Binding Kinetics of the Intrinsically Disordered p53 Family

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Binding Kinetics of the Intrinsically Disordered p53 Family Transactivation Domains and MDM2 Emma Åberg, O. Andreas Karlsson, Eva Andersson, and Per Jemth J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03876 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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The Journal of Physical Chemistry

Binding Kinetics of the Intrinsically Disordered p53 Family Transactivation Domains and MDM2

Emma Åberg, O. Andreas Karlsson, Eva Andersson, and Per Jemth*

Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.

*Corresponding author; E-mail: [email protected]

1 Environment ACS Paragon Plus

The Journal of Physical Chemistry 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 Because of their prominent roles in cell cycle regulation and cancer the interaction between MDM2 and the intrinsically disordered transactivation domain (TAD) of p53 is exceptionally well studied. However, while there are numerous computational studies on the interaction mechanism there is a paucity of experimental data regarding the kinetics and mechanism. We have used stopped flow fluorescence to investigate the binding reaction between MDM2 and the TAD from p53 as well as from its paralogs p63 and p73, and in particular focussed on the salt dependence of the interaction. The observed kinetics are consistent with a two-state mechanism within the time frame of the stopped flow methodology, thus, any conformational changes including the previously identified MDM2 lid dynamics must occur on a time-scale < 5 ms at 10°C. The association rate constants are similar for the three TADs and differences in the dissociation rate constants determine the various affinities with MDM2. In contrast to previous studies, we found a relatively small ionic strength dependence for all three interactions, highlighting the large variation in the role of electrostatics among binding reactions of intrinsically disordered proteins. The basal association rate constants in the absence of electrostatic interactions were relatively high (≥2 × 106 M-1s-1 at 10°C), suggesting that a large number of initial contacts may lead to a productive complex. Our findings support an emerging picture of 'conformational funneling' occuring in the initial stages of interactions involving intrinsically disordered proteins and that these early binding events can rely on hydrophobic as well as charge-charge interactions.


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Introduction

The p53 protein is a central transcription factor in humans, regulating key cellular processes1. Because a functional p53 protects against cancer, it is incapacitated in around 50% of all cancers and reactivation of p53 is a general current strategy for drug design2,3. In a healthy cell, p53 is downregulated by MDM2, which interacts via its p53 binding domain with the p53 transactivation domain (p53 TAD) (Fig. 1). p53 has two paralogs in vertebrates called p63 and p73, respectively. The three proteins, p53, p63 and p73 display overlapping as well as distinct functions in cell regulation4. MDM2 binds to all three proteins, but the result of the interaction is different where for example p53 is ubiquitinylated by MDM2 and subsequently degraded, but p63 and p73 are not5. We recently investigated the evolutionary history of the p53 family and its co-evolution with MDM26, and it is clear that MDM2 proteins interact with the p53/63/73 family of proteins across the animal kingdom.

The p53 TAD is contained within the first 61 amino acid residues of p53 and contains two binding motifs, TAD1 in the N-terminal part and TAD2 in the C-terminal part, respectively. A number of previous studies7–10 have measured the affinity between TADs from p53, p63 and p73 and MDM2. Within TAD1 there are three conserved hydrophobic residues in an eight-residue motif (FxxxWxxL). This motif interacts directly with the p53-binding pocket in MDM2 and provides much of the binding energy through hydrophobic interactions11,12.

Despite the large body of data on this interaction including theoretical mechanistic studies13,14, there are few experimental kinetic studies addressing the binding



mechanism15,16. To learn more about the mechanistic aspects of this binding reaction, we subjected MDM2 and the TADs of p53, p63 and p73, respectively, to a kinetic analysis using the stopped-flow technique. The salt dependence of the rate constants was found to be low and thus distinct from that of other IDPs, corroborating the notion of a wide spectrum of interactions and mechanisms governing IDP interactions17. On the other hand, the relatively large basal association rate constants support the emerging picture of great flexibility in forming the inital contact(s) of IDP interactions18 (often referred to as the pre-complex). Finally, lid dynamics of MDM2 have been proved important for its binding to p53 TAD19. Our kinetic data put an upper limit for the time constant of lid dynamics in the low ms range.


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Results

The binding motif of TAD1 contains a Trp residue, which fits into a hydrophobic pocket of the p53-binding domain of MDM2 (hereafter referred to as MDM2)11 (PDB code: 1YCR) thus providing a good fluorescent probe for monitoring binding in stopped-flow experiments that we utilized in the present study (Fig. 1). In order to record reliable kinetic traces the experiments were performed at 10°C where the observed rate constant kobs for the TAD/MDM2 interaction is in an accessible range for the stopped-flow technique. We used five peptide constructs, p53 TAD13-61, which contains both TAD1 and TAD2, and the following four peptides, which contains TAD1 of the respective p53/p63/p73 protein: p53 TAD15-29, p53 TAD15-26, p63 TAD51-65, and p73 TAD11-25.

