compete for the same

binding site in a monomer of p53 (Hansson et al., 2002).is shown in Figures 3C and 3D. Spectra at pH 12 withand without salt were still those of an unfolded protein. We assayed the binding of p53 core to HIF-1� 403–603,

which contains these two binding sites, and to HIF-1�At pH 2, in both cases, a clear change can be observedby the appearance of a broad “U” shaped peak starting 530–698, which contains only one. The binding affinities

of the two constructs of HIF-1� with p53 core wereat 230 nm and finishing around 200 nm. It is difficultto distinguish the type of secondary structure being measured by fluorescence anisotropy at ionic strengths

of 20 and 56 mM, and by analytical ultracentrifugationformed, and it could be a mixture of � helices, � strands,and/or � turns since no typical CD spectrum shape for (AUC) at ionic strengths of 150 and 200 mM. The affinities

varied from the nanomolar to the high micromolar rangeany of the various individual secondary structures ispresent. The intensity of the signal, however, was lower depending on the ionic strength of the buffer used (Table

1). In all cases the HIF-1� constructs were fluorescein-than the normal values expected for well-defined sec-ondary structures. Further, the linear shape of the CD labeled, and the experiments were set up to detect the

fluorescent probe. For HIF-1� 530–698, at an ionicspectra with increasing temperature at this pH showedthat the protein behaves predominantly as a random strength of 200 mM, no binding was detected, and the

data best described a single species of Mr 21,000, corre-coil (data not shown).Addition of trimethylamine N-oxide (up to 3 M) did not sponding to HIF-1� 530–698 alone. At physiological


Table 1. Dissociation Constants for the Different HIF-1� Proteins Binding to p53 Core or p53 flQM at Different Ionic Strengths

I (mM)a HIF1-� 530–698 � p53 core HIF1-� 403–603 � p53 core HIF1-� 403–603 � p53 flQMb

200 NB 613 � 120 �M 13 � 2 �M150 690 � 90 �M 200 � 18 �M 9 � 1 �M56 30 � 1 �M 2 � 0.1 �M —20 162 � 5 nM KdML1 61 � 9 nM; KdML2 21 � 1 nM —

All experiments were done in duplicate, and the values presented in this table correspond to their averages. NB, no binding.a Binding experiments were conducted in 50 mM HEPES (pH 7.2), 1 mM DTT for I 20 mM; 25 mM NaPi (pH 7.2), 1 mM DTT for I 56 mM; 25mM NaPi (pH 7.2), 90 mM KCl, 1 mM DTT for I 150 mM; and 25 mM NaPi, 150 mM KCl, 1 mM DTT for I 200 mM, at 10�C.b Values correspond to apparent dissociation constants. Experiments at ionic strengths of 20 and 56 mM were not done due to extensiveaggregation of p53 flQM.

ionic strength (I 150 mM), the interaction was very weak different sites for binding p53 core located at either endof the molecule; the one at the C terminus is also present(Kd 690 �M). The data were fitted to a two-exponential

model in which the species in the equilibrium had Mr of in the HIF-1� 530–698 construct. The binding site at theN terminus end of the HIF-1� 403–603 construct has a23,000 � 2,000 and 41,000 � 3,000, the first correspond-

ing to free HIF-1� 530–698 and the second to a complex higher affinity for p53 core than the one at the C terminusand thus overshadows its binding, except in the casewith p53 core with 1:1 stoichiometry (Figures 4A and

4B). Using fluorescence anisotropy (Figures 5A and 5B), at lower ionic strength when it was possible to measurebinding at both sites. Differences in the dissociationat lower ionic strengths the binding was tighter, with

dissociation constants 30 and 0.16 �M at I 56 and 20 constant obtained for the interaction at the C terminussite in the two different constructs (HIF-1� 530–698 andmM. respectively.

