Deciphering the Dynamic Interaction Profile of an Intrinsically

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Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy Elise Delaforge, Jaka Kragelj, Laura Tengo, Andrés Palencia, Sigrid Milles, Guillaume Bouvignies, Nicola Salvi, Martin Blackledge, and Malene Ringkjøbing Jensen J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12407 • Publication Date (Web): 25 Dec 2017 Downloaded from http://pubs.acs.org on December 25, 2017

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Journal of the American Chemical Society

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy

Elise Delaforge†, ‡, Jaka Kragelj†, ‡, Laura Tengo†, Andrés Palencia§, Sigrid Milles†, Guillaume Bouvignies||, ^, Nicola Salvi†, Martin Blackledge† and Malene Ringkjøbing Jensen†, *

†

Univ. Grenoble Alpes, CNRS, CEA, IBS, F-38000 Grenoble, France

§

Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, Univ. Grenoble Alpes,

Grenoble, France ||

Laboratoire des Biomolécules, Département de chimie, École normale supérieur, UPMC Univ. Paris

06, CNRS, PSL Research University, 24 rue Lhomond, 75005 Paris, France ^

Sorbonne Universités, UPMC Univ. Paris 06, École normale supérieur, CNRS, Laboratoire des

Biomolécules (LBM), Paris, France

‡

These authors contributed equally to this work

* To whom correspondence should be addressed

Dr. Malene Ringkjøbing Jensen E-mail: [email protected] Tel: +33 4 57 42 86 68

1 ACS Paragon Plus Environment

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ABSTRACT Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance in order to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R1r, Carr-Purcell-Meiboom Gill (CPMG) relaxation dispersion as well as chemical exchange saturation transfer (CEST) to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.


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INTRODUCTION Intrinsically disordered proteins (IDPs) are implicated in a wide range of biological functions, among them signal transduction, transcription, cell cycle regulation or chaperoning.1 To carry out their functions, IDPs rely on linear motifs i.e. short sequence segments that mediate interactions with partner proteins.2 IDPs display a large number of interaction modes including folding-upon-binding,3 binding without major structural transitions or binding through highly dynamic complexes where several conformations are sampled on the surface of the partner (fuzziness).4,5 The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with peptides corresponding to linear motifs of IDPs.6,7 However, only short IDP sequences that remain sufficiently rigid and bind to their partner proteins with moderate-to-high affinity are likely to crystallize. In addition, crystal structures provide a mainly static view of the complexes and do not give information about conformational dynamics, or the associated time scales, experienced by the IDP in the complex. Knowledge of conformational dynamics of IDP complexes is of fundamental importance in order to understand the molecular details of IDP binding modes and how IDPs engage in highly specific interactions without concomitantly high binding affinity.8 Nuclear magnetic resonance (NMR) spectroscopy remains the only experimental technique that can access both structure and dynamics of IDP complexes at atomic resolution, even for interactions displaying low to moderate affinities. However, chemical shift titrations of IDPs with their binding partners often lead to excessive line broadening of the NMR resonances due to conformational exchange occurring on the micro- to millisecond (µs-ms) time scale. In order to overcome this problem, NMR exchange techniques have been used in a few cases to visualize IDP interaction trajectories and to determine the structure of the bound complex by creating a low-populated bound state of the IDP (detectable by the exchange experiments) by adding small



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sub-stoichiometric amounts of the binding partner.9–12 Yet, these studies have not concentrated directly on the site-specific dynamics of the bound IDP. Here, we study a dynamic signaling complex formed within the mitogen-activated protein kinase (MAPK) cell signaling pathways.13–15 These pathways feature a kinase module consisting of a MAPK kinase kinase (MKKK) that phosphorylates and thereby activates a MAPK kinase (MKK) that in turn activates a MAPK by phosphorylation. The MKKs specifically activate the MAPKs according to the following scheme: MKK1/2/5 activate ERK, MKK3/4/6 activate p38 and MKK4/7 activate JNK. This specificity is mediated by intrinsically disordered N-terminal regulatory domains of the MKKs that recruit their cognate MAPKs. The interaction is facilitated by docking site motifs composed of two to three basic residues followed by a sub-motif containing three hydrophobic residues FL, FA, FB (K/R2-3-X1-6-FLX1-3-FA-X-FB, where X is any amino acid type).16–18 The docking site motifs bind to the docking groove on the MAPK, where the three hydrophobic residues insert into three pockets, while the basic residues contact the negatively charged common docking (CD) groove (Figure 1).19–24 Docking site motifs are present not only in MKKs, but are also found within intrinsically disordered regions of a large number of proteins including MAPK substrates, phosphatases, and scaffold proteins and therefore represent important regulatory motifs in cell signaling.

Figure 1: Structural features of eukaryotic MAPKs showing the N- and C-terminal lobes with the position of the catalytic site (orange) and the activation loop (green). The docking groove (beige) accommodates docking site motifs of the MKKs, MAPK substrates, phosphatases and scaffold proteins. The zoom shows the structural basis of the interaction between JNK1 and one of the conformations of the bound docking site motif of MKK7 (PDB 4UX9).


