Site-directed allostery perturbation to probe the negative regulation of hypoxia inducible factor-1α

The interaction between the intrinsically disordered transcription factor HIF-1α and the coactivator proteins p300/CBP is essential in the fast response to low oxygenation. The negative feedback regulator, CITED2, switches off the hypoxic response through a very efficient irreversible mechanism. The negative cooperativity with HIF-1α relies on the formation of a ternary intermediate that leads to allosteric structural changes in p300/CBP, in which the cooperative folding/binding of the CITED2 sequence motifs plays a key role. Understanding the contribution of a binding motif to the structural changes in relation to competition efficiency provides invaluable insights into the molecular mechanism. Our strategy is to site-directedly perturb the p300–CITED2 complex's structure without significantly affecting binding thermodynamics. In this way, the contribution of a sequence motif to the negative cooperativity with HIF-1α would mainly depend on the induced structural changes, and to a lesser extent on binding affinity. Using biophysical assays and NMR measurements, we show here that the interplay between the N-terminal tail and the rest of the binding motifs of CITED2 is crucial for the unidirectional displacement of HIF-1α. We introduce an advantageous approach for evaluating the roles of the different sequence parts with the help of motif-by-motif backbone perturbations.


Introduction
][10][11] In hypoxia, the a subunit of HIF-1 (HIF-1a) translocates to the nucleus and interacts with the HIF-1b subunit, forming HIF-1. 128][19] In a negative feedback loop, the CREB-binding protein/p300-interacting transactivator with ED-rich tail (CITED2) terminates the hypoxic response through the competition with HIF-1a for binding to p300/ CBP. 20,21Even though CITED2 and HIF-1a have similar binding affinities to p300/CBP, the competition is so efficient that CITED2 completely displaces HIF-1a in a switch-like, irreversible mechanism, allowing the precise regulation of the hypoxic response. 22Several experimental [22][23][24][25] and molecular modelling studies [26][27][28][29] investigated the underlying structural mechanism of this competition.These works agreed on basic concepts but highlighted different key determinants or provided alternative explanations for the virtually irreversible competition.
CITED2 and HIF-1a bind with their disordered C-terminal activation domain (CTAD) to the TAZ1 (or CH1) domain of p300/CBP 20,21,30,31 and form folded structures comprising several binding motifs (Fig. 1).Both display high-affinity interactions with p300/CBP, 22,25,30 show slow dissociation kinetics, 22,32 and compete through a partially overlapping binding site (Fig. 1e).The competition mechanism involves the formation of a transient ternary complex in which the CITED2 a A helix and the C-terminal helix/helices (a B and a C ) of HIF-1a are simultaneously bound to p300/CBP.][24][25][26][27][28][29] Several factors play an important role in this complex inhibitory mechanism.These include differences in binding thermodynamics, 25 displacement kinetics, 22,32 and intramolecular cooperativity between binding motifs. 22,23,29Changes in p300/CBP dynamics 33 and electrostatic forces 28,29 also contribute to effective inhibition.26,29 The binding of the CITED2 a A helix alters the conformation of the a4 helix of p300/CBP and destabilises the binding of HIF-1a a C . 24The conserved LPEL motif replacing HIF-1a LPQL is a key step in the displacement process; 22,26 mutations in this region significantly decrease competition efficiency. 23,34he binding of a A and LPEL is not sufficient for effective inhibition, the absence of the C-terminal hydrophobic region diminishes the competition efficiency of CITED2, suggesting that the C-terminus induces unidirectionality. 23The removal of the C-terminus, however, also results in a significantly lower binding affinity (2 orders of magnitude in K D , Table S1, ESI †), 23,25 which can affect the detected competition efficiency and mask the role of these residues in mediating allostery.Furthermore, our previous ITC data showed that CITED2 224-259 , comprising all three binding motifs (a A , loop, and C-terminus) is not an efficient competitor, suggesting that a fourth binding motif, the N-terminal tail of CITED2 (residues 218-223) plays an important role, 25 but the exact function of the N-terminal tail and how it cooperates with the rest of the CITED2 binding motifs is currently unclear.
Because the formation of the transient ternary complex is key to the negative cooperativity between the ligands, probing  30 and (b) to p300 TAZ1 domain (PDB: 1L3E). 31The helices of the TAZ1 domain are annotated a1-a4 from N to C terminus, and the spheres represent Zn atoms.HIF-1a helices are annotated a A -a C from N to C terminus; a A was not observed for the p300-bound structure.Structures of CITED2 CTAD (red) bound to (c) CBP TAZ1 domain (wheat, PDB: 1R8U) 21 and (d) to p300 TAZ1 domain (PDB:1P4Q). 20CITED2 binding motifs are annotated as N-tail, a A , loop, and C-term.(e) Overlayed structures of CITED2 and HIF-1a bound to TAZ1.Only one protein domain is shown for clarity.(f) Overlayed structures of p300-HIF-1a and p300-CITED2 complex showing the conserved LPE/QL motif.(g) Sequences of HIF-1a (residues 776-826 used in ITC, NMR measurements and as the competitor in FA titrations; residues 786-826 was used as the fluorescent tracer in FA titrations) and CITED2 peptides used in this study, with their binding motifs indicated above.Flu: 5,6-carboxyfluorescein, Ahx: aminohexanecarboxylic acid.

Paper
RSC Chemical Biology the extent to which each binding motif contributes to the intermediate formation would enhance our understanding of the underlying molecular mechanism.Furthermore, identifying binding sites that can tolerate structural perturbations with conserved competition mechanisms would provide useful insights into the mimicry and/or inhibition of the system.To this end, we set out to understand the role of the N-terminal tail of CITED2 in the competition mechanism and probe its interplay with the rest of the binding motifs leading to the formation of the ternary intermediate and the negative cooperativity with HIF-1a.
Our approach is to induce site-directed structural changes in p300 by altering the bound conformation of CITED2 and linking those changes to the competition mechanism.Because the effective competition between CITED2 and HIF-1a depends both on high-affinity binding and allostery, it is important to use only modifications that minimize the effect of direct binding.In this way, the observed competition efficiency of a modified ligand is mostly dependent on the induced structural change, rather than on its binding affinity.Consequently, we avoid such modifications that substantially decrease binding affinity (i.e.sequence truncations) and maintain the native-like properties (intrinsic disorder, charge, side-chains, and binding thermodynamics) of CITED2 variants.The plasticity of p300/ CBP allows their adaptation to various disordered ligands and participation in fuzzy interactions. 35We hypothesised that a similar adaptation would occur to the local conformational perturbations of the bound CITED2, resulting in close to native binding parameters, which we achieve through motif-by-motif backbone modifications.A small number of a to b 3 -amino acid replacements in CITED2 allowed us to produce native-like CITED2 variants and induce binding-site dependent structural changes in p300.Using a combination of direct binding, competition assays, and NMR measurements, here we provide further details on the underlying molecular mechanism of the p300/HIF-1a competition.

Results
The N-terminal residues of CITED2 drive the unidirectional replacement of HIF-1a The structure of the p300 and CBP TAZ1 domains is similar, but their reported complexes with CITED2 have differences in the more flexible N-and C-terminal regions (Fig. 1c-d).Considering both structures, we synthesised the shortest possible CITED2 sequence (CITED2 218-256 , Fig. 1g) comprising residues reported to be in contact with p300/CBP. 20,21CITED2 218-256 therefore contains the following binding motifs: an extended N-terminal region (N-tail, residues 218-223), an a-helix (a A , residues 224-235) that is connected through a loop (residues 236-253) to an aromatic/hydrophobic amino acid rich C-terminus (residues 253-256).To probe the role of the N-terminal tail we compared the competition efficiency CITED2 218-256 to a shorter sequence lacking the N-terminus (CITED2 224-256 , which we term CITED2DN Fig. 1g).Fluorescence anisotropy (FA) measurements indicated that CITED2 218-256 is more potent than CITED2DN in competing for binding to p300 330-420 (referred to as p300) with fluorescently labelled HIF-1a 786-826 (Flu-HIF-1a, Fig. 2a).The fitted apparent competition K D value of CITED2DN is similar to the K D,app of HIF-1a displacing itself (86 AE 5 and 57 AE 4 nM, respectively), indicating the absence of negative cooperativity.CITED2 218-256 , however, displays an inhibition with significantly higher efficacy (K D,app = 2.0 AE 0.9 nM).This is in line with previous observations, but the K D,app deviates from reported values 22 (Table S1, ESI †), which may be due to different experimental setups or protein/competitor sequences used.
To characterise the negative cooperativity between the ligands and the stability of the ternary intermediate, the following ITC titrations were performed: direct titration of HIF-1a and CITED2 variants to p300 and competition titration of CITED2 variants to a preformed p300-HIF-1a complex.7][38] From this model we extracted the direct binding affinities K D and DH for CITED2 variants and the parameters Dg and Dh, which represent the additional Gibbs energy and enthalpy due to cooperative interactions when CITED2 is bound to the Fitted parameters are listed in Table 1.

RSC Chemical Biology
Paper p300-HIF-1a complex relative to CITED2 binding to free p300.We observed a similar enthalpically favoured high affinity direct interaction with p300 with K D = 10.6 nM for CITED2 218-256 and 50.5 nM for CITED2DN (Fig. 2b, Fig. S1 (ESI †), Table 1).In line with the anisotropy data the two CITED2 sequences showed very different competition efficiencies (Fig. 2c and Table 1).The small positive Dg (1.2 kcal mol À1 ) and the Dh of 26 kcal mol À1 fitted for the competition of CITED2 218-256 indicated an entropy-driven negative cooperativity with HIF-1a through the formation of a transient ternary intermediate.The competition of CITED2DN against p300-HIF-1a resulted in Dg value greater than 4 kcal mol À1 (and Dh = 0, Table 1.) indicating that the ternary complex formation is unlikely, resulting in the loss of the allosteric communication between the ligands.The effect of the N-terminal CITED2 residues on the conformation of p300 was characterised by solution NMR using 13 C-, 15 N-labelled p300 330-424 (referred to as p300) in complex with the different CITED2 peptides.The calculated chemical shift perturbations using the assigned 1 H and 15 N resonances show that the two peptides induce different structural changes (Fig. 3 and Fig. S4, ESI †).The absence of the N-terminal CITED2 residues had a major effect on the amide resonances of p300 a1 residues 347-357, a3 residues 401-407, and a4 residues 418-420 (Fig. 3a and b).The observed changes were spatially close to the expected binding site of the N-terminal residues, suggesting that these residues interact with the protein surface.Since the core of p300 is tightly packed with hydrophobic amino acids, the CH 3 chemical shifts of these residues can indicate the overall structural change.Significant differences in CH 3 chemical shifts were observed for residues L342, L346 and L417 (Fig. S5, ESI †).These are located at the a1/a4 interface of p300 (Fig. 3b), suggesting that the presence of CITED2 N-terminal residues alters the relative orientation of these helices.
To investigate the reversibility of the competition, samples containing p300 with equimolar amounts of HIF-1a and CITED2 218-256 or CITED2DN were prepared.For the p300:HIF-1a:CITED2DN sample, we observed chemical shifts that correspond to the binary complexes of p300-HIF-1a and p300-CITED2DN (Fig. 3c and Fig. S6, ESI †), indicating that both complexes are present in the solution.For p300:HIF-1a:CITED2 218-256 , only resonances corresponding to the CITED2-bound state were detected (Fig. 3d and Fig. S6, ESI †).These data strongly supported that the presence of the N-terminal residues is not only required for the negative cooperativity through the transient ternary complex formation, but also results in the conformational lock that renders the competition unidirectional.To rule out the effect of the slightly decreased affinity of CITED2DN on the competition results we repeated the FA and NMR experiments with CITED2 224-259 which has a longer C terminus and a K D similar to CITED2 218-256 25 (Table S1, ESI †).
This modification, however, did not influence competition efficiency or unidirectionality (Fig. S7, ESI †), further confirming that the absence of the N-terminal tail is detrimental to the negative cooperativity with HIF-1a.