Although the binding of the fragment HIF-1� 403–603 HIF-1� 403–603) arise from the cooperative effect.and p53 core showed the same dependency on ionicstrength, the effect was not so pronounced (Table 1). p53 flQM Binds to HIF-1� 403–603

in a Cooperative MannerIn contrast with HIF-1� 530–698, at the highest ionicstrength (I 200 mM) it was possible to detect weak bind- The effect of oligomerization on the ability of p53 to bind

specifically to HIF-1� 403–603 was examined by AUCing (Kd 600 �M) between HIF-1� 403–603 and p53 core,while at I 150 mM the binding was slightly tighter, Kd and fluorescence anisotropy. We used AUC to deter-

mine the stoichiometry of the complex. The data best200 �M. In both cases, however, the AUC data describedspecies of 23 � 1.2 and 48 � 1 kDa, suggesting that described a single species in equilibrium with Mr

100,400 � 3,300, which compares with the 109.4 kDaeven when the HIF-1� 403–603 construct contains thetwo binding sites predicted by Hansson et al. (2002), expected for a complex formed by one HIF-1� 403–603

and a dimer of p53 flQM (data not shown). It was notthe stoichiometry of the complex is 1:1 (Figures 4C and4D). More revealing are the fluorescence anisotropy re- possible to determine the dissociation constant value

by AUC, because the binding was too tight for thatsults at lower ionic strengths. At ionic strength 56 mM,the data fitted a model of one binding site (Kd 2 �M), method. The binding measured by florescence anisot-

ropy was highly cooperative. We fitted the data frombut at I 20 mM the shape of the graph clearly changedfrom hyperbolic to sigmoidal (Figures 5C and 5D). For the fluorescence anisotropy experiments to the Hill

equation (data not shown), which gave Hill coefficientsthe latter, the data best described a model of two non-identical noninteracting binding sites where binding to of 1.6 � 0.1 and 1.5 � 0.06 at ionic strengths of 150 and

200 mM, respectively. Accordingly, the binding curvesthe first site does not change the anisotropy value (Ef-tink, 1997). The dissociation constants obtained from approximated to a simple binding isotherm (Figure 6A)

with mean apparent dissociation constants for HIF-1�the fit correspond to KdML1 61 nM and KdML2 21 nM, andthe anisotropy values associated with each complex 403–603 of 9.1 � 1 and 12.6 � 1.7 �M, respectively

(Table 1). Thus, oligomerization of p53 increased thewere rML1 �0.00621 � 0.006418 and rML2 0.12 � 0.001.Fluorescence anisotropy uses polarized excited light affinity of binding to HIF-1� 403–603 20-fold relative to

p53 core at an ionic strength of 150 mM and 50-foldand polarized detection to monitor rotational diffusionevents. It is most likely that the first binding event occurs in the case of an ionic strength of 200 mM. Further,

comparison of the binding curves of p53 flQM to HIF-1�at the site located at the C terminus of the protein faraway from the fluorophore whose tumble is, therefore, 530–698 and to HIF-1� 403–603 highlights the impor-

tance of having the two binding sites present within theless affected and thus exhibits a very low anisotropy.The second binding event, however, has a predicted same construct (Figure 6A). At the same final concentra-

tion of p53 flQM, it was not possible to reach saturationdissociation constant smaller than that associated withthe first, suggesting that the binding is cooperative; when binding to HIF-1� 530–698 compared with HIF-1�

403–603, a reflection of the higher affinity of the latterbinding at one site causes an increase in affinity at theother. The Hill coefficient (Fersht, 1999) obtained from due to the cooperative effect. Although the mutations

in the core domain of p53 flQM do not alter its overallthe Hill plot analysis was 1.6 � 0.02 (Figures 5C, inset),corroborating the fact that binding of two p53 core mole- structure (Joerger et al., 2004), we performed the same

binding experiments using a construct of wild-type p53cules to HIF-1� 403–603 at ionic strength 20 mM isa positively cooperative event. Altogether, the binding that comprises the core and tetramerization domain

(residues 94–360) and confirmed that these mutationsexperiments showed that HIF-1� 403–603 contains two

Molecular Cell16

Figure 4. Equilibrium Sedimentation Analysis of the Interaction of HIF-1� 530–698 and HIF-1� 403–603 with p53 Core

(A and B) HIF-1� 530–698 with p53 core at I 150 and 200 mM, respectively. (C and D) HIF-1� 403–603 with p53 core at I 150 and 200 mM,respectively. Upper panels show concentration distribution plots. Continuous lines correspond to the fit assuming two species of unknownmass in equilibrium, except for (B), where data best described a single species present. Lower panels show the plots of residual deviations.

do not have an effect on the binding (data not shown). DiscussionFinally, we performed a competition assay to corrobo-rate that HIF-1� 403–603 bound to p53 flQM in its DNA HIF-1� ODD and Inhibitory Domains

Are Natively Unfoldedbinding region. Figure 6B illustrates that unlabeled DNAdisplaced HIF-1� 403–603 from the previously formed The hydrodynamic properties of HIF-1� 530–698 and

HIF-1� 403–603 inferred from gel filtration and analyticalcomplex with p53 flQM. The HIF-1� 403–603/p53 flQMcomplex exhibited a large anisotropy value due to slow ultracentrifugation show that they behave like extended

monomeric proteins. NMR and far-UV CD of HIF-1� 530–tumble, and as DNA was added, anisotropy decreasedbecause of the appearance of free HIF-1� 403–603 that 698 and far-UV CD of HIF-1� 403–603 indicate that both

are essentially unstructured and lack detectable sec-tumbles fast. The intersection of the two linear parts ofthe binding isotherm revealed stoichiometric equiva- ondary structure content. In addition, sequence analysis

and secondary structure prediction of the fragment oflence of 2.8 �M DNA concentration, which comparesfavorably with the 2.5 �M DNA concentration value ex- HIF-1� spanning residues 403–698 confirmed the above

experimental results. The parameters used to recognizepected for the complex of one p53 tetramer binding toone molecule of DNA. lack of structure in proteins such as the mean hydropho-


Figure 5. Binding of HIF-1� 530–698 and HIF-1� 403–603 with p53 Core Examined by Fluorescence Anisotropy

(A and B) HIF-1� 530–698 with p53 core at I 20 and 56 mM, respectively. (C and D) HIF-1� 403–603 with p53 core at I 20 and 56 mM,respectively. (Inset in [C]) Hill plot of HIF-1� 403-603 with p53 core at I 20 mM. The Hill coefficient (slope) value corresponds to 1.9 � 0.06.The continuous line in each plot corresponds to a single site binding model fit, except for plot (C), where data were fitted to a two nonidentical,noninteracting binding site model.

bicity �H and the mean net charge �R were calcu- Conformation can also be induced by changes in theenvironment such as pH, temperature, ionic strength,lated as in Uversky et al. (2000). The values are 0.4227

and 0.091, respectively, which are comparable to those or desiccation (reviewed in Wright and Dyson, 1999).Changes in the environment tested within this studypredicted for natively unfolded proteins of �H 0.39 �

0.05 and �R 0.12 � 0.09. The average deviation of did not alter the unfolded structure of HIF-1� 530–698.Trifluoroethanol (TFE) is useful in revealing regions thatamino acid composition of the HIF-1� ODD and inhibi-

tory domains compared with those in the SWISS-PROT have a propensity for unfolding-folding transitions. Atthe maximum concentration at which the protein re-database indicates that it contains few order-promoting

residues (W, C, F, Y, I, and L) and is enriched in most mained soluble (40% TFE), the fractional helicity wascalculated (Chen et al., 1974) to be only 9.4%.disorder-promoting residues (R, Q, S, and E) (Dunker et

al., 2001; Williams et al., 2001). Further, the PONDRpredictor of intrinsically disordered regions (Gold- Binding of Natively Unfolded HIF-1�

ODD Domain to p53enberg, 2003; Romero et al., 2001) predicts an overallpercentage of disorder of 76.01%. Finally, 5.1% of � Two peptides contained within the HIF-1� ODD domain

were found to bind p53 core and to contact specificallyhelix, 5.9% of � sheet, and 89.1% of random coil waspredicted using the Psi-PRED server (http://bioinf. the residues involved in DNA binding (Hansson et al.,

2002). As with the peptides used in this study, the HIF-1�cs.ucl.ac.uk/psiform.html). This suggests that up to 35%of the full-length HIF-1� is unfolded. Moreover, there is ODD domain is unfolded and also binds to the DNA

binding region of p53. Further, the ODD domain is veryevidence that the C terminus might also be unfolded(Dames et al., 2002; Freedman et al., 2002), implying acidic in nature, and binding to p53 core is ionic strength

dependent. We speculate that the natively unfolded na-that up to 50% of the protein may be completely unstruc-tured and that only the N terminus, which contains the ture of HIF-1� ODD domain can mimic DNA and thus

mediate the interaction with p53 core. The binding ofDNA binding region (bHLH-PAS domain), is structured.Interestingly, these natively unfolded proteins can adopt p53 core (as with any other DNA binding protein) to

DNA is also affected by ionic strength, implying thata defined conformation upon binding to specific ligands,which include other proteins, DNA, RNA, and cofactors. electrostatic interactions of basic residues with the

Molecular Cell18

fected by ionic strength, it suggests that other forcesrather than just electrostatic ones are driving the inter-action.