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In addition to docking site motifs, it has been shown in the case of phosphatases that kinase specificity sequences (KIS), located immediately C-terminally to the docking site motifs, contribute to binding of cognate MAPKs.25–27 The role of the KIS domain in the formation of MKK-MAPK complexes, however, remains elusive. Here, we characterize the regulatory domain of MKK4 and its interaction with p38α at atomic resolution. We combine rotating-frame R1r,28–31 Carr-Purcell-Meiboom Gill (CPMG) relaxation dispersion32–34 as well as chemical exchange saturation transfer (CEST)35,36 to decipher the dynamic interaction profile of MKK4. Interestingly, our results show how the docking site motif remains rigid in the complex with p38α and thereby functions as an anchor point, while the KIS domain undergoes fast dynamics and samples several conformational states on the surface of p38α in a fuzzy complex.

RESULTS Residual Structure and Dynamics of the Regulatory Domain of MKK4 We expressed and purified the intrinsically disordered regulatory domain of MKK4 (residues 1-86, MKK41-86) (Figure 2A, B) and obtained the complete backbone assignment using a series of BEST-type triple resonance experiments37 (Figure S1A). The secondary 13Cα chemical shifts show that this domain is devoid of transiently populated secondary structures (Figure 2C). Nuclear relaxation rates can be used to probe the dynamics of IDPs on the pico- to nanosecond time scale including correlated motions.38–42 We measured 15N transverse relaxation rates, R2, 15

N transverse chemical shift anisotropy (CSA) / 15N-1H dipole-dipole (DD) cross-correlated

relaxation rates, hxy, as well as {1H}-15N nuclear Overhauser enhancements (nOes) (Figure 2D, E). All rates follow the same overall pattern with smaller rates for residues 1-20 followed by more elevated rates for the central region of the protein and finally the largest rates for the region encompassing residues 72-78. The profile is consistent with the local dynamics being



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dominated by the bulkiness of the amino acids along the chain (Figure S2).43 The similar overall profile of R2 and hxy, show that contributions are negligible from conformational exchange occurring on the micro- to millisecond (µs-ms) time scale (Figure 2D), except residue H31 that shows a small exchange contribution, potentially due to protonation/deprotonation of the imidazole side chain.

Figure 2. The regulatory domain of MKK4 is intrinsically disordered. (A) Domain organization of MKK4 with its intrinsically disordered regulatory domain (residues 1-86), docking site motif for JNK and p38 (residues 40-48, blue) and the KIS domain (residues 49-62, green). (B) Sequence of the regulatory domain of MKK4 with the docking site and KIS domain highlighted in blue and green, respectively. (C) Secondary chemical shifts of MKK41-86 calculated on the basis of the experimental Cα chemical shifts. (D) Experimental 15N transverse relaxation rates, R2, and CSA/DD cross-correlated relaxation rates, hxy. (E) Experimental {1H}-15N nOes of MKK41-86. All relaxation rates were obtained at 5°C and a 1H frequency of 850 MHz.


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In order to decrease signal overlap, notably within the serine/glycine rich repeat region (residues 7-19), we also obtained the backbone assignment of a smaller sub-construct of the regulatory domain comprising residues 12-86. The spectrum of this construct (MKK412-86) superimposes almost perfectly on the spectrum of MKK41-86 (Fig. S1B), and was, therefore, used for further studies.

Ensemble Description of MKK4 from Chemical Shifts and Residual Dipolar Couplings In order to precisely map the conformational sampling of MKK412-86, we measured multiple residual dipolar couplings (RDCs) (1DHN, 1DCaHa, 1DCaC’, 2DHNC’ and 4DHNHa). The statistical coil generator Flexible-Meccano44,45 was used in combination with the genetic algorithm ASTEROIDS46 to select representative structural ensembles (five ensembles of 200 conformations each) in agreement with experimental 1H,

15

N and

13

C chemical shifts and

RDCs.47–49 We used 1DNH, 1DCαHα and 1DCαC’ as active parameters in the ensemble selections and 4DHNHα and 2DHNC’ were retained for cross validation. The selected ASTEROIDS ensembles are in excellent agreement with the experimental data and, importantly, are able to reproduce the RDCs that were not included in the selections providing quantifiable evidence that the local conformational sampling contained in the ASTEROIDS ensembles is genuine and accurate (Figure 3).50 Analysis of the conformational sampling in terms of populations in different regions of Ramachandran space shows an elevated population of poly-proline II (PPII) within the MKK4 docking site motif and at the C-terminal end of the regulatory domain (Figure 3D). Other regions of MKK4 essentially adopt statistical coil conformations and are devoid of transiently populated secondary structures (Figure 3C, D).