First, we compared the binding kinetics of p53 TAD13-61 with p53 TAD15-29. All experimental traces were well described by single exponential kinetics (Fig. 2a). By varying the TAD concentration at a constant concentration of MDM2 we determined association rate constants (kon) from the linear dependence of the observed rate constant kobs versus the concentration of TAD (Fig. 2b) by fitting an equation for the association of two molecules under non pseudo first order conditions20. Dissociation rate constants (koff) were either determined from the curve fitting using the same data set or in a separate displacement experiment. The experimental data were in perfect agreement with a two-state reaction, i.e., an apparent one step association of the TAD and MDM2 under the experimental conditions. However, these data do not rule out more complex mechanisms. For example, simulations suggest that the p53 TAD/MDM2 interaction proceeds via an unstable encounter complex(es)13,14,21,22.



Moreover, the NMR structure of free MDM2 shows that it must adjust upon TAD binding to accommodate the peptide23. Other NMR experiments24,25 and simulations26 interpreted in the light of the crystal structure11 demonstrate that residues 19-23 in MDM2 partially covers the p53 TAD-binding site and obstructs the interaction. The opening and closing of this helix were detected in NMR experiments at 25°C19. Our measured kobs values were linear at least up to 200 s-1 at 10°C (τ ∼ 5 ms) showing that lid dynamics or other first-order transitions related to a multistep mechanism must occur on a shorter time scale (< 5 ms), in agreement with computational investigations of the binding mechanism14. The binding kinetics of the p53 TAD13-61 (kon = 6.4 µM-1s-1, koff = 0.6 s-1) were similar to those of the shorter p53 TAD15-29 (kon = 9.9 µM-1s-1, koff = 1.0 s-1). Thus, the interactions with the high affinity TAD1 apparently dominate the observed kinetics. The small difference we observe in kon might be related to the notion that residues 19-23 in the N-terminus of MDM2 are able to form an interaction with part of the TAD binding pocket that is not competed out by short TAD peptides25, explaining the observed higher affinity of shorter peptides (i.e., p53 TAD17-26) as opposed to longer TAD variants15. Indeed, the association rate constant for a p53 TAD15-26 peptide was found to be 23 µM-1s-1 (Fig. 2). The lower kon values for p53 TAD15-29 and p53 TAD13-61 could be explained by the fast pre-equilibrium between open and closed MDM2 in which the longer peptides bind less well or not at all to the closed state.

Next, we compared the binding of MDM2 to TAD1 from p53, p63 and p73, respectively. To learn more about the interaction, we performed these kinetic experiments at different ionic strengths. Such experiments shed light on the electrostatic contribution to the interaction as well as the fraction of productive


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collisions between the proteins as mirrored by the basal association rate constant17,27– 29

. Similarly to the p53 TAD/MDM2 interaction, the experiments with p63 and p73

TAD were consistent with a two-step reaction. Experimentally determined association rate constants (Figure 2, Table 1, Supplementary Fig. S1) showed a surprisingly small dependence on the ionic strength, varying only 2-fold in the measured ionic strength interval (0.05-1 M) (Fig. 3). This is similar to the salt dependence of the disordered cMyb and KIX29 but distinct from several other IDP interactions where a stronger ionic

strength

dependence

was

observed,

for

example

for

MLL/KIX30,

NCBD/ACTR28, WASP/Cdc4231,32, PUMA/Mcl-133 and TAD-STAT2/TAZ234. By measuring rate constants at different ionic strength, we could also estimate by extrapolation the basal association rate constant for the respective TAD with MDM2 (Fig. 4). We found that kon,basal for p53 and p73 TAD1 with MDM2 was similar to those of cMyb/KIX, MLL/KIX35 and NCBD/ACTR28,29 and slightly higher than WASP/Cdc4232, PUMA/Mcl-133 and TAD-STAT2/TAZ234, if considered at a similar temperature. Interestingly, the p63 TAD1/MDM2 interaction displayed a relatively high kon,basal value of (35±15) × 106 M-1s-1 (for example, the reportedly large kon,basal for cMYb/KIX was determined as 7.7 × 106 M-1s-1 at 10°C29). Furthermore, the koff value of the p63 TAD1/MDM2 interaction decreased slightly more with ionic strength than koff for p53 and p73 TAD1 such that the equilibrium dissociation constant Kd at extrapolated infinite ionic strength approaches those of the p53 and p73 TAD1/MDM2 interactions (Fig. 4, Table 2). Thus, the low affinity p63 TAD1/MDM2 complex at zero ionic strength experiences a slight increase in affinity at infinite ionic strength. On the other hand, the complexes between p53 TAD1/MDM2 and p73 TAD1/MDM2 display an approximately 10-fold reduction in affinity going from zero to infinite ionic strength. We note that all experiments on p63 TAD1 suffer from a



lower signal-to-noise with regard to kinetic amplitudes as compared to p53 and p73 TAD1. This results in larger errors in measured rate constants, but in particular in the extrapolated rate constants at zero and infinite salt concentration. Nevertheless, the trend of a low and even reverse salt dependence of p63 TAD1 is present within the measured (intrapolated) region of the data.