So far it is not clear whether the functional p53 DNAbinding unit is the monomer, dimer, or tetramer (Nichollset al., 2002). Notably, the results presented here showthat HIF-1� ODD domain binds to a dimer of p53 despitethe fact that p53 is generally thought to exist as a dimerof dimers and that it would, therefore, also bind to thetetrameric form. The results presented in this manuscriptsuggest that two p53 core domains contained withinone p53 dimer bind to two different binding sites in asingle molecule of HIF-1� ODD domain. The oligomericstate of p53 affects the binding to HIF-1�, making ittight and cooperative simply by the chelate effect.

Biological ImplicationsThe HIF-1� ODD domain plays a dual role in the contextof the full-length protein: (1) it functions as a transcrip-tional activator which stimulates gene expression viaprotein-protein interactions with the basal machinery,and (2) it senses changes in oxygen levels through enzy-matic hydroxylation of two of its prolines, resulting insubsequent ubiquitination by the VHL-elongin B-elonginC (VCB) complex followed by degradation. The HIF-1�ODD domain is a very versatile domain, such that, be-sides recognizing the VCB complex and the basal tran-scriptional machinery, it also interacts with ARD1 (Jeonget al., 2002), with three different PHDs (Schofield andRatcliffe, 2004), and, as reported here, with p53. Thismultiple binding property may be achieved due to itsunfolded nature, which allows the protein to recognizedifferent biological partners with different specificitiesby using diverse mechanisms. Binding to VCB is entropi-cally driven by the formation of two hydrogen bondswith the hydroxyl group (Hon et al., 2002; Min et al.,Figure 6. Binding of HIF-1� ODD and Inhibitory Domain to Full-

Length p53 2002) or as reported here by making use of the chelate(A) Binding of HIF-1� 530–698 and HIF-1� 403–603 to p53 flQM effect when binding to a p53 homodimer. Multiple com-examined by fluorescence anisotropy. Binding of HIF-1� 403–603 binations of different posttranslational modifications, to-to p53 flQM at (�) 150 and (�) 200 mM ionic strength. Solid lines gether with coupling molecular recognition with binding,represent the fitting to a single binding site model with a drift. Bind- may confer to HIF-1� the plasticity needed to performing of HIF-1� 530–698 to p53 flQM at (�) 150 and (�) 200 mM

its intricate roles in the cell. Most natively unfolded pro-ionic strength.teins, including HIF-1�, seem to be proteins involved in(B) Fluorescence anisotropy competition assay between unlabeled

p53 consensus DNA and fluorescein-labeled HIF-1� 403–603 for cell signaling and cancer, where finely tuned regulationp53 flQM. Unlabeled DNA was added to a solution containing a is needed. Moreover, unfolded regions are commonlypreformed complex of 0.1 �M fluorescein-labeled HIF-1� 403–603 found among domains responsible for protein degrada-and 10 �M p53 flQM (initial anisotropy �0.1; see [A]). Consecutive tion (Hochstrasser, 1996), as is the case for the HIF-1�additions of unlabeled DNA resulted in the displacement of the

ODD domain. An example of the versatility of nativelyfluorescein-labeled HIF-1� 403–603 and formation of a new complexunfolded proteins is the binding of the kinase-induciblebetween p53 flQM and DNA. As more DNA is added, free HIF-1�

403–603 is generated until anisotropy reaches a value correspond- activation domain (KID) of CREB and of c-Myb to theing to that of the free molecule. KIX domain of CBP. The phosphorylated form of KID is

disordered in the free state but folds upon binding to theKIX domain of CBP while isolated c-Myb spontaneouslyadopts helical structure (Zor et al., 2002).acidic backbone of DNA contribute to the interaction.