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Figure 3. Structural ensemble description of MKK412-86 from chemical shifts (1HN, 15N, 13Cα, 13Cβ and 13 C’) and RDCs (1DNH, 1DCαHα, 1DCαC’). (A) Agreement between experimental (red) and back-calculated secondary chemical shifts (blue) from one of the selected ASTEROIDS ensembles. (B) Agreement between experimental (red) and back-calculated RDCs (blue). The 1DNH, 1DCαHα and 1DCαC’ RDCs were included in the ASTEROIDS selections (active parameters), while 4DHNHα and 2DHNC’ were retained for cross-validation of the resulting ensembles (passive parameters). (C, D) Site-specific a-helical (aR) and PPII populations of MKK412-86 derived from five independently selected ASTEROIDS ensembles (blue) compared to statistical coil populations (red). Blue and green shadings highlight the location of the docking site motif and the KIS domain, respectively.

Interaction of the Regulatory Domain of MKK4 with p38a The interaction between MKK412-86 and p38a was initially studied by isothermal titration calorimetry (ITC) showing the formation of a 1:1 complex with a dissociation constant of KD = 4.1 µM (Figure S3). Subsequently, we monitored the interaction by chemical shift titrations. The NMR signals of MKK412-86 display only small chemical shift perturbations (CSPs) and undergo extensive line-broadening, even for small sub-stoichiometric amounts of p38a (Figure


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4A). The observed line broadening may arise due to an increase in the local correlation time due to the larger size of the p38a kinase (41 kDa) and from conformational exchange occurring on the µs-ms time scale between free and p38a-bound MKK412-86. The HSQC intensity profile of MKK412-86 in the presence of p38a reveals that the interaction is not only localized to the docking site motif, but implicates the KIS domain of MKK4, with the largest line broadening observed for the residues within the docking site motif (Figure 4B). This interaction profile is also supported by the small 15N CSPs observed in the initial steps of the chemical shift titration (Figure 4C).

Figure 4. Chemical shift titration of 15N-labeled MKK412-86 with p38a. (A) Regions of the 1H-15N HSQC spectrum of MKK412-86 with different admixtures of p38a: 0 % (gray), 10 % (green), 15 % (red). (B) HSQC intensity profile (I/I0) of isolated MKK412-86 (I0) compared to MKK412-86 with different admixtures of p38a (I): 6 % (blue), 10 % (green), 15 % (red). (C) 15N chemical shift perturbations in MKK412-86 upon addition of different molar ratios of p38a: 6 % (blue), 10 % (green), 15 % (red).



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Figure 5. Interaction of MKK412-86 with p38a as probed by 15N nuclear relaxation rates. (A) R2 relaxation rates of isolated MKK412-86 (black) and of MKK412-86 in the presence of different admixtures of p38a: 2.5 % (green), 6 % (blue) and 10 % (red) obtained at a 1H frequency of 850 MHz. (B) R2 relaxation rates of MKK412-86 with 6 % (molar fraction) of p38a obtained at 600 (gray) and 850 MHz (blue). (C) CPMG RD data showing the difference between the effective R2 rates at high (1 kHz) and low (31 Hz) CPMG frequencies obtained at 600 (gray) and 850 MHz (blue). (D) Comparison of effective R2 rates from the CPMG RD experiments and R1r-derived R2 rates at a 1H frequency of 850 MHz. Data are shown of MKK412-86 with 6 % p38a. All relaxation rates were obtained at 5°C in the presence of 5 % glycerol.

To obtain further insight into the interaction profile and dynamics of the MKK4:p38a complex, we measured 15N rotating frame relaxation rates, R1r, of the free form of MKK412-86 as well as in the presence of three different admixtures of p38a (Figure 5A). The R1r rates were converted


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into transverse relaxation rates, R2, by taking into account off-resonance effects.31 Large increases in the 15N R2 rates are observed even for small molar ratios of p38a for all residues of the docking site motif and the KIS domain, as well as for residues immediately adjacent to these regions. These results confirm the extended nature of the MKK4 interaction site with p38a. To reveal the origin of the increases in relaxation rates, we measured R1r rates at two different magnetic field strengths of MKK412-86 containing 6 % (molar ratio) of p38a. The profiles of these rates closely match each other at the two magnetic field strengths without clear deviations for single residues or stretches of residues, demonstrating that the spin-lock field (1.5 kHz) has effectively quenched contributions to R2 arising from exchange between the free and bound state of MKK412-86. Therefore, the R2 rates of MKK412-86, R2sub, measured at the different admixtures of p38a (Figure 5A) probe an increasing contribution from the R2 rates of the complex. The relaxation rates, R2sub, are given by51,52 𝑅"#$% = 𝑅"'()) +

+,- ./0123 45

eq 1

6789:

This expression is valid for exchange between two states of unequal transverse relaxation rates (DR2 = R2bound – R2free) and pbound is the population of MKK412-86 in complex with p38a, while kex is the rate of exchange between the free and bound state. The dynamic interaction profile (R2bound) of MKK412-86 in complex with p38a can therefore be obtained from eq. 1 provided that the exchange rate, kex, and the population, pbound, can be accurately determined, for example from NMR exchange techniques as detailed below.

Interaction of MKK4 with p38a Detected by CPMG Relaxation Dispersion and CEST 15

N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) in MKK412-86 with three

different admixtures of p38a (2.5, 6 and 10 %) were measured at 1H frequencies of 600 and



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850 MHz. Conformational exchange contributions are observed for residues within the docking site motif and the KIS domain (Figure 5C). Significant errors in derived exchange parameters from RD can be obtained if the two exchanging species display unequal intrinsic transverse relaxation rates.53 With this in mind, we complemented our dataset with chemical exchange saturation transfer (CEST) data at 600 MHz (for the same admixtures as the RD data) that are sensitive to changes in the transverse relaxation rate between the exchanging states. Several observations can be made on the basis of the experimental data (Figure 6): 1) The RD curves of MKK412-86 are amplified (increasing exchange contributions) with increasing molar ratio of p38a demonstrating that the excited state detected by these experiments most likely correspond to the p38a-bound state. 2) The RD curves asymptote to the R1r-derived R2 rates at high CPMG frequencies (Figure 5D), and the increase in the plateau values with the molar fraction of p38a therefore reflects an increasing contribution from the relaxation rate, R2bound, in the complex. 3) The 15N CEST data reveal that the chemical shift difference between the free and bound state of MKK412-86 is relatively small (|Dw| < 1.5 ppm) as all residues within the docking site motif and the KIS domain show merged, asymmetric CEST peaks, with the exception of residue K45 that display two nicely separated peaks (|Dw| » 4 ppm). We selected a subset of residues displaying large exchange contributions in the RD data (residues R41, K45, A49, F53, K54, Figure 5C), and we analyzed the RD and CEST data simultaneously across all admixtures according to a two-state exchange model using ChemEx (Figure 6).12,36 For each admixture, global values (across all residues) for kex and pbound were optimized, while the chemical shift difference, Dw, between free and bound MKK412-86 as well as the rates R2free and R2bound were obtained on a per-residue basis with common values across all admixtures (Table S1).


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Figure 6. Interaction between MKK412-86 and p38a detected by 15N CPMG RD and CEST. Each row presents data from a single residue: CPMG (left) and CEST (right). The dispersion and CEST data were acquired in MKK412-86 at three different admixtures of p38a: 2.5 % (green), 6 % (blue) and 10 % (red). The CPMG dispersion experiments were recorded at two different magnetic field strengths (600 and 850 MHz), while the CEST experiments were recorded at 600 MHz using a B1 saturating field of 26 Hz. Experimental data are displayed as points, while full drawn lines correspond to a simultaneous analysis of all the data according to a two-site exchange model (see text). All exchange data were obtained at 5°C in the presence of 5 % glycerol.

The simultaneous analysis of the RD and CEST data gives populations, pbound, of bound MKK412-86 of 2.6, 6.1 and 9.6 % for the three different admixtures in good agreement with the expected values according to the measured dissociation constant of the complex (Figure S3). The fitted exchange rates were 231 ± 5, 249 ± 5 and 269 ± 8 s-1 for the 2.6, 6.1 and 9.6 % admixtures, respectively, showing that the exchange rates are almost entirely dominated by the off-rate of the complex. We note that the fitted values for R2free (Table S1) correspond within experimental error to those measured independently in isolated MKK412-86 (Figure 5A, black data) showing that the NMR exchange data indeed detect conformational exchange departing from the free state of MKK412-86. Importantly, the fitted values of R2bound (Table S1) range from



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70 s-1 up to 155 s-1 depending on the residue and the magnetic field strength showing that the excited state corresponds to the p38a-bound state. To investigate whether residues within the docking site motif and the KIS domain experience the same exchange parameters, we also carried out individual fits for residues in these two regions. The pbound value derived for residues within the docking site motif is identical within experimental error to the value derived for residues within the KIS domain (Table S2). The analysis of the experimental data does therefore not show evidence for a different exchange behavior of the KIS domain compared to the docking site motif. Dynamic Interaction Profile of the MKK4-p38α Signaling Complex The analysis of the RD and CEST data shows that the formation of the MKK4:p38α signaling complex can be described by a simple two-site exchange between the free state of MKK412-86 and the p38a-bound state. The dynamic interaction profile (R2bound) of MKK412-86 can therefore be calculated for all residues from the R2 values (Figure 5A) measured for the different admixtures of p38a using eq. 1. The R2bound values calculated for the individual admixtures closely resemble each other showing consistency between the different datasets (Figure 7). The R2bound values decay to the experimentally measured values of R2free as we approach the Nterminus of MKK412-86, showing that these residues remain dynamically unaffected by the interaction with p38a. The largest R2bound values are observed for residues within the docking site motif, while residues of the KIS domain show lower rates. This observation is consistent with the docking site motif mediating a specific interaction with p38a, while the KIS domain experiences fast dynamics (nanosecond motions) in the complex by sampling a number of different conformations on the surface of p38a. We note that as the NMR exchange experiments show nearly identical populations of bound MKK4 for residues within the docking site motif and the KIS domain (Table S2), the experimental data support a model, where the


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KIS domain is persistently in contact with the surface of p38 rather than undergoing exchange between a single ordered (on-surface) and multiple disordered (off-surface) conformations. The residues located C-terminally of the KIS domain also show slightly elevated R2bound rates. Interestingly, this region shows increased rigidity in free MKK412-86 (Figure 2D, E), and subsequently experiences further restriction of motion upon interaction with p38a, according to the calculated R2bound relaxation rates.

