Enhanced Binding Affinity via Destabilization of the Unbound State: A

Subscriber access provided by Purdue University Libraries

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

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

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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


Page 2 of 18

Page 3 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

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


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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


Page 4 of 18

Page 5 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

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


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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


Page 11 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

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-


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 18

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 /


Page 13 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

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


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 18

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


Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

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.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References (1) Goh, C.-S., Milburn, D., and Gerstein, M. (2004) Conformational changes associated with proteinprotein interactions. Curr. Opin. Struct. Biol. 14, 104–109. (2) Perkins, J. R., Diboun, I., Dessailly, B. H., Lees, J. G., and Orengo, C. (2010) Transient protein-protein interactions: structural, functional, and network properties. Struct. Lond. Engl. 1993 18, 1233–1243. (3) Jones, S., and Thornton, J. M. (1996) Principles of protein-protein interactions. Proc. Natl. Acad. Sci. U. S. A. 93, 13–20. (4) Neduva, V., and Russell, R. B. (2005) Linear motifs: evolutionary interaction switches. FEBS Lett. 579, 3342–3345. (5) Meek, D. W. (1998) Multisite phosphorylation and the integration of stress signals at p53. Cell. Signal. 10, 159–166. (6) Di Lello, P., Jenkins, L. M. M., Jones, T. N., Nguyen, B. D., Hara, T., Yamaguchi, H., Dikeakos, J. D., Appella, E., Legault, P., and Omichinski, J. G. (2006) Structure of the Tfb1/p53 complex: Insights into the interaction between the p62/Tfb1 subunit of TFIIH and the activation domain of p53. Mol. Cell 22, 731– 740. (7) Boehr, D. D., Nussinov, R., and Wright, P. E. (2009) The role of dynamic conformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5, 789–796. (8) Carroll, M. J., Mauldin, R. V., Gromova, A. V., Singleton, S. F., Collins, E. J., and Lee, A. L. (2012) Evidence for dynamics in proteins as a mechanism for ligand dissociation. Nat. Chem. Biol. 8, 246–252. (9) Chang, J., Kim, D. H., Lee, S. W., Choi, K. Y., and Sung, Y. C. (1995) Transactivation ability of p53 transcriptional activation domain is directly related to the binding affinity to TATA-binding protein. J. Biol. Chem. 270, 25014–25019. (10) Van Orden, K., Giebler, H. A., Lemasson, I., Gonzales, M., and Nyborg, J. K. (1999) Binding of p53 to the KIX domain of CREB binding protein. A potential link to human T-cell leukemia virus, type Iassociated leukemogenesis. J. Biol. Chem. 274, 26321–26328. (11) Jenkins, L. M. M., Durell, S. R., Mazur, S. J., and Appella, E. (2012) p53 N-terminal phosphorylation: a defining layer of complex regulation. Carcinogenesis 33, 1441–1449. (12) Ashcroft, M., Kubbutat, M. H., and Vousden, K. H. (1999) Regulation of p53 function and stability by phosphorylation. Mol. Cell. Biol. 19, 1751–1758. (13) Blau, J., Xiao, H., McCracken, S., O’Hare, P., Greenblatt, J., and Bentley, D. (1996) Three functional classes of transcriptional activation domain. Mol. Cell. Biol. 16, 2044–2055. (14) Joerger, A. C., and Fersht, A. R. (2016) The p53 Pathway: Origins, Inactivation in Cancer, and Emerging Therapeutic Approaches. Annu. Rev. Biochem. 85, 375–404. (15) Okuda, M., and Nishimura, Y. (2014) Extended String Binding Mode of the Phosphorylated Transactivation Domain of Tumor Suppressor p53. J. Am. Chem. Soc. 136, 14143–14152. (16) Rob, T., Gill, P. K., Golemi-Kotra, D., and Wilson, D. J. (2013) An electrospray ms-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labelling reveals allosteric effects in enzyme inhibition. Lab. Chip 13, 2528–2532. (17) Rob, T., Liuni, P., Gill, P. K., Zhu, S., Balachandran, N., Berti, P. J., and Wilson, D. J. (2012) Measuring dynamics in weakly structured regions of proteins using microfluidics-enabled subsecond H/D exchange mass spectrometry. Anal. Chem. 84, 3771–3779. (18) Resetca, D., and Wilson, D. J. (2013) Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry. FEBS J. 280, 5616–5625. (19) Keppel, T. R., Howard, B. A., and Weis, D. D. (2011) Mapping Unstructured Regions and Synergistic Folding in Intrinsically Disordered Proteins with Amide H/D Exchange Mass Spectrometry. Biochemistry (Mosc.) 50, 8722–8732.