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Discussion

The rate of association between two proteins into a complex depends on the concentrations of the proteins and the rate constants of the interaction27,36. These parameters govern the frequency of productive collisions. Because the interaction surface of a protein usually comprises only a small fraction of the entire solventaccessible surface area the frequency of productive collisions in protein-protein interactions is reduced compared to two theoretical spheres with 100% reactive surfaces. This is reflected in a decreased association rate constant. Proteins are however not static spheres and the plasticity of protein surfaces likely increases the fraction of productive collisions by allowing for a range of 'misaligned' associations, resulting in a high-energy 'encounter complex', which can rearrange into the most stable protein-protein complex in an induced fit type or 'dock-and-coalesce' mechanism32. It is becoming accepted that such plasticity is particularly prevalent for protein-protein interactions involving IDPs with a striking recent example in which prothymosin-α associates with histone H1 to form an apparently fully disordered complex relying solely on non-specific charge-charge interactions37. In fact, electrostatics often play an important role in modulating the protein-protein association rate constants of IDPs as well as globular proteins by steering the association of the proteins into the correct positions38. This would mask the contribution of plasticity to the interaction. kon,basal is the extrapolated 'basal' association rate constant at infinite ionic strength in absence of electrostatic interactions27,28. Experimentally, the plasticity can therefore be assessed by comparing kon,basal values for different protein-protein interactions. The hypothesis is that IDPs in general have a higher frequency of productive associations, which would result in a



higher association rate constant if everything else were equal. Such relaxed dependence of specific conformations for initial contacts is captured in the conformational funneling model18. Indeed, the basal association rate constants for the p53 family TADs are similar compared to those of other IDPs, i.e., around 106-107 M1 -1

s around 25°C, as compared to 105 M-1s-1 for the barnase/barstar interaction, a well

studied protein-protein interaction involving folded domains39.

Because IDPs in general are enriched in polar residues and depleted in hydrophobic ones it may be assumed that electrostatics in general play an important role in their interactions. However, our present data show that electrostatic interactions have a rather small effect on the TAD/MDM2 interaction for p53, p63 and p73. This is consistent with the conserved hydrophobic binding motif in the TAD, FxxxWxxL. Based on the crystal structure11, there are putative charge-charge and charge-polar interactions in the p53TAD/MDM2 interface (p53TADGlu17/MDM2His73, p53TADGlu17/MDM2Lys94, p53TADGlu28/MDM2Lys51, p53TADLeu25 backbone/MDM2His96) but apparently they do not contribute much to the affinity in comparison to hydrophobic interactions, which may be enhanced by high ionic strength. MDM2His73 and MDM2Lys94, which are both within 5 Å of p53TADGlu17, are also in the vicinity of each other. Thus, a non-favorable electrostatic interaction between the Lys and a protonated His could contribute to the weak salt dependence by counteracting the potentially favorable interactions between p53TADGlu17/MDM2His73, p53TADGlu17/MDM2Lys94, and p53TADGlu28/MDM2Lys51. In the NMR structure of p73TAD/MDM2 40 there are putative favorable electrostatic interactions between MDM2Lys51 and p73TADGlu27 as well as p73TADAsp27 and the (non-native) Cterminus of the peptide, but no obvious non-favorable ones.


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Conclusions

This study adds to the growing body of kinetic data on IDPs that can be used for mechanistic interpretation17. Our present data suggest that while generalizations about electrostatics in IDP interactions are conceptually attractive, each one must be individually assessed. p53, p63 and p73 are paralogs, which diverged from a common vertebrate ancestor around 440 million years ago6. Their interactions with MDM2 vividly illustrates how divergent evolution within a species can result in distinct affinities and rate constants of closely related protein-protein interactions under physiological pH and salt concentration. Nevertheless, the range of basal association rate constants for p53, p63 and p73 TAD is similar to those of other IDP interactions suggesting that general principles related to conformational funneling, i.e., "degree of disorderness" of the initial contacts may be derived.