Figure 7. Calculated transverse relaxation rates, R2bound, of MKK412-86 in complex with p38a at a 1H frequency of 850 MHz. The rates were obtained from the relaxation data presented in Figure 5A for each admixture separately using eq. 1. The values for pbound and kex were extracted for each admixture from the RD and CEST experiments (Figure 6 and Table S1). The dashed line corresponds to the expected relaxation rate of free p38a: Global analysis of 15N relaxation rates in p38a at 25°C resulted in a rotational correlation time of 21.8 ns.54 Under our experimental conditions (5°C and 5% glycerol), we estimate a correlation time of 47.1 ns by taking into account the change in viscosity55 using the Stokes-Einstein equation. Using this value for the correlation time, we estimate an average R2 rate of 85 s-1 (850 MHz) for p38a taking into account dipolar and CSA contributions.

Structure and Dynamics of the MKK4-p38α Signaling Complex The RD and CEST experiments of MKK412-86 with different admixtures of p38a provide clear evidence that the KIS domain of MKK4 is directly involved in complex formation (Figure 5C, D). In order to map the binding site of this domain on p38a, we recorded TROSY spectra of perdeuterated p38a with saturating amounts of MKK4 peptides of different lengths (Figure 8). Addition of the canonical docking site peptide of MKK4 (residues 39-50) leads to CSPs primarily in three different regions of p38a involving residues 110-129, 160-164 and 304-320 that are all located at or in close proximity to the docking groove. Upon addition of the MKK4



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peptide encompassing both the docking site motif and the KIS domain (residues 33-62), additional CSPs are observed in p38a for the residues 216-221 and 269-280. No additional CSPs were observed upon addition of the entire regulatory domain (Figure S4) showing that the residues located C-terminally of the KIS domain of MKK412-86 do not make specific contacts with p38a.

Figure 8. Titration of p38a (1H, 15N, 2H) with different unlabeled MKK4 peptides. (A) Two different regions of the 1H-15N TROSY spectra of p38a (red) with five times excess of the canonical docking site peptide of MKK4 (residues 39-50, blue) and a peptide encompassing both the docking site motif and the KIS domain (residues 33-62, green). (B) Combined 1H and 15N CSPs (15N shifts divided by a factor of 8 compared to 1H shifts) in p38a upon addition of the docking site peptide (blue) and the peptide encompassing both the docking site motif and the KIS domain (green). All spectra were recorded at 25°C in the absence of glycerol.

On the basis of this information, we propose a model of the MKK4:p38a signaling complex that encodes the dynamic interaction profile of MKK412-86 obtained from the NMR relaxation


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experiments (Figure 9). The docking site motif of MKK4 binds at the docking groove of p38a, while the KIS domain makes contacts with a region located at the bottom of the C-lobe of p38a, which is largely composed of surface-exposed hydrophobic residues such as V273, I275, G276, A277, P279 and L280. We note that while the docking site motif adopts a specific, rigid conformation within the complex and anchors the regulatory domain to p38a, the KIS domain experiences fast dynamics in the complex and samples a number of conformations in a dynamic equilibrium consistent with the relaxation rates, R2bound, within the complex (Figure 9).

Figure 9. Structural model of the MKK4:p38a signaling complex encoding the dynamic interaction profile of MKK412-86 derived from NMR relaxation. The structure of p38a is shown in cartoon representation (beige) with Ca atoms shown as spheres for the residues displaying CSPs upon interaction with the canonical docking site peptide of MKK4 (residues 39-50, blue) and the peptide encompassing both the docking site motif and the KIS domain (residues 33-62, green). The regulatory domain of MKK4 is represented by a single structure which is in agreement with the experimental CSPs in both MKK4 and p38a. MKK4 is color coded according to the transverse relaxation rate, R2bound, in the complex (Figure 7). Gray residues indicate proline residues for which no data could be obtained. For clarity, the first 30 N-terminal amino acids of the regulatory domain of MKK4 are not displayed as they remain dynamically unaffected by the interaction with p38a.

DISCUSSION In the MAPK pathways, signaling specificity is controlled by intrinsically disordered domains of kinases, phosphatases and scaffold proteins that mediate the assembly and co-localization of



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multiple enzymes into large signaling complexes. The seven human MKKs, all have disordered regulatory domains that specifically recruit their cognate MAPKs. While numerous structures of folded, catalytic domains of MAPKs and MKKs have been solved by X-ray crystallography, we have very little information about the structural propensities and dynamics of the regulatory domains of the MKKs and their interaction mechanisms with MAPKs. Here we have obtained an atomic resolution description of the disordered domain of MKK4 showing that this region behaves as a statistical coil with the exception of slightly elevated populations of PPII conformations within its docking site motif and at the C-terminus (Figure 3). While elevated PPII conformations were also observed within the second docking site motif of MKK7, the statistical coil behavior of MKK4 is in contrast to the regulatory domain of MKK7 that contains a 30-residue helical molecular recognition element at its N-terminus.23 The disordered domains of the MKKs therefore display a large structural diversity encoded in their primary sequences. In general docking site motifs are thought to determine signaling specificity by varying the number and position of hydrophobic and basic residues. However, it has been impossible to establish general rules for pathway discrimination by docking site motifs. Analysis of crystal structures and affinity measurements pointed towards an essential role for the intervening region between the consensus positions of the docking site as the key determinants for specificity,22 while a recent study, using substrate competition assays, suggested that the nature of the hydrophobic residues within the docking site governs specificity.56 Here, we provide evidence that KIS domains are key players in the recognition of MAPKs by MKKs and should be considered, in addition to docking site motifs, as mediators of specificity. The role of KIS domains in molecular recognition has previously been noted in the binding of p38α to the regulatory domains of different phosphatases. It was found that the docking site motif is essential for binding to p38α, while the specificity towards different phosphatases is


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encoded in the KIS domain.25 In addition, Peti, Page and co-workers directly proved the implication of the KIS domain in binding to p38α on the basis of NMR titrations of labeled p38α with unlabeled constructs of the regulatory domain of hematopoietic tyrosine phosphatase (HePTP).26 Our structural model of the MKK4:p38α complex show striking similarity to the HePTP:p38α complex (Figure S5) suggesting that p38α in general rely on KIS domains for molecular recognition in order to enhance affinity and specificity. Interestingly, despite such high similarity between the complexes, the KIS domains of MKK4 and HePTP show essentially no sequence identity (Figure S5). This observation is remarkable from a structural point of view and may only be understood by accounting for the dynamics of the KIS domain on the surface of p38α as described below. In this study, we have deciphered the dynamic interaction profile of the disordered domain of MKK4 in complex with p38α using a combination of R1r, CPMG RD and CEST. The RD and CEST experiments show that the docking site motif and the KIS domain experience the same exchange process, revealing identical parameters for the exchange rate and the population of bound MKK4 (Table S2). This demonstrates a concerted binding of these two regions upon formation of the complex and that the KIS domain makes persistent contacts with the surface of p38α. The experimental R1r rates measured for different admixtures allowed us to derive the dynamic profile, R2bound, of MKK4 in the complex. The docking site motif shows values that are elevated above those estimated for free p38α under the same experimental conditions (Figure 7). This is in agreement with a significant contribution to the total correlation time of the complex from the N- and C-terminal regions of the regulatory domain that remain dynamically unaffected by the interaction with p38α. Disordered segments are known to significantly increase the correlation time of folded domains by exerting a so-called “drag” effect.57 In addition, we note that the R2bound values are significantly lower for the basic cluster



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(K40-R41-K42) of the docking site motif suggesting that these residues do not make stable contacts with p38α, in contrast to the hydrophobic cluster. Surprisingly, the KIS domain shows R2bound values that are significantly lower than the docking site motif. This can be explained by fast dynamics of the KIS domain on the surface of p38α, presumably controlled by fluctuating hydrophobic contacts between the KIS domain and p38α. This interaction mode would also explain how the KIS domains of both MKK4 and HePTP retain the same binding mode to p38α (Figure 9 and Figure S5) despite low or absent sequence identity in the KIS domains. The KIS domains of both MKK4 and HePTP contain a number of hydrophobic residues that could be responsible for mediating this dynamic interaction (in particular F53 and F59 in MKK4 that show high R2bound values). In general, our study demonstrates that IDPs can employ a combination of interaction modes leading to a complex displaying significantly different dynamics across the bound regions. This feature may ensure that interactions become highly specific due to the use of an extended linear motif for binding, while at the same time retaining the dynamic behavior necessary for rapid dissociation characteristic of signaling pathways, where components are linked by a multitude of linear motifs. In addition, the possibility of describing the conformational dynamics of IDP complexes at atomic resolution will be of fundamental importance in the rational conception of drug design for IDPs.

EXPERIMENTAL SECTION Expression and Purification of the Regulatory Domain of MKK4 The intrinsically disordered regulatory domain of MKK4 (residues 1-86 or 12-86), were subcloned into a modified pET-28a vector with an N-terminal thioredoxin and a 6xHis tag followed by a tobacco etch virus (TEV) cleavage site. Escherichia coli (E. coli) BL21(DE3) cells were transformed with one of the MKK4 constructs and grown in lysogeny broth (LB) medium at


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37°C until the optical density (OD) at 600 nm reached 0.6. Protein expression was induced by addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The cultures were grown while shaking at 37°C for an additional three hours or overnight at 18°C, and the cells were harvested by centrifugation. Isotopically 15N/13C and 15N labeled samples were produced by growing transformed E. coli BL21(DE3) cells according to the protocol described previously.58 The proteins were purified using Ni affinity chromatography followed by size-exclusion chromatography. For cell lysis, inhibitor cocktail (Roche) was added to the purification buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol (BME)). The elution buffer was the same as the purification buffer, but with 250 mM imidazole. After removing thioredoxin and the 6xHis tag with the TEV protease and after a second Ni affinity column, size-exclusion chromatography was performed on a Superdex 75 column (GE Healthcare) equilibrated with NMR buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 2 mM dithiothreitol (DTT)).