The interaction between proteins and highly charged Here we demonstrated that HIF-1� ODD domain bindsto p53 via its core domain in a cooperative way, whichpolymers is entropically driven by the release of mono-

valent counterions from the latter (deHaseth et al., 1977; may abolish the ability of p53 to bind DNA and thushamper gene transactivation. The results presentedRecord et al., 1976). Further, there are two other iso-

forms of HIF-�, HIF-2� and HIF-3�. They are homolo- here corroborate the idea that the interaction of p53 withdifferent corepressors, such as HIF-1�, can modulategous proteins with similar domain organization but dif-

ferent spatial expression patterns; this may have an its transcriptional activity, resulting in decreased p53-dependent apoptosis under hypoxic conditions (Kou-impact on their binding to p53. Also, since the interaction

of HIF-1� with a dimer of p53 is not so extensively af- menis et al., 2001). The existence of an alternative HIF-1�


Size-Exclusion Chromatographydegradation mechanism in which p53 recruits Mdm2The following procedure was carried out for both HIF-1� 530–698has also been proposed (Ravi et al., 2000). The directand HIF-1� 403–603 proteins. 200 �l of purified protein was injectedinteraction between the HIF-1� ODD domain and p53,onto an analytical Superdex 200 HR10/30 column (Pharmacia) and

however, does not exclude the possibility of a ternary run at a flow rate of 0.5 ml/min at room temperature in an AKTAcomplex with Mdm2 where they all contact each other design XT Explorer 900 Kit (Pharmacia) instrument equipped with

a Monitor UV-900 detector and the Unicorn 3.10 software package.rather than Mdm2 acting as a bridge in between the twoThe buffer used was 25 mM NaPi (pH 7.2), 150 mM KCl, 10 mMas previously suggested (Chen et al., 2003).2-mercaptoethanol. Molecular weight standards (440, 232, 158, 67,43, and 25 kDa; Pharmacia) were run under the same conditions,and their elution volumes were used to create a calibration curveExperimental Procedures(Equation 1) from which the apparent molecular weight (Mr) of theproteins under study were calculated. The theoretical Stokes radiusBacterial Strains and Media

The Escherichia coli strain DH5� was used for manipulation of the (Rs) of a native (RaN) and fully unfolded (Rs

Urea) protein was determinedas described in Uversky (2002a) using Equations 2 and 3, respec-DNA constructs. The E. coli strain C41 was used for expression of

the recombinant proteins. Cells were grown in 2 TY media. tively.

log(Mr) � 8.1614 � 0.264(Ve) (1)PlasmidsThe plasmids pRSETHisLipoTEV-HIF1�530–698 and pRSETHisLi- log(RN

s ) � �(0.254 � 0.002) � (0.369 � 0.001) log(Mr) (2)poTEV-HIF1�403–603 contain the sequence coding for residues530–698 and 403–603 of the human wild-type HIF-1�, respectively.

log(RUreas ) � �(0.657 � 0.004) � (0.524 � 0.001) log(Mr) (3)The clones were obtained from Dr. Alex Buchberger (Max Planck

Institute, Germany) and reamplified by PCR and digested withBamHI and EcoRI. The amplified fragments were subsequently puri-

Far UV Circular Dichroismfied and ligated into the plasmid pRSETHisLipoTEV (Weinberg et

Temperature dependence of ellipticity was followed with a JASCOal., 2004) (containing a short histidine tag, the first 100 residues of

J-720 spectropolarimeter equipped with a Jasco PTC-348WI tem-the human lypoyl domain, and a TEV [tobacco etch virus] protease

perature controller. CD spectra were recorded using a 1 mm cuvetterecognition site) at the BamHI and EcoRI sites immediately down-

and protein concentration of 30 �M for HIF-1� 530–698 and 10 �Mstream of the TEV site.