Materials and Methods

Protein expression and purification The DNA encoding human MDM2 (17-125) was purchased from GenScript in a pSY10 plasmid with an N-terminal NusA domain followed by a TEV protease site, a His-tag, a PreScission protease site and the human MDM2. The plasmid was transformed into Escherichia coli BL21 (DE3) pLys cells (Invitrogen) using heatshock. The cells were grown in LB medium at 37°C and overexpression of the fusion protein was induced with 1 mM isopropyl-β-D-thiogalactopyranoside when the



optical density at 600 nm reached 0.7-0.8 whereafter the cultures were incubated at 18°C overnight. Cells were harvested through centrifugation and the pellet resuspended in binding buffer (400 mM sodium chloride, 50 mM sodium phosphate, pH 7.8, 10% glycerol) followed by sonication. Thereafter, cells were centrifuged at 4°C to remove cell debris and the supernatant was filtered and loaded onto a Nickel Sepharose Fast Flow column (GE Healthcare). The fusion protein was eluted using binding buffer with 250 mM imidazole and then further purified using size-exclusion chromatography on a Hi load 16/60 Sephacryl S-100 column (GE Healthcare) in the binding buffer with pH adjusted to 7.4. The fusion protein was then cleaved with PreScission protease overnight at 4°C followed by a second run on the size-exclusion chromatography column to remove the NusA protein. Purity was checked with SDSPAGE and MALDI-TOF mass spectrometry and pure samples were dialysed against 150 mM sodium chloride, 20 mM sodium phosphate, pH 7.4. Protein concentration was determined by measuring the absorbance at 280 nm and using extinction coefficients calculated from the respective amino acid sequence.

The 15-mer p53TAD (15-29), p63TAD (51-65) and p73TAD (11-25) were ordered as acetylated peptides (Synpeptide Co. Ltd) and dissolved in 20 mM sodium phosphate. The concentration was determined by measuring Trp absorbance at 280 nm.

Kinetic experiments

All experiments were performed on an upgraded SX-17 MV stopped-flow spectrometer (Applied Photophysics). The experiments were performed at 10°C in 20 mM sodium phosphate, pH 7.4, 1 mM TCEP at different ionic strengths (0.05, 0.1, 0.2, 0.5, 1.0 M) adjusted with sodium chloride. The conserved tryptophan in the


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FxxxWxxL motif which binds in a hydrophobic pocket of MDM2 was used to monitor the change in fluorescence upon binding. The tryptophan was excited at 280 nm and the emission was recorded at 330 nm using a band-pass filter. MDM2 was held at a constant concentration of 0.5-2 µM and mixed with p53 TAD, p63 TAD and p73 TAD peptides at concentrations between 1-10 µM. For each reported experimental point, 4-8 experimental traces were averaged and fitted to a single exponential. The fitting errors for kobs of such averages from technical replicates are typically low, in the present case 3-7 % (p53 TAD), 6-7 % (p63 TAD) and 4-7 % (p73 TAD). The kobs values at different TAD concentrations were plotted and fitted to an equation for a reversible bimolecular interaction20, where the slope at high ligand concentration equals the association rate constant, kon. Curve fitting also estimates the dissociation rate constant, koff, which was used for p63 TAD. kobs = ((kon2 ([MDM2]0-[TAD]0)2 + koff2+2 konkoff ([MDM2]0+[TAD]0))0.5

[MDM2]0 (non-varied species ) and [TAD]0 (varied species) are the total concentrations of MDM2 and TAD, respectively, after mixing in the experiment. For p53 and p73 TAD, the koff values were ≤ 5 s-1 and thus associated with large errors in curve fitting of binding data. Therefore, we used displacement experiments in which a pre-formed complex of TAD and MDM2 (1 µM:1 µM) was mixed with high concentrations (10, 20 µM) of a dansylated p53 TAD peptide. The experimental traces were fitted to a single exponential equation and the average of the resulting kobs values were taken as a good estimate of koff. The accuracy and precision in these displacement experiments are very high since the obtained kobs values at high concentration of displacer is not sensitive to errors in concentrations. The equilibrium



dissociation constant was then calculated as Kd = koff /kon and the propagated error was calculated from the fitting errors of koff and kon.

The basal association rate constant, kon,basal, is the kon in absence of electrostatic effects. The relation between ionic strength and kon can be described by a DebyeHückel-like approximation27:

ln ݇௢௡ = ln ݇௢௡,௕௔௦௔௟ − ൬

ܷ 1 ൰ ܴܶ 1 + ߢߙ

where U corresponds to the electrostatic interaction energy, R is the gas constant and T is the temperature. κ is a constant that contains the term for ionic strength: (2NAe2I/e0erkBT)1/2; NA, Avogadro’s constant; e, elementary charge; I ionic strength; e0, the vacuum permittivity; er, the dielectric constant of water; kB, the Boltzmann constant; T, temperature; α, the minimal distance of approach approximated as 6 Å. A plot of ln kon versus (1 + κα)−1 is linear and the intercept of the line at (1 + κα)−1= 0 is ln kon,basal, from which the basal rate constant kon,basal can be determined.

Supporting Information Supplementary Figure 1 showing observed rate constants as a function of TAD concentration for all data sets.

Acknowledgements This work was funded by the Swedish Research Council (Grant no 2016-04134).