Page 16 of 18

Page 17 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(20) Zhu, S., Shala, A., Bezginov, A., Sljoka, A., Audette, G., and Wilson, D. J. (2015) Hyperphosphorylation of intrinsically disordered tau protein induces an amyloidogenic shift in its conformational ensemble. PloS One 10, e0120416. (21) Ferguson, P. L., Pan, J., Wilson, D. J., Dempsey, B., Lajoie, G., Shilton, B., and Konermann, L. (2007) Hydrogen/Deuterium Scrambling during Quadrupole Time-of-Flight MS/MS Analysis of a Zinc-Binding Protein Domain. Anal. Chem. 79, 153–160. (22) Bai, Y., Milne, J. S., Mayne, L., and Englander, S. W. (1993) Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86. (23) Zhang, Y.-Z. (2000) Protein and peptide structure and interactions studied by hydrogen exchange and NMR. PhD Thesis, Structural Biology and Molecular Biophysics, University of Pennsylvania, PA, USA.. (24) Shieh, S. Y., Ahn, J., Tamai, K., Taya, Y., and Prives, C. (2000) The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289–300. (25) Loughery, J., and Meek, D. (2013) Switching on p53: an essential role for protein phosphorylation? Biodiscovery. (26) Lanucara, F., Holman, S. W., Gray, C. J., and Eyers, C. E. (2014) The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nat. Chem. 6, 281–294. (27) Glover, M. S., Dilger, J. M., Acton, M. D., Arnold, R. J., Radivojac, P., and Clemmer, D. E. (2016) Examining the Influence of Phosphorylation on Peptide Ion Structure by Ion Mobility Spectrometry-Mass Spectrometry. J. Am. Soc. Mass Spectrom. 27, 786–794. (28) Ruotolo, B. T., Gillig, K. J., Woods, A. S., Egan, T. F., Ugarov, M. V., Schultz, J. A., and Russell, D. H. (2004) Analysis of Phosphorylated Peptides by Ion Mobility-Mass Spectrometry. Anal. Chem. 76, 6727– 6733. (29) Daughdrill, G. W., Kashtanov, S., Stancik, A., Hill, S. E., Helms, G., Muschol, M., Receveur-Bréchot, V., and Ytreberg, F. M. (2012) Understanding the structural ensembles of a highly extended disordered protein. Mol. Biosyst. 8, 308–319. (30) Lee, H., Mok, K. H., Muhandiram, R., Park, K. H., Suk, J. E., Kim, D. H., Chang, J., Sung, Y. C., Choi, K. Y., and Han, K. H. (2000) Local structural elements in the mostly unstructured transcriptional activation domain of human p53. J. Biol. Chem. 275, 29426–29432. (31) Homaeian, L., Kurgan, L. A., Ruan, J., Cios, K. J., and Chen, K. (2007) Prediction of protein secondary structure content for the twilight zone sequences. Proteins 69, 486–498. (32) Lin, K., Simossis, V. A., Taylor, W. R., and Heringa, J. (2005) A simple and fast secondary structure prediction method using hidden neural networks. Bioinforma. Oxf. Engl. 21, 152–159. (33) Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and Sternberg, M. J. E. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858. (34) Horn, J. R., Kraybill, B., Petro, E. J., Coales, S. J., Morrow, J. A., Hamuro, Y., and Kossiakoff, A. A. (2006) The role of protein dynamics in increasing binding affinity for an engineered protein-protein interaction established by H/D exchange mass spectrometry. Biochemistry (Mosc.) 45, 8488–8498.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only

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


Page 18 of 18

Enhanced Binding Affinity via Destabilization of the Unbound State: A

Recommend Documents