for HIF-1� 403–603. Thermal denaturation was followed at 222 nm,with an increase of 1� per min, a time response of 10, and a band-width of 1 nm. Circular dichroism wavelength scan measurementsProtein Expression and Purificationwere followed at 20�C with an AVIV 2025F stopped flow circularThe plasmids pRSETHisLipoTEV-HIF1�530–698 and pRSETHisLi-dichroism spectrometer equipped with a Peltier temperature con-poTEV-HIF1�403–603 were each transformed into E. coli C41, platedtroller. CD spectra were recorded using a 1 mm cuvette at the sameonto 2 TY/ampicillin plates, and grown overnight at 37�C. A singleprotein concentrations as for thermal denaturation. Scan wave-colony was used to inoculate 50 ml of 2 TY medium containinglength was followed from 260 to 190 nm, with an increase of 0.5100 �g/ml of ampicillin and was grown for 2 hr at 37�C. 10 ml ofnm per step, an averaging time of 5 and a bandwidth of 1 nm. Allthis culture were used to inoculate 1 liter of 2 TY medium plussamples were dialyzed against a buffer containing 25 mM NaPi (pHantibiotic. At A600 �0.8, gene expression was induced with 1 mM7.2), 150 mM KCl, 1 mM DTE. Ellipticity was calculated from theisopropyl-1-thio-�-D-galactoside (IPTG), and the cells incubated forinstrument units with Equation 4.a further 4 hr at 37�C. Cells were harvested by centrifugation, and

the pellet was resuspended in lysis buffer (50 mM sodium phosphate [�](degree cm2 dmol�1) �buffer [NaPi] [pH 8.0], 300 mM NaCl, 10 mM imidazole) supple-mented with complete protease inhibitor (Roche). The cell suspen- CDobs 106

Pathlength [Protein](�M) No. residues(4)sion was sonicated, and cell debris was removed by centrifugation.

The supernatant was applied to a fast-flow Ni column (Qiagen) usinga FPLC system (Pharmacia) preequilibrated with lysis buffer. The

Analytical Ultracentrifugationbound protein was eluted with elution buffer (50 mM NaPi [pH 8.0],Experiments were performed at 10�C with a Beckman Optima XLI300 mM NaCl, 250 mM imidazole). Protein fractions were pooledanalytical ultracentrifuge using an An60Ti rotor. Data were collectedand digested with His-TEV (Lucast et al., 2001) protease while dialyz-at 280 nm for unlabeled protein and 492 nm for fluorescein-labeleding in lysis buffer. The digested protein was reintroduced onto theprotein. To assess the oligomeric state of the proteins, we used 100Ni column. The protein HIF-1� 530–698 was concentrated and finally�M unlabeled samples. The buffer consisted of 25 mM NaPi (pHpurified by gel filtration on a HiLoad 26/60 Superdex 75 column7.2), 150 mM KCl, 1 mM DTT. When testing the interaction between(Pharmacia) with 25 mM NaPi (pH 7.2), 150 mM KCl and stored atHIF-1� 530–698 and HIF-1� 403–603, both fluorescein-labeled, with�70�C. The protein HIF-1� 403–603 was purified in a Q-columnp53 core and p53 flQM, the protein concentrations used were 5 �Musing a gradient of 0.05–1 M NaCl over 12CV prior to gel filtrationfor the two former and 100 �M for the two latter. Buffers used wereon a HiLoad 26/60 Superdex 200 column (Pharmacia). The purity ofthe same as before but ionic strength (I ) was adjusted to either 150the proteins was analyzed by sodium dodecyl sulfate polyacrilamideor 200 mM by varying the salt concentration. All samples weregel electrophoresis (SDS-PAGE), and its identity was confirmed byloaded into six-sector 12 mm pathlength cells. Scans were collectedMALDI-TOF (Voyager-DE Biospectrometry Workstation). The pro-at 24 hr intervals until equilibrium was reached as judged by thetein concentration was determined spectrophotometrically usingfact that there were no further changes in subsequent scans. Datathe molar extinction coefficients 280 � 2560 M�1 cm�1 and 280 � 5120were analyzed using UltraSpin software (www.mrc-cpe.cam.ac.uk/M�1 cm�1 for HIF-1� 530–698 and HIF-1� 403–603, respectively,ultraspin) and Kaleidagraph program version 3.6 (Abelbeck Soft-obtained from the ProtParam program on the EXPASY server. Fluo-ware, Reading, PA) for graph plotting. Data were fitted to a single-rescein-labeled protein was produced using the Fluorescein-EXor double-exponential model indicating equilibrium of one or twoProtein Labeling kit from Molecular Probes following the manufac-species of unspecified mass.turer’s instructions. The p53 core domain (residues 94–312) and p53

core�tet protein (residues 94–360) were purified as described inWeinberg et al. (2004), and p53 full-length quadruple mutant (p53 Fluorescence Anisotropy

Different constructs of p53 were titrated into 0.1 �M fluorescein-flQM) was a kind gift from Caroline Blair (Centre for Protein Engi-neering, Cambridge, UK). p53 flQM is a superstable mutant (M133L/ labeled HIF-1� 530–698 or HIF-1� 403–603 in buffers with different

ionic strength at 10�C. At ionic strength of 20 mM, a 50 mM HEPESV203A/N239Y/N268D) of the full-length p53 protein (Joerger et al.,2004). (pH 7.2), 1 mM DTT buffer was used, and for ionic strengths of 56,

Molecular Cell20

150, and 200 mM, a 25 mM NaPi (pH 7.2), 1 mM DTT with 0, 90, gelstein, B. (1992). Definition of a consensus binding site for p53.Nat. Genet. 1, 45–49.and 150 mM NaCl buffer was used. Measurements were made in

a Perkin Elmer LS55 luminescence spectrometer equipped with a Fersht, A. (1999). Structure and Mechanism in Protein Science: AHamilton microlab dispenser controlled by laboratory software at Guide to Enzyme Catalysis and Protein Folding (New York: W.H.an excitation wavelength of 480 nm and emission wavelength of Freeman and Company).525 nm. For each data point, the mixture was incubated for 1 min

Freedman, S.J., Sun, Z.Y., Poy, F., Kung, A.L., Livingston, D.M.,with 30 s of stirring before measuring the fluorescence. Dissociation

Wagner, G., and Eck, M.J. (2002). Structural basis for recruitmentconstants were obtained by fitting anisotropy data to equations for

of CBP/p300 by hypoxia-inducible factor-1 alpha. Proc. Natl. Acad.one binding site or two nonidentical, noninteracting binding sites

Sci. USA 99, 5367–5372.as described (Eftink, 1997). In the competition binding assay, a stock

Goda, N., Ryan, E.H., Khadivi, B., McNulty, W., Rickert, C.R., andsolution of 80 �M unlabeled double-stranded p53 consensus DNAJonhson, S.R. (2003). Hypoxia-inducible factor 1� is essential for(5�-GAGCCCAGCATGCTTAGACATGTTCTGCTC-3�) (el-Deiry et al.,cell cycle arrest during hypoxia. Mol. Cell. Biol. 23, 359–369.1992) was added stepwise into 800 �l of preincubated mixture ofGoldenberg, D.P. (2003). Computational simulation of the statistical10 �M p53 flQM and 0.1 �M fluorescein-labeled HIF-1� 403–603 inproperties of unfolded proteins. J. Mol. Biol. 326, 1615–1633.the aforementioned buffer of 150 mM ionic strength.

Graeber, T.G., Peterson, J.F., Tsai, M., Monica, K., Fornace, A.J.,Acknowledgments Jr., and Giaccia, A.J. (1994). Hypoxia induces accumulation of p53

protein, but activation of a G1-phase checkpoint by low-oxygenWe thank Caroline Blair for p53 core and p53 flQM protein purifica- conditions is independent of p53 status. Mol. Cell. Biol. 14, 6264–tion, Dr. Rosario Fernandez-Fernandez for valuable discussions and 6277.helpful comments on this manuscript, and Dr. Stefan M.V. Freund for Graeber, T.G., Osmanian, C., Jacks, T., Housman, D.E., Koch, C.J.,the NMR experiment. N.S.-P. was supported by a Gates Cambridge Lowe, S.W., and Giaccia, A.J. (1996). Hypoxia-mediated selectionTrust scholarship. of cells with diminished apoptotic potential in solid tumours. Nature

379, 88–91.Received: August 24, 2004 Green, S.L., and Giaccia, A.J. (1998). Tumor hypoxia and the cellRevised: October 10, 2004 cycle: implications for malignant progression and response to ther-Accepted: October 27, 2004 apy. Cancer J. Sci. Am. 4, 218–223.Published: January 6, 2005

Hansson, L.O., Friedler, A., Freund, S., Rudiger, S., and Fersht, A.R.(2002). Two sequence motifs from HIF-1alpha bind to the DNA-

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Binding of Natively Unfolded HIF-1α ODD Domain to p53