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References (1) Napoli, M.; Flores, E. R. The P53 Family Orchestrates the Regulation of Metabolism: Physiological Regulation and Implications for Cancer Therapy. Br. J. Cancer 2017, 116, 149–155. (2) Joerger, A. C.; Fersht, A. R. The Tumor Suppressor P53: From Structures to Drug Discovery. Cold Spring Harb. Perspect. Biol. 2010, 2, a000919. (3) Merkel, O.; Taylor, N.; Prutsch, N.; Staber, P. B.; Moriggl, R.; Turner, S. D.; Kenner, L. When the Guardian Sleeps: Reactivation of the P53 Pathway in Cancer. Mutation Research - Reviews in Mutation Research 2017, 773, 1–13. (4) Yang, A.; McKeon, F. P63 and P73: P53 Mimics, Menaces and More. Nat. Rev. Mol. Cell Biol. 2000, 1, 199–207. (5) Harms, K. L.; Chen, X. The Functional Domains in P53 Family Proteins Exhibit Both Common and Distinct Properties. Cell Death and Differentiation 2006, 13, 890–897. (6) Åberg, E.; Saccoccia, F.; Grabherr, M.; Ying, W.; Ore, J.; Jemth, P.; Hultqvist, G. Evolution of the P53-MDM2 Pathway. BMC Evol. Biol. 2017, 17, 177. (7) Teufel, D. P.; Bycroft, M.; Fersht, A. R. Regulation by Phosphorylation of the Relative Affinities of the N-Terminal Transactivation Domains of P53 for P300 Domains and Mdm2. Oncogene 2009, 28, 2112–2118. (8) Lee, C. W.; Ferreon, J. C.; Ferreon, A. C. M.; Arai, M.; Wright, P. E. Graded Enhancement of P53 Binding to CREB-Binding Protein (CBP) by Multisite Phosphorylation. Proc. Natl. Acad. Sci. USA 2010, 107, 19290–19295. (9) Shan, B.; Li, D.-W.; Brüschweiler-Li, L.; Brüschweiler, R. Competitive Binding between Dynamic P53 Transactivation Subdomains to Human MDM2 Protein: Implications for Regulating the P53·MDM2/MDMX Interaction. J. Biol. Chem. 2012, 287, 30376–30384. (10) Shin, J.-S.; Ha, J.-H.; Lee, D.-H.; Ryu, K.-S.; Bae, K.-H.; Park, B. C.; Park, S. G.; Yi, G.-S.; Chi, S.-W. Structural Convergence of Unstructured P53 Family Transactivation Domains in MDM2 Recognition. Cell Cycle 2015, 14, 533– 543. (11) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.; Pavletich, N. P. Structure of the MDM2 Oncoprotein Bound to the P53 Tumor Suppressor Transactivation Domain. Science 1996, 274, 948–953. (12) Estrada-Ortiz, N.; Neochoritis, C. G.; Dömling, A. How to Design a Successful P53-MDM2/X Interaction Inhibitor: A Thorough Overview Sased on Crystal Structures. ChemMedChem 2016, 11, 757–772. (13) Dastidar, S. G.; Lane, D. P.; Verma, C. S. Multiple Peptide Conformations Give Rise to Similar Binding Affinities: Molecular Simulations of P53-MDM2. J. Am. Chem. Soc. 2008, 130, 13514–13515. (14) Saglam, A. S.; Wang, D. W.; Zwier, M. C.; Chong, L. T. Flexibility vs Preorganization: Direct Comparison of Binding Kinetics for a Disordered Peptide and Its Exact Preorganized Analogues. J. Phys. Chem. B 2017, 121, 10046–10054. (15) Schon, O.; Friedler, A.; Bycroft, M.; Freund, S. M. V.; Fersht, A. R. Molecular Mechanism of the Interaction between MDM2 and P53. J. Mol. Biol. 2002, 323, 491–501. (16) Crabtree, M. D.; Borcherds, W.; Poosapati, A.; Shammas, S. L.; Daughdrill, G. W.; Clarke, J. Conserved Helix-Flanking Prolines Modulate Intrinsically



(17)

(18)

(19)

(20) (21)

(22)

(23)

(24)

(25)

(26)

(27) (28)

(29)

(30)

Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex. Biochemistry 2017, 56, 2379–2384. Gianni, S.; Dogan, J.; Jemth, P. Coupled Binding and Folding of Intrinsically Disordered Proteins: What Can We Learn from Kinetics? Current Opinion in Structural Biology. 2016, 36, 18–24. Schneider, R.; Maurin, D.; Communie, G.; Kragelj, J.; Hansen, D. F.; Ruigrok, R. W. H.; Jensen, M. R.; Blackledge, M. Visualizing the Molecular Recognition Trajectory of an Intrinsically Disordered Protein Using Multinuclear Relaxation Dispersion NMR. J. Am. Chem. Soc. 2015, 137, 1220– 1229. Showalter, S. A.; Bruschweiler-Li, L.; Johnson, E.; Zhang, F.; Brüschweiler, R. Quantitative Lid Dynamics of MDM2 Reveals Differential Ligand Binding Modes of the P53-Binding Cleft. J. Am. Chem. Soc. 2008, 130, 6472–6478. Malatesta, F. The Study of Bimolecular Reactions under Non-Pseudo-First Order Conditions. Biophys. Chem. 2005, 116, 251–256. Zwier, M. C.; Pratt, A. J.; Adelman, J. L.; Kaus, J. W.; Zuckerman, D. M.; Chong, L. T. Efficient Atomistic Simulation of Pathways and Calculation of Rate Constants for a Protein–peptide Binding Process: Application to the MDM2 Protein and an Intrinsically Disordered P53 Peptide. J. Phys. Chem. Lett. 2016, 7, 3440–3445. Zhou, G.; Pantelopulos, G. A.; Mukherjee, S.; Voelz, V. A. Bridging Microscopic and Macroscopic Mechanisms of P53-MDM2 Binding with Kinetic Network Models. Biophys. J. 2017, 113, 785–793. Uhrinova, S.; Uhrin, D.; Powers, H.; Watt, K.; Zheleva, D.; Fischer, P.; McInnes, C.; Barlow, P. N. Structure of Free MDM2 N-Terminal Domain Reveals Conformational Adjustments That Accompany P53-Binding. J. Mol. Biol. 2005, 350, 587–598. McCoy, M. A.; Gesell, J. J.; Senior, M. M.; Wyss, D. F. Flexible Lid to the P53Binding Domain of Human Mdm2: Implications for P53 Regulation. Proc. Natl. Acad. Sci. USA 2003, 100, 1645–1648. Showalter, S. A.; Bruschweiler-Li, L.; Johnson, E.; Zhang, F.; Brüschweiler, R. Quantitative Lid Dynamics of MDM2 Reveals Differential Ligand Binding Modes of the P53 Binding Cleft. J. Am. Chem. Soc. 2008, 130, 6472–6478. Mukherjee, S.; Pantelopulos, G. A.; Voelz, V. A. Markov Models of the ApoMDM2 Lid Region Reveal Diffuse yet Two-State Binding Dynamics and Receptor Poses for Computational Docking. Sci. Rep. 2016, 6, 31631. Schreiber, G.; Haran, G.; Zhou, H.-X. Fundamental Aspects of Protein-Protein Association Kinetics. Chem. Rev. 2009, 109, 839–860. Dogan, J.; Jonasson, J.; Andersson, E.; Jemth, P. Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains. Biochemistry 2015, 54, 4741–4750. Shammas, S. L.; Travis, A. J.; Clarke, J. Remarkably Fast Coupled Folding and Binding of the Intrinsically Disordered Transactivation Domain of CMyb to CBP KIX. J. Phys. Chem. B 2013, 117, 13346–13356. Rogers, J. M.; Oleinikovas, V.; Shammas, S. L.; Wong, C. T.; De Sancho, D.; Baker, C. M.; Clarke, J. Interplay between Partner and Ligand Facilitates the Folding and Binding of an Intrinsically Disordered Protein. Proc. Natl. Acad. Sci. 2014, 111, 15420–15425.


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(31) Hemsath, L.; Dvorsky, R.; Fiegen, D.; Carlier, M.-F.; Ahmadian, M. R. An Electrostatic Steering Mechanism of Cdc42 Recognition by Wiskott-Aldrich Syndrome Proteins. Mol. Cell 2005, 20, 313–324. (32) Ou, L.; Matthews, M.; Pang, X.; Zhou, H.-X. The Dock-and-Coalesce Mechanism for the Association of a WASP Disordered Region with the Cdc42 GTPase. FEBS J. 2017, 284, 3381–3391. (33) Rogers, J. M.; Steward, A.; Clarke, J. Folding and Binding of an Intrinsically Disordered Protein: Fast, but Not “Diffusion-Limited.” J. Am. Chem. Soc. 2013, 135, 1415–1422. (34) Lindström, I.; Dogan, J. Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 Are Not a Requirement. Biochemistry 2017, 56, 4145–4153. (35) Shammas, S. L.; Travis, A. J.; Clarke, J. Allostery within a Transcription Coactivator Is Predominantly Mediated through Dissociation Rate Constants. Proc. Natl. Acad. Sci. USA 2014, 111, 12055–12060. (36) Gianni, S.; Jemth, P. How Fast Is Protein-Ligand Association? Trends Biochem. Sci. 2017, 42, 847–849. (37) Borgia, A.; Borgia, M. B.; Bugge, K.; Kissling, V. M.; Heidarsson, P. O.; Fernandes, C. B.; Sottini, A.; Soranno, A.; Buholzer, K. J.; Nettels, D.; et al. Extreme Disorder in an Ultrahigh-Affinity Protein Complex. Nature 2018, 555, 61–66. (38) Schreiber, G.; Haran, G.; Zhou, H.-X. Fundamental Aspects of Protein-Protein Association Kinetics. Chem. Rev. 2009, 109, 839–860. (39) Schreiber, G.; Fersht, A. R. Rapid, Electrostatically Assisted Association of Proteins. Nat. Struct. Biol. 1996, 3, 427–431. (40) Shin, J.-S.; Ha, J.-H.; Lee, D.-H.; Ryu, K.-S.; Bae, K.-H.; Park, B. C.; Park, S. G.; Yi, G.-S.; Chi, S.-W. Structural Convergence of Unstructured P53 Family Transactivation Domains in MDM2 Recognition. Cell Cycle 2015, 14, 533– 543.



