Deciphering the Dynamic Interaction Profile of an IntrinsicallyDisordered Protein by NMR Exchange SpectroscopyElise Delaforge,†,⊥ Jaka Kragelj,†,⊥ Laura Tengo,† Andres Palencia,‡ Sigrid Milles,†

Guillaume Bouvignies,§,∥ Nicola Salvi,† Martin Blackledge,† and Malene Ringkjøbing Jensen*,†

†Universite Grenoble Alpes, CNRS, CEA, IBS, F-38000 Grenoble, France‡Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, Universite Grenoble Alpes, F-38000 Grenoble, France§Laboratoire des Biomolecules, Departement de Chimie, Ecole Normale Superieur, UPMC Universite Paris 06, CNRS, PSL ResearchUniversity, 24 rue Lhomond, 75005 Paris, France∥Sorbonne Universites, UPMC Universite Paris 06, Ecole Normale Superieur, CNRS, Laboratoire des Biomolecules (LBM), 75005Paris, France

*S Supporting Information

ABSTRACT: Intrinsically disordered proteins (IDPs) display a largenumber of interaction modes including folding-upon-binding, bindingwithout major structural transitions, or binding through highlydynamic, so-called fuzzy, complexes. The vast majority ofexperimental information about IDP binding modes have beeninferred from crystal structures of proteins in complex with shortpeptides of IDPs. However, crystal structures provide a mainly staticview of the complexes and do not give information about theconformational dynamics experienced by the IDP in the bound state.Knowledge of the dynamics of IDP complexes is of fundamentalimportance to understand how IDPs engage in highly specificinteractions without concomitantly high binding affinity. Here, wecombine rotating-frame R1ρ, Carr−Purcell−Meiboom Gill relaxation dispersion as well as chemical exchange saturation transferto decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamicsignaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disorderedregulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interactionmodes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

■ INTRODUCTION

Intrinsically disordered proteins (IDPs) are implicated in awide range of biological functions, among them signaltransduction, transcription, cell cycle regulation, and chaperon-ing.1 To carry out their functions, IDPs rely on linear motifs,i.e., short sequence segments that mediate interactions withpartner proteins.2 IDPs display a large number of interactionmodes including folