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Enhanced Binding Affinity via Destabilization of the Unbound State: A Millisecond Hydrogen / Deuterium Exchange Study on the Interaction Between p53 and a Pleckstrin Homology Domain Shaolong Zhu, Rahima Khatun, Cristina Lento, Yi Sheng, and Derek J. Wilson Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00193 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 13, 2017

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Enhanced Binding Affinity via Destabilization of the Unbound State: A Millisecond Hydrogen / Deuterium Exchange Study on the Interaction Between p53 and a Pleckstrin Homology Domain

Shaolong Zhu1, Rahima Khatun2, Cristina Lento1, Yi Sheng2, and Derek J. Wilson1,3*

1

Departmentof Chemistry, York University, Toronto, Ontario, Canada M3J 1P3 2

3

Departmentof Biology, York University, Toronto, Ontario, Canada M3J 1P3

Centre for Research in Mass Spectrometry, York University, Toronto, Ontario, Canada M3J 1P3

*Corresponding authors: Derek Wilson E-mail: [email protected]; Phone: (416) 7362100 x20786

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Abstract The incorporation of intrinsically disordered domains enables proteins to engage a wide variety of targets, with phosphorylation often modulating target specificity and affinity. Although phosphorylation can clearly act as a chemical driver of complexation in structured proteins, e.g. by abrogating or permitting new charge/charge interactions, the basis for enhancement of the hydrophobically-driven interactions that are typical of disordered protein / target complexation is less clear. To determine how phosphorylation can positively impact target recruitment in disordered domains, we have examined the interaction between the disordered N-terminal TransActivation Domain (TAD) of p53 and the pleckstrin homology (PH) domain of p62. Using Time-Resolved ElectroSpray Ionisation with Hydrogen Deuterium eXchange (TRESI-HDX), we demonstrate that phosphorylation has little effect on the conformation of the p53 TAD when it is bound to PH, but instead increases conformational disorder in the unbound state. We propose that this increase in disorder creates a wider free-energy gap between the free and bound states, providing a target-independent mechanism for enhanced binding when the phosphorylated and unphosphorylated p53:target complexes have similar free energies.

Keywords: tumor suppressor p53, checkpoint kinase 2, pleckstrin homology domain, protein dynamics, time-resolved electrospray ionization, hydrogen-deuterium exchange, binding affinity


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Protein-protein interactions (PPIs) are critical to virtually all biological processes including signal transduction, catalysis, and cell cycle regulation.1–3 Many of the proteins that act as hubs in PPI networks have an abundance of disordered domains whose function is modulated by post-translational modifications (PTMs) acting as non-binary molecular switches.4,5,6 These covalent chemical modifications can either function by changing local chemistry of global structure or their protein targets. Whereas the importance of structural changes due to PTMs have long been appreciated, there is increasing evidence that changes in protein dynamics upon post-translational modification can significantly affect PPI dissociation constants (Kd) without an appreciable change in the ground-state structure.7,8 Moreover, it would be difficult to account for some PTM-driven changes in Kd in intrinsically disordered proteins (or domains) based on conventional structural or chemical arguments. The p53 N-Terminal transactivation Domain (NTD) is intrinsically disordered and contains many sequence motifs that are known to drive interactions with various transcriptional regulatory factors such as MDM2, TATA binding protein, CBP/p300, replication protein A (RPA) and the p62 subunit of general transcription factor II H (GTFIIH), among many others.6,9,10 In unstressed cells, the tumor-suppressor p53 is expressed at low levels and is short-lived. However, upon exposure to cellular stress, p53 becomes activated and stabilised through a series of post-translational modifications.5,11,12 Once activated, it regulates a wide range of cellular responses such as apoptosis, cell cycle arrest, and/or DNA repair depending on the nature of the stress. Phosphorylation of the NTD tightly regulates the activity and stability of p53. For example, phosphorylation can regulate PPI either positively (e.g., CBP and p62), negatively (e.g., MDM2), or neutrally (e.g. RPA).11 Interaction of p53 NTD with p62 is important for the recruitment of p53 to the TFIIH complex to initiate the transcriptional elongation process.13 Specifically, the p53 NTD binds to the pleckstrin homology (PH) domain of the p62 subunit.6 The apparent Kd for this interaction is 3.2 µM when p53 is unmodified.6 However when phosphorylated at either position Ser46 or Thr55, the Kd decreases by 6 fold to approximately 500nM, and dual phosphorylation at both sites further decreases the Kd to 100nM.6 Some residues critical to binding have been determined for the p53 NTD/PH


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domain interaction, with mutagenesis identifying Lys18 of the PH domain as being necessary for tighter binding when the p53 NTD is phosphorylated.6 From a structural point of view, binding to MDM2 in particular appears to involve stabilization of amphipathic helices in p53 driven by hydrophobic interactions involving (on p53) Phe19, Trp23, and Leu26.14 However, since helix stabilization upon binding can occur in this region in the presence or absence of phosphorylation, this observation does not in itself provide a strong rationale for a phosphorylation-driven modulation of Kd.7,8 Recent work has also shown that a subsection of the p53 NTD (transactivation domain 2) binds the PH domain from human p62 in an extended ‘string’ configuration upon phosphorylation.15 To examine the mechanism for phosphorylation-enhanced binding in an intrinsically-disordered domain, we examined the changes in conformation and dynamics that occur in the p53 NTD upon phosphorylation and upon binding to the p62 PH domain using Time-Resolved Electrospray Ionization Hydrogen Deuterium Exchange (TRESI-HDX).16–18 This technique measures slight changes in the rate of deuterium uptake at backbone amides on the millisecond timescale,19 making it a highly sensitive probe of residual structure in disordered proteins/domains and solvent accessibility in binding interactions.17,20 Our aim is to experimentally examine residual structure in the unmodified p53 NTD and to reveal the structural / dynamic basis for recruitment of cofactors upon phosphorylation.

Materials and Methods

Reagents. pDONR223 plasmid was supplied by Addgene. Ni Sepharose 6 Fast Flow and Glutathione Sepharose 4B resins were purchased from GE Life Sciences. Zeba desalting columns, titanium dioxide phosphopeptide enrichment kit, Tris, magnesium chloride (MgCl2), and dithiothreitol (DTT) were purchased from Thermo Fisher. Adenosine triphosphate (ATP) and bovine serum albumin (BSA) were purchased from New England Biolabs. Ammonium acetate and acetic acid were purchased from Sigma Aldrich. BioSep-SEC-S4000 HPLC column was supplied by Phenomenex. Poly(methyl methacrylate) was purchased from Professional Plastics. Polyimide coated glass capillaries (O.D. 0.152mm) and metal


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capillaries (I.D. 0.178mm) were purchased from Polymicro Technologies and McMaster-Carr respectively.

Protein expression and purification. p53 NTD- pET28a construct and Chk2- pET28a plasmids were kindly provided by Dr. Yi Sheng (York University). PH domain was cloned from pDONR223 plasmid encoding general transcription factor II H, polypeptide I gene into pGEX4T2 plasmid. The proteins were bacterially expressed and further purified using Ni Sepharose 6 Fast Flow resin (p53 NTD and Chk2) and Glutathione Sepharose 4B resin (PH-GST construct). Subsequently, the proteins were concentrated using Vivaspin concentrators and buffer exchanged using zeba desalting columns to remove high concentrations of imidazole or reduced glutathione.

In vitro p53 NTD phosphorylation. p53 NTD phosphorylation was carried out by incubating 70uM of p53 NTD with 10uM of Chk2 kinase in phosphorylation buffer, 50mM Tris-HCl (pH 7.5), 10mM MgCl2, 5mM DTT and 3mM ATP for two hours. The kinase was deactivated by boiling the reaction at 80°C for 5 minutes, the precipitated kinase was then centrifuged and discarded.

Phosphopeptides identification and localization. The reaction was either digested on the microfluidic chip using pepsin agarose (for HDX experiments) or tryptically digested overnight (for phosphopeptide ID). In phosphopeptide ID experiments, phosphopeptides from digested samples were enriched using titanium dioxide resins and were identified using MS/MS or LC-MS/MS.

In vitro binding studies. To determine the binding of p53 NTD to PH domain, proteins were incubated in equimolar concentrations (50µM:50µM) overnight in 4°C. 30uL of the reaction was then loaded on


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BioSep-SEC-S4000 HPLC column (5µm resin) coupled to MS along with 5uM of internal BSA standard. Separation was performed at a flow rate of 0.2ml/min in SEC buffer (200mM ammonium acetate, pH 7.5) with a method time of 60 minutes.

TRESI-HDX. The microfluidic device was constructed as previously described.17 The rapid mixing device and acid quenching lines were connected using valco t-mixer externally. The output of the valco tmixer was then connected to the PMMA chip for infusion into the digestion chamber. Protein and deuterium oxide were mixed at a 1:1 ratio at flow rates of 2:2 ul/min. Acid flow rate was 12 ul/min. All of the experiments were conducted in triplicates. HDX data were analyzed using an in-house built program that uses distribution fitting to determine the uptake levels for individual peptides.21 Intrinsic rates which are dependent on the primary sequence were determined using the SPHERE web server.22,23 Back exchange was negligible ( 2 regions highlighted in blue. (Bottom) Averaged protection factor for phospho-p53 NTD. In this case, all PFs are far below the threshold set for the unmodified


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Regions with PF > 2 align closely with the regions predicted to exhibit residual secondary structures in the 1D protein structure prediction server as well as PHYRE server. For unmodified p53, residues 18-21, 32-43, and 53-59 meet this criterion. These are also the regions that exhibited more pronounced lowered exchange at the earliest HDX mixing time of 0.2s (Figure 2). For the phospho-p53 NTD, on the other hand, all the regions exhibited PFs below 1.3, which indicates a conformational ensemble that is nearly completely unstructured. While there was an increase in the rate of deuterium uptake overall, the C-terminal helix exhibited partial protection from exchange that persisted beyond 2 s of labelling time, appearing in our kinetics data as a decrease in uptake amplitude (Figure 4). These data indicate the formation of a small number of relatively stable hydrogen bonds (PF > 10) in approximately 30% of the C-terminal segment, while the reminder of the sequence is destabilized compared to the unphosphorylated state. Such local stabilization could allow for the exposure of other linear motifs within the NTD that facilitate binding with target cofactors.

Figure 4. Kinetic plots for three sample peptides: 3-29 (3xPhos), 18-47 (3xPhos), and 44-52. Dashed lines represent the calculated intrinsic rate of HDX for the peptide. The effect of covalently bound phosphate groups on the intrinsic HDX rates is unknown but should be negligible for large peptides. Solid lines represent the experimental data.

Ultimately, the HDX and IMS data combined show unabiguously that unmodified and phosphorylated p53 NTD exhibit substantially different dynamic behaviour. Unmodified p53 NTD adopts a partially-collapsed conformation with the presence of residual helical structures and phosphorylated p53 NTD undergoes a global conformational extension with partial stabilisation of a small segment of the C-


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terminal region.29 These distinctive dynamics may represent the basis of Kd modulation when binding to the PH domain and other p53 NTD binding partners. p53 and phospho-p53 NTD complexation with the PH domain of p62. In order to further investigate the role of phosphorylation in p53 NTD complexation, we examined the interaction between unmodified and phospho-p53 NTD and the PH domain of p62. The complex is not easily detected in the native mass spectrum, though minor peaks corresponding to three charge states of the complex are observed (Figure S2). To ensure that the p53 NTD and the PH domain were forming a complex under our conditions in solution, an equimolar mixture (50µM each) of the two proteins was examined by SEC-MS. Using a BioSep SEC s4000 column and a flow rate of 0.2ml/min, free p53 NTD and PH eluted at a retention time of 53.4 and 51.4 minutes respectively while p53 of the complex eluted at 51.2 minutes (Figure 5A). Complex formation was further validated by running the complex on a native agarose gel as shown in Figure 5B.

Figure 5. A. SEC-MS chromatogram for free p53 NTD, PH domain, and p53 NTD of the complex. Free p53 NTD and PH domain eluted at 53.4 and 51.4 minutes respectively, whereas the bound p53 NTD eluted at 51.4 minutes. B. Native agarose gel showing migrations of free p53 and PH domain as well as complexes at variable concentration ratios as shown.

Next, TRESI-HDX was used to provide a structural picture of the p53 or phospho-p53 complex with the PH domain, under identical experimental conditions to those employed in the unmodified p53 /


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phosphop53 analysis described above. Equimolar concentrations of 50µM were used to form the complex. Figure 6 shows difference plots of deuterium uptake (bound vs. unbound) for unmodified p53

Figure 6. Segment-averaged difference HDX data for PH-bound and unbound p53 NTD. The unmodified and phosphorylated species (black trace and grey trace, respectively) show similar uptake profiles when bound to PH. (black trace) and phospho-p53 (grey trace) at 0.95s of HDX mixing. Of the seven common peptides encompassing 75% sequence coverage, all showed negligible differences (±3%) except for short regions at the N- and C-termini.6


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Ultimately, this suggests that, in contrast to the unbound species, the ‘in-complex’ structure of the p53 NTD is not dependent on phosphorylation. Interestingly, data from NMR studies conducted by Okuda and Nishimura,15 suggested a similar finding where binding of the p53 transactivation domain 2 (TAD2, residues 41-62) to the PH domain of human p62 subunits yielded similar bound structures of TAD2 in phosphorylated and unphosphorylated states. However, they reported a string-like (extended conformation) binding mode as opposed to binding through an amphipathic helix. At the same time, Di Lello et al.,6 reported that p53 NTD was binding to PH domain of yeast homologue of p62 (Tfb1) occurs through an amphipatic helix (residues 47-55). It is important to note that although there are significant structural similarities, there is virtually no shared sequence identity between the two pH domain homologues, which might explain why the binding mode of the p53 NTD to the two proteins may very well be different. TRESI-HDX and IMS analysis of the unbound state indicates a general increase in disorder upon phosphorylation, which is likely to correspond to an elevated free energy relative to the unmodified species. Since the structure and dynamics of the bound-state appear to be independent of phosphorylation, increased disorder in the unbound state would result in a widened free energy gap between the bound and unbound species, resulting ultimately in a more favorable interaction through a substantial conformational change in p53 NTD upon binding to PH domain.. While not previously proposed for disordered proteins, the concept of increased affinity induced by destabilization of the unbound state is not without precedent. For instance, Horn et al. reported a similar scenario for human growth hormone, in which a particular mutant exhibited a 400-fold increase in affinity for its receptor due to destabilization of one of its four helices.34 In the intrinsically disordered protein Tau, phosphorylation is known to cause a loss of compactness in the structural ensemble and is used to modulate target binding affinity.20 Taken together, our results provide multiple lines of evidence for a disorder-driven mechanism of p53 NTD co-factor recruitment upon phosphorylation, at least for the positive regulations such as p62. It would be interesting to investigate other negative regulators such as MDM2 and RPA PPI as these exhibit a decrease and no


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change in binding affinity upon phosphorylation respectively. Results from such studies would shed light on the role of p53 NTD phosphorylation in regulation of cellular events from a structural perspective.

Conclusions Despite the unstructured nature of unmodified p53 NTD, our data indicate that it adopts a partially collapsed conformational ensemble with the presence of transient helices. TRESI-HDX analysis of the unphosphorylated structure locates the residual helices in good agreement with computational predictions. Upon phosphorylation, the p53 NTD adopts a more extended conformational ensemble that lacks residual structure except for a small region of the C-terminus. This structural feature may provide access to linear motifs important for co-factor recruitment, but the main thermodynamic driver for complexation appears to be destabilization of the unbound state, a conclusion that is supported by virtually identical HDX profiles for unmodified and phospho-p53 in the bound state. Ultimately, these results highlight the importance of protein dynamics in PTM-modulated molecular recognition, especially for disordered proteins and/or domains. More specifically, they reveal a target-agnostic mechanism for enhanced co-factor recruitment, through destabilization of the ‘native’ conformational ensemble.

FUNDING. D.W. and Y.S. are supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant program (DG-03860). D.W. is also supported by a Krembil Foundation Biomedical grant.

SUPPORTING INFORMATION. MS/MS spectra identifying p53 phosphorylation sites. Native mass spectrum of p53-NTD, PH and the PH:p53-NTD complex.


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Enhanced Binding Affinity via Destabilization of the Unbound State: A Millisecond Hydrogen / Deuterium Exchange Study on the Interaction Between p53 and the Pleckstrin Homology Domain Shaolong Zhu, Rahima Khatun, Cristina Lento, Yi Sheng, and Derek J. Wilson


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