Figures

Figure 1. Structure of TAD and MDM2. A) Domain structure of the p53 family of proteins and MDM2. B) Sequence alignment of the transactivation domains from p53, p63 and p73, with numbering according to p53. C) Crystal structure of the complex between the p53-binding domain in MDM2 and p53 TAD1. The three conserved hydrophobic residues in TAD1, Phe19, Trp23 and Leu26 are shown in stick representation (PDB code: 1YCR).


Page 18 of 29

Page 19 of 29 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


Figure 2. Binding kinetics for MDM2 and p53 TAD. A) Kinetic traces for the interaction between MDM2 (0.5 µM) and, from left to right, p53 TAD13-61, p53 TAD15-29 and p53 TAD15-26 (7 µM), respectively, at an ionic strength of 0.2 M. B) Observed rate constants for p53 TAD13-61, p53 TAD15-29 and p53 TAD15-26 at an ionic strength of 0.2 M. C) Observed rate constants for p53 TAD15-29 compared to those for p63 TAD and p73 TAD at an ionic strength of 0.2 M.



Figure 3. The dependence of rate and equilibrium constants on ionic strength. A) Association (kon), B) dissociation (koff) rate constants and C) equilibrium constants (Kd = koff/kon) were determined by stopped flow spectroscopy at different ionic strengths in 20 mM sodium phosphate, pH 7.4. The rate and equilibrium constants for all data points of p63 and p73 at 0.5 M are averages from 2-5 repetitions. See Table 1 for errors.


Page 20 of 29

Page 21 of 29 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


Figure 4. Estimation of the rate and equilibrium constants at zero and infinite ionic strength. A) Association (kon), B) dissociation (koff) rate constants and C) equilibrium constants were plotted versus (1+κα)-1, at each data point the respective ionic strength is indicated above the plot. The intercept at zero is the basal constant at infinite ionic strength (i.e., in the absence of electrostatic interactions) whereas the intercept at unity (where κ = 1) represents the constant at zero ionic strength. Errors and extrapolated rate constants are shown in Table 1 and 2.



Page 22 of 29

Tables Table 1. Experimentally determined rate constants at different ionic strengths with fitting errors. The Kd value was calculated as the ratio of koff and kon with a propagated fitting error. Average rate and equilibrium constants from 2-5 repetitions with

one standard error of the mean, performed for data sets with individual traces having low signal to noise. Other errors are fitting errors from curve fitting. kon (µM-1s-1)

koff (s-1)

Kd (µM)

Binding partner

I (M)

p5313-61

0.05

25

±

0.8

0.4

±

0.0002 0.015

±

0.0005

0.1

12

±

0.3

0.4

±

0.0001 0.034

±

0.001

0.2

6.4

±

0.1

0.6

±

0.02

0.10

±

0.003

0.5

7

±

0.1

0.4

±

0.0004 0.06

±

0.001

1

8.1

±

0.2

0.4

±

0.0002 0.05

±

0.001

0.05

14.4

±

0.2

0.8

±

0.01

0.06

±

0.001

0.1

15

±

0.1

0.9

±

0.02

0.06

±

0.001

0.2

9.9

±

0.1

1.0

±

0.02

0.10

±

0.002

0.5

10.8

±

0.2

1.2

±

0.002

0.11

±

0.002

1

7.5

±

0.5

1.2

±

0.02

0.16

±

0.01

0.06

21.6*

±

1.8

72*

±

2

3.35*

±

0.2

0.1

15.8*

±

2.8

90*

±

3

5.91*

±

1.2

0.2

17.9*

±

3.3

85*

±

4

5.0*

±

1.2

0.5

27.6*

±

2.7

52*

±

8

2.51*

±

0.8

±

8.9

43

*

±

1

2.14

*

±

0.8

p5315-29

p6351-65

1

26.6

*

p7311-25 0.05

22

±

0.5

5.1

±

0.1

0.23

±

0.01

0.1

23.2

±

0.3

4.9

±

0.2

0.21

±

0.01

0.2

14.2

±

0.3

4.9

±

0.02

0.34

±

0.01

0.5

10.1*

±

0.9

4.7

±

0.2

0.47*

±

0.04

1

11.4

±

0.7

4.1

±

0.1

0.36

±

0.02


Page 23 of 29 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


*

Average rate and equilibrium constants from 2-5 repetitions with one standard error of the mean, performed for data sets with individual traces having low signal to noise. Other errors are fitting errors from curve fitting.

Table 2. Basal constants and constants at zero ionic strength. The large errors for some of the parameters are due to long extrapolations as shown in Fig. 4. Thus, while these parameters could not be determined accurately they are tabulated to stress the large errors, since they are not obvious from the graphs in Fig. 4. p5313-61

p5315-29

p6351-65

p7311-25

kon, basal (µM-1s-1)

2.4

±

1.7

4.4

±

1.2

35

± 15

3.9

± 1.1

kon, I=0 (µM-1s-1)

41

±

62

27

±

16

13

±

12

52

± 32

koff, basal (s-1)

0.4

±

0.2

1.9

±

0.1

21

±

7

3.5

± 0.3

koff, I=0 (s-1)

0.5

±

1.2

0.6

±

0.1

190

± 140

6

Kd, basal (µM)

0.18 ± 0.22 0.42 ±

0.12

0.6

± 0.45

0.91

± 0.33

Kd, I=0 (µM)

0.01 ± 0.03 0.02 ±

0.01

15

±

0.12

± 0.1


25

±

1



Page 24 of 29

APage 25 of 29

p53/p63/p73 TAD


DNA BD

OD

C

SAM

Phe19

Leu26

1 2 MDM2 3 4 B5 6 7 p53 13 - P L 8 p63 51 9 11 p73 10

Trp23

p53 BD

Acidic

Zn finger

RING

S Q E T F S D L W K L L P E N N V L S P L P S Q A M D D L M L S P D D I E Q W F T E D P G P D - 61 ACS Paragon Plus Environment S P E V F Q H I W D F L E Q P - 65 G G T T F E H L W S S L E P D - 25

A


6.74

p5313-61

5.05

p5315-29

Page 26 of 29

3.4

p5315-26

6.72

5

kobs (s-1)

Fluorescence

1 2 3 4 5 6 7 8 9 B 10 11 12 13 14 15 16 17 18 19 20

3.35 6.7

4.95 6.68

3.3 4.9 6.66

6.64

0

0.1

0.2

0.3

0.4

0.5

0.6

4.85

0

0.05

0.1

0.15

0.2

0.25

Time (s)

C 250

250 p5315-29

p5315-26

p6351-65

p5315-29

200

150

150

100

100

ACS Paragon Plus Environment

50

0

p7311-25

200

p5313-61

50

0

2

4

6

8

10

0

0

Concentration (μM)

2

4

6

8

10

3.25

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

A

B

5 Page 27 of 29

5 The Journal of Physical Chemistry

p5315-29

p5315-29

3

3

2

p5313-61

6

p6351-65 p7311-25

2

ln Kd / nM

p6351-65 p7311-25

ln koff / s-1

ln kon / μM-1 s-1

1 2 3 4 5 6 7 8

8

4

p5313-61

4

C

1 0

1

4

p5315-29

2

p5313-61


p6351-65 p7311-25

-1

0

0

-2 0

0.2

0.4

0.6

0.8

1

0

0.2

0.4

0.6

Ionic strength (M)

0.8

1

0

0.2

0.4

0.6

0.8

1


A

B 1

5

0.5

0.2 0.1 0.05

C

Ionic strength (M) 1

6

0.5

0.2 0.1 0.05

1

10

0.5

0.2 0.1 0.05

5 8 4 3

2 p5315-29

p5313-61

3

p6351-65 p7311-25

2 1

p5315-29 p6351-65 p7311-25

0

-2 0.4

0.6

p5313-61

-1

0 0.2

4

2

p6351-65 p7311-25

0

6

0

p5313-61

1

p5315-29

ln Kd / nM

ln koff / s-1

M-1 s-1

4

ln kon /

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 28 of 29

0.8

1

0

0.2

0.4

(1 +

0.6

0.8

1

)-1


0

0.2

0.4

0.6

0.8

1

The Journal of- Physical Chemistry -

Complex MDM2 ACS Paragon Plus Environment p73 -

-

koff +

p63

kon -

-

-

p53 Ionic strength (M)

1 2 3 4 5 6 7 80

+ - - + + + + - + + + + - + + + - + + + - + - + + + - + + + - - + + + -+ + + + - + + - + - + - - + + + + + + + + - - + - + + + - + - - + - +

1 29 of 29 Page

Binding Kinetics of the Intrinsically Disordered p53 Family

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