Expression and Purification of p38α Synthetic codon-optimized p38α corresponding to residues 1-360 (Uniprot Q16539) was subcloned into a pET vector with a N-terminal 6-His-Tag followed by a TEV cleavage site. Transformed E. coli BL21(DE3) cells were grown in LB medium at 37°C and protein expression was induced at 18°C with addition of IPTG at OD600 of 0.6. Cells were harvested 12-14 h later by centrifugation and were frozen at −80°C. Harvested cells were resuspended in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 % (vol/vol) glycerol, 10 mM imidazole, 2 mM BME, protease inhibitor mixture (Roche)), sonicated on ice, applied to Ni affinity chromatography column, and washed with lysis buffer without protease inhibitor mixture. Protein was eluted in elution buffer (50 mM Tris pH 8.0, 150 mM NaCl, 250 mM imidazole, 2



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mM BME). The eluted protein was dialysed against 50 mM HEPES pH 7.0, 150 mM NaCl, 5 % (vol/vol) glycerol, 2 mM BME and digested using TEV protease. The protein was loaded on a size exclusion chromatography column equilibrated with 50 mM HEPES pH 7.0, 150 mM NaCl, 5% (vol/vol) glycerol, 2 mM DTT. All purification steps were performed at 4 °C. 13C, 15

N, 2D-labeled p38α was obtained by growing the cells in D2O and supplementing the growth

medium with deuterated glucose.

NMR Spectroscopy Spectral assignments of the regulatory domain of MKK4 (residues 1-86 and 12-86) were obtained at 5°C in NMR buffer using a set of BEST-type triple resonance spectra.37 All spectra were processed in NMRPipe 59, visualized in Sparky 60 and the program MARS 61 was used for automatic assignment of spin systems followed by manual verification. Random coil values for the calculation of secondary chemical shifts were obtained from the neighbour corrected intrinsically disordered protein library.62 Residual dipolar couplings (RDCs) were obtained at 5°C by aligning 15N, 13C-labeled MKK41286

in a liquid crystal composed of poly-ethylene glycol (PEG C8E5, Sigma) and 1-octanol

giving rise to a 2H quadrupole splitting of 23 Hz.63 The RDCs were obtained using BEST-type three-dimensional HNCO- and HN(CO)CA experiments as described previously.64 The

15

N relaxation rates R1, R1ρ and {1H}-15N heteronuclear Overhauser effects (nOes) of

MKK41-86 were measured in NMR buffer at 5°C and a 1H frequency of 850 MHz using HSQCdetected pulse sequences.65 In addition, CSA-DD transverse cross-corrected cross-relaxation rates (hxy) were measured under the same conditions.66 The measured R1ρ and R1 rates were converted into transverse relaxation rates, R2, by taking into account off-resonance effects.31 Chemical shift titrations of 15N labeled MKK412-86 with unlabeled p38α were carried out at 5°C in NMR buffer supplemented with 5% glycerol (vol/vol). The concentration of MKK412-86 was


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kept constant (247 µM), while varying the concentration of p38α (0, 2.5, 6, 10 and 15 % molar ratio). A 1H-15N HSQC spectrum was recorded at a 1H frequency of 950 MHz at each step of the titration. In addition, 15N R1 and R1ρ were recorded at a 1H frequency of 850 MHz and 5°C for the samples containing 0, 2.5, 6 and 10 % p38α. In addition,

15

N R1 and R1ρ was also

measured for the 6 % sample at a 1H frequency of 600 MHz at 5°C. The 15N relaxation dispersion CPMG experiments34 were carried out at 5°C at 1H frequencies of 600 and 850 MHz on the samples of 15N-labeled MKK412-86 containing 2.5, 6 and 10 % of unlabeled p38α. A constant-time relaxation delay of 32 milliseconds (ms) was employed with CPMG frequencies ranging between 31 and 1000 Hz. The 15N CEST experiments36 were carried out at a 1H frequency of 600 MHz at 5°C for the same samples as the CPMG relaxation dispersion. An 15N B1 field strength of 26 Hz was applied for all samples during a constant period of 300 ms. All NMR exchange data were fitted simultaneously as described in the main text using the program ChemEx.36 Interaction of perdeuterated p38α with different unlabeled constructs of MKK4 was carried out in 50 mM HEPES, pH 6.8, 150 mM NaCl, 5 mM DTT at 25°C in order to match the conditions used for the spectral assignment of p38α.26 Peptides (>98% purity) corresponding to the docking site of MKK4 (residues 39-50) or the docking site and the KIS domain (residues 3362) were obtained from CASLO Laboratory ApS (Denmark). All peptides were soluble and their concentration was estimated using a BCA assay (Thermo). The extended peptide was dialyzed extensively against the p38α buffer before interaction studies, whereas the canonical peptide was dissolved directly in the p38α buffer followed by a verification of the pH. 1H-15N TROSY spectra of perdeuterated p38α (200 µM) with five times excess of the two peptides were recorded and compared to the spectrum of free p38α.

Isothermal Titration Calorimetry



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ITC measurements were performed on a MicroCal iTC200 (GE Healthcare, PA) at 20°C. Injections of 3 µl were carried out every 180 seconds, 13 in total at a stirring speed of 800 rpm. Prior to the experiments, MKK412-86 and p38α were dialyzed into ITC buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 5 % glycerol (vol/vol), 2 mM BME). MKK412-86 (1.0 mM) was titrated into a solution of p38α with a concentration of 50 µM. Experiments were done in triplicates. The resulting binding isotherms were analyzed by a nonlinear least square fit of the experimental data to a single site model using the Microcal Origin 7.0 software.67,68 Experiments were done in triplicates, and the variability estimated to be 5 % in the binding enthalpy and 10 % in both the binding affinity and the number of sites.

Ensemble Selections of MKK412-86 A pool of conformers (10000 structures) of MKK412-86 was initially generated on the basis of the statistical coil library implemented in the program Flexible-Meccano.44,45 This library closely resembles alternative statistical coil libraries bearing the same local sampling features.69,70 Side chains were added using SCCOMP71 and chemical shifts were predicted using SPARTA72. Five sub-ensembles of 200 conformers each were selected from the pool using ASTEROIDS47,73,74 on the basis of the experimental chemical shifts using the following estimated errors: 1HN (0.04 ppm), 15N (0.2 ppm), 13Ca (0.1 ppm), 13C’ (0.1 ppm) and 13Cb (0.1 ppm). As the experimental chemical shifts were obtained at 5°C, a correction was applied using previously determined temperature coefficients in order to match the temperature (25°C) at which most proteins were assigned in the SPARTA database.75 The backbone dihedral angles were subsequently extracted from the five representative ensembles giving 1000 f/y pairs for each residue. These dihedral angle distributions were used to generate a new pool of conformers from which a new round of selections of five ensembles


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was carried out. This procedure was repeated five times (iterations) until convergence with respect to the experimental chemical shifts was achieved. In a final step, experimental 1DNH, 1DCαHα and 1DCαC’ RDCs were included in the target function to select sub-ensembles in agreement with both experimental chemical shifts and RDCs starting from a pool of structures (25000 conformers) created using the 1000 f/y pairs extracted from the fifth chemical shift iteration (20000 conformers) and a standard statistical coil ensemble (5000 conformers). RDCs were calculated for each member of the ensemble using PALES76 using a local alignment window of 15 amino acids in length77 combined with a baseline taking into account the chain-like nature of the protein.73 Two types of RDCs (4DHαHN and 2DHNC’) were retained for cross-validation of the resulting ensembles. The final ensembles were analyzed in terms of site-specific populations in four distinct regions of Ramachandran space defined as: aL {f > 0°}; aR {f < 0, -120° < ψ < 50°}; bP {-100° < f < 0°, ψ > 50° or ψ < 120°}; bS {-180° < f < -100°, ψ > 50° or ψ < -120°}.

Modeling the Complex of p38a:MKK4 A model of the docking site peptide of MKK4 in complex with p38a was obtained on the basis of the X-ray structure of the p38a:MEF2A complex (PDB 1LEW). The sequences of the docking sites of MKK4 and MEF2A were aligned, and PyMol was used to change the residues of the MEF2A peptide into the corresponding residues of the MKK4 peptide. Appropriate side chain rotamers devoid of steric clashes with p38a were selected. The missing activation loop in the X-ray structures of p38a were modelled using the ARCH_Pred server.78 The chains Cand N-terminally of the docking site motif of MKK4 were subsequently built using FlexibleMeccano45 on the basis of the amino acid specific conformational sampling derived from the experimental NMR chemical shifts and RDCs. Steric clashes of the disordered parts of MKK4 with p38a were avoided. Structures of the complex were sorted by minimizing the distance



Page 26 of 30

from any residue in the KIS domain to any residue experiencing CSPs in p38a. These structures are then expected to fulfill the CSPs observed both within the KIS domain (Figure 4) and within p38a (Figure 8). Figure 9 presents one of these structures color coded according to the dynamics in the complex.

SUPPORTING INFORMATION Assigned HSQC spectra of MKK4; the bulkiness profile of MKK4; ITC data of the p38a:MKK4 complex; CSPs in p38a upon interaction with MKK412-86; comparison of the p38a:MKK4 complex with p38a:HePTP; results of the analysis of NMR exchange data. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS The authors thank Prof. Wolfgang Peti for providing the ensemble of the p38a:HePTP complex. This work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). Financial support is acknowledged from the French Agence Nationale de la Recherche through ANR JCJC NMRSignal (to M.R.J.). J.K. acknowledges support from the IDPbyNMR Marie Curie action of the European commission (Contract no 264257). We thank Caroline Mas for assistance and access to the biophysical platform.

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Deciphering the Dynamic Interaction Profile of an Intrinsically

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