Backbone perturbation strategy to probe the interplay between binding motifs
The above analysis highlighted a determining role of the N-terminus; however, the literature results clearly indicate that a fine interplay between CITED2 segments are responsible for the negative regulation of HIF-1a.To probe cooperativity between the N-terminus and the rest of the binding motifs (a A helix, loop and C-terminus), our goal was to induce sitedirected structural changes in p300 by perturbing the bound conformation of CITED2 motif-by-motif.The induced structural changes are then correlated with the ability of the modified CITED2 to inhibit p300-HIF-1a.To separate the effect of direct binding affinity on the competition efficiency we set out to prepare native-like CITED2 variants, maintain the intrinsic disorder of the ligand, the original side-chain functionality, and the enthalpy-driven high-affinity interaction with p300.A backbone modification strategy fulfils all these requirements, for which we selected b 3 -amino acid replacements.0][41][42][43] The extra methylene group in the backbone influences the local conformational preferences, thus structural adaptation of the target protein can be expected.Based on the above considerations, six CITED2 variants were synthesized (1-4, Fig. 4a) by modularly incorporating two to three b 3 -residues.To keep the perturbation effects of the modifications at a controlled level, Paper RSC Chemical Biology our aim was to replace amino acids that have little contact with p300 in the bound state.This was supported by computational alanine scanning using BAlaS, 44,45 and solvent-exposed amino acids whose side chains have only a small contribution to the binding free energy (o4 kJ mol À1 ) were selected for replacement or removal (Fig. S8, ESI †).To tune the distance between the aA helix and the C-terminus, three loop-region variants were prepared (3a-c), including deletions of amino acids to compensate for the backbone elongation caused by the additional methylene group of the b 3 -amino acids.To ensure that the modifications do not significantly affect the overall conformation of the CITED2 variants, circular dichroism (CD) measurements were carried out.CD spectra recorded for the free peptides 1-4 showed random coil conformation, with a slight helical content indicated by the minimum at 220 nm (Fig. 4b).This pointed toward that the presence of b 3 -amino acids did not induce unwanted folding, and the modified peptides retained their disordered nature.The binding and competition efficiency of the CITED2 variants was examined using the same ITC method described

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above, from which the direct binding and cooperative parameters were derived (Fig. 5, Table 1; Fig. S9-S10, ESI †).Regarding their direct interaction with p300 sequences 1, 2, and 4 displayed a similar enthalpy-driven interaction with affinities (K D in the range of 16-19 nM) comparable to the native CITED2 sequence (Table 1.).Modifications in the loop, however, resulted in more pronounced effects.Replacement in the conserved LPEL motif (3a) resulted in a 50-fold increase in K D with an excessively different thermodynamic profile.b-amino acid replacements were more tolerated when the conserved region was not directly involved.The thermodynamic signatures of the binding of 3b and 3c to p300 resembled more to the native CITED2 with K D values 98 nM and 29 nM, respectively (Fig. 5 and Table 1).Overall, the less favourable binding enthalpy of the CITED2 variants pointed to loss of noncovalent interactions, which is compensated by decreased entropic cost of binding.This change may be attributed to the presence of the additional methylene groups, and can be the result of multiple contributing factors, such as a more favourable desolvation during folding, 46 increased local flexibility of the bound structure 25 or favourable displacement of surface waters. 47It should be noted that global conformational changes of p300 can also affect the detected thermodynamic profiles and cannot be ruled out based on these data.
The global analysis revealed that compounds 1, 2, and 4 maintained their ability to form the transient ternary intermediate indicated by the small positive Dg values and exhibited negative cooperativity with HIF-1a (Table 1).The lower Ds values of these variants point to decreased entropic stabilisation of the ternary intermediate, which can be the result of their less unfavourable direct binding entropy.Modification of the tail region (1) resulted in slightly higher Dg (2.6 kcal mol À1 ) indicating that the transient intermediate is less stable, and this region is more sensitive to alterations, further underlining the role of the N-terminal tail in mediating effective competition.CITED2 variants containing loop modifications (3a-c) completely lost their ability to cooperate negatively with HIF-1a, even when the direct binding affinity was high (3c).Similar results were obtained using fluorescence anisotropy (Fig. S11 and Table S2, ESI †) where CITED2 variants 2 and 4 exhibited inhibitory efficiency comparable to the native sequence.
To elucidate the effect of the b-amino acid modifications on the structure of p300, we recorded 1 H-15 N HSQC spectra using 13 C-, 15 N-labelled p300 in complex with all the peptides.The weighted average chemical shift differences were calculated relative to the native p300-CITED2 218-256 complex (Fig. 6, Fig. S12-S17, ESI †).Generally, the observed chemical shift differences were low (Dd o 0.2 ppm), indicating that the overall folds of the complexes are similar, but the location of the observed changes was highly dependent on the modification site.b-amino acid replacements in the N-terminal tail of CITED2 (1) resulted in the change in the terminal regions of a1, a3, and a4 of p300, which was similar to what was observed for the p300-CITED2DN complex, further supporting the importance of the N-terminus in binding.The modified a A helix (2) and C-terminal hydrophobic region (4) also had only local effects on the structure of p300, corresponding to the interaction sites of these binding motifs.Modifications in the loop region (3a-c), however, resulted in a more distributed effect, and significant chemical shift differences were observed for amino acids distant from the loop interaction site (Fig. 6).This suggested that the loop region is critical for connecting the recognition elements, and local modification of the loop might influence the placement of the other binding motifs.
The CH 3 resonance changes corresponded well with the amide proton chemical shifts (Fig. 6 red arrows, Fig. S18-S19,  ESI †).Methyl protons of L346 and L417 at the interface of a1 and a4 are mainly affected by the binding of 1, while L342 shifted the most upon interaction with 3a-c, resulting in chemical shift values close to the CITED2DN.
Overall, structural changes associated with modifications in the a A helix and C-terminus of CITED2 can be tolerated, and the negative cooperativity with HIF-1a is retained.The N-terminus is more sensitive to backbone modifications, but to some extent retains the ability to act through the allosteric process.On the other hand, modifications in the loop of CITED2 are detrimental to the competition efficiency, which is accompanied by distributed structural changes, indicating strong cooperativity between these sequence motifs.

Discussion
Understanding an effective competition mechanism to which several factors contribute, such as the high binding affinity of the competing ligands, the cooperativity between the binding motifs, and allosteric structural changes, can be challenging.Several studies investigated the molecular details by which CITED2 disrupts the p300/CBP-HIF-1a complex, [22][23][24][25][26][27][28][29]33 often highlighting different key determinants for the unidirectional competition. Her, we clarify the role of a crucial sequence part of CITED2 that is necessary for negative cooperativity with HIF-1a.We also introduce a strategy with which the interplay between binding motifs and their individual contribution to allostery can be efficiently probed.The two sequence variants, CITED2 218-256 and CITED2 224-256 (CITED2DN) display an enthalpy-driven high-affinity interaction with p300, but their ability to displace HIF-1a significantly differs (Fig. 2 and Table 1).The presence of N-terminal residues (CITED2 218-223) is required for negative cooperativity through the formation of transient ternary complex (Fig. 2).Our NMR data indicate that these residues interact with p300 and induce conformational changes that affect the a1/ a4 interface (Fig. 3).This is in line with the previously reported mechanism but reveals a different effector.It has been shown that the conformational change of the a4 helix of p300/CBP mediates the negative cooperativity between CITED2 a A and HIF-1a a C .24 Still, our data indicate that without the N-terminal tail, the competition is reversible (Fig. 3c).Therefore, N-terminal residues are essential to lock the conformation into the CITED2-bound state.Although it has not been explicitly stated, an indication of such a mechanism could be seen from the NMR data of a fusion peptide comprising CITED2 residues 216-246 connected to HIF-1a residues 796-826.24 An L822 to alanine mutation in the fusion peptide, which impairs the binding of the HIF-1a a C helix, resulted in significant chemical shift changes of residues corresponding to the CITED2 N-terminal tail, suggesting that these residues are involved in the displacement of the HIF-1a a C helix.
To probe the interplay between the N-terminal tail with all the binding motifs, we introduce a strategy that maintains the native properties of CITED2 but allows binding site-targeted investigation of individual sequence parts.Using b-amino acid replacements in a native ligand is a well-known approach for designing peptidomimetics with improved properties. 40,42,43,48elical and loop structures containing b 3 -amino acids closely resemble the native fold and reproduce the binding contacts with the target protein. 39,41,49,50We expected that native-like CITED2 variants could be obtained with a to b 3 amino acid replacements, to which p300 would respond with binding-site dependent structural adaptation.Using a motif-by-motif screen, we prepared CITED2 variants that bind with high affinity to p300 and show similar thermodynamic fingerprints (Fig. 5) that helped us to probe the correlation between structural adaptation and competition efficiency.
Backbone modifications of the a A helix (2) and the hydrophobic C terminus (4) of CITED2 cause local structural changes in p300 that are accompanied by retained negative cooperativity with HIF-1a (Fig. 5 and 6, Table 1).This indicates that these motifs are not directly involved in the structural change that renders the competition unidirectional.Taking into account that the contribution of the N-terminal tail to p300 binding affinity is not significant (Table 1), it suggests that the role of the simultaneously bound a A and C terminus is to provide high affinity, thus a strong tether for the tail to bind.This is in line with the reported sequence truncation data.Modifications that decrease the p300/CBP binding affinity of CITED2, such as removing the hydrophobic C-terminus, 23 can lead to a weakened tether for the N-terminal tail; the conformational lock is lost, and the competition becomes reversible.Molecular dynamic simulations suggested that the effector in the allosteric process is the highly conserved charged residues of the CITED2 helix immediately after the N-terminal tail. 29In conjunction with our data, the role of this charged patch is more likely to contribute to the strong tether for the N-terminal tail.
The N-terminal tail is more sensitive to backbone modifications.The location of structural changes in p300 upon binding to the a/b-modified N-terminus matches with those that we observed for CITED2DN, but the changes are less pronounced (Fig. 6, 3b).This is in good agreement with the ability of compound 1 to partially retain its function in forming the transient intermediate and acting as the allosteric effector.This also holds promise that with careful design the function of this key sequence part can be mimicked.
On the other hand, the loop region of CITED2 is highly sensitive to modifications leading to decreased p300-binding affinity (3a-b), and loss of competition efficiency even when binding parameters closer to the native CITED2 are retained (3c, Fig. 5).Several studies highlighted that CITED2 binding motifs act synergistically in which the loop region is functionally important. 23,26Our data indicate a similar role; the loss in competition efficiency accompanied by the distributed structural changes in p300 point toward that all binding motifs are affected by the modified loop that leads to weak cooperativity or the misplacement of important binding motifs.
In conjunction with previous studies, our data provide a more detailed picture of the molecular mechanism of the competition between CITED2 and HIF-1a.CITED2 218-256 is the minimal sequence required for the irreversible competition with HIF-1a, in which the concerted action of the binding motifs that leads to the conformational lock provided by the N-terminal tail is crucial.][53][54] An inhibitory ligand that exploits this unidirectional competition with HIF-1a would be highly advantageous.Our data provide insights for modification sites developing such ligand.
Sensitive regulation of the hypoxic response essentially relies on the intrinsic disorder of CITED2 and HIF-1a; since IDPs are prevalent in eukaryotic cells, determining crucial factors that contribute to their regulatory mechanisms would provide insight into their function.7][58][59] Such complexity often hinders understanding of the underlying processes in molecular detail.The site-directed conformational perturbation strategy presented in this work provides a general approach to gain mechanistic insights for IDP-mediated interactions with multiple, cooperatively acting binding motifs and inform inhibitor design for interactions of therapeutic relevance.

Fig. 1
Fig.1Structure of p300/CBP bound to HIF-1a and CITED2.(a) Structure of HIF-1a CTAD (blue) bound to CBP TAZ1 domain (wheat) (PDB: 1L8C)30 and (b) to p300 TAZ1 domain (PDB: 1L3E).31The helices of the TAZ1 domain are annotated a1-a4 from N to C terminus, and the spheres represent Zn atoms.HIF-1a helices are annotated a A -a C from N to C terminus; a A was not observed for the p300-bound structure.Structures of CITED2 CTAD (red) bound to (c) CBP TAZ1 domain (wheat, PDB: 1R8U)21 and (d) to p300 TAZ1 domain (PDB:1P4Q).20 CITED2 binding motifs are annotated as N-tail, a A , loop, and C-term.(e) Overlayed structures of CITED2 and HIF-1a bound to TAZ1.Only one protein domain is shown for clarity.(f) Overlayed structures of p300-HIF-1a and p300-CITED2 complex showing the conserved LPE/QL motif.(g) Sequences of HIF-1a (residues 776-826 used in ITC, NMR measurements and as the competitor in FA titrations; residues 786-826 was used as the fluorescent tracer in FA titrations) and CITED2 peptides used in this study, with their binding motifs indicated above.Flu: 5,6-carboxyfluorescein, Ahx: aminohexanecarboxylic acid.

Fig. 4
Fig. 4 Sequences and overall structure of the b-amino acid modified CITED2 variants.(a) Sequences of modified CITED2 variants with binding motifs indicated above.ab 3 substitutions are highlighted in red, and deletions are represented by a line.(b) CD curves for CITED2 and variants in 20 mM Na-phosphate, 1 mM DTT, pH 7.4 at 20 mM concentration at room temperature.

Fig. 5
Fig. 5 Direct binding to p300 and competition with p300-HIF-1a of the b-amino acid modified CITED2 variants.Direct binding (circles) and competition (squares) titrations were fitted globally to a model that involved the formation of ternary intermediate (Fig. S20, ESI †).Titrations were carried out in 40 mM sodium phosphate, pH 7.5 100 mM NaCl, 1 mM DTT buffer using 5 mM protein in the cell and 60 mM ligand in the syringe at 35 1C.Error bars represent raw data integration errors.Raw data and fitted thermograms are included in the ESI † (Fig. S9-S10).

Fig. 6
Fig. 6 NMR chemical shift changes of p300 upon binding to the b-amino acid modified CITED2 variants.Weighted average 1 H-15 N chemical shift differences between the p300-CITED2 variants and p300-CITED2 218-256 complexes.Dd = [(Dd H ) 2 + (Dd N /5) 2 ] 1/2 .The significance level was determined using the average Dd and standard deviation for all the CITED2 variants, residues above the significance level (Dd 4 0.9 Dd average + s) highlighted blue in the bar graph and mapped onto the p300-CITED2 structure, CITED2 residues in red, b 3 -amino acid modifications sites represented as yellow spheres, removed amino acids highlighted grey.Black diamonds on the bar graphs indicate unassigned or proline residues.Red arrows indicate residues for which significant methyl proton chemical shift differences were detected (Fig. S18-S19, ESI †).

Table 1
Thermodynamic parameters for direct binding and competition with p300-HIF-1a.ITC data were fitted globally to a model that included ternary intermediate formation (Fig.S20, ESI) using constrained K D and DH for HIF-1a (Fig.S2, ESI).Dg (and Dh) represent the additional Gibbs energy (and enthalpy) due to cooperative interactions when CITED2 is bound to the p300-HIF-1a complex relative to CITED2 binding to free p300.Positive Dg and Dh values indicate negative cooperativity with an unfavourable contribution to enthalpy.68% confidence intervals of the fitting are included in brackets DH (kcal mol À1 ) DS (cal mol À1 K À1 ) Dg (kcal mol À1 ) Dh (kcal mol À1 ) Ds (cal mol À1 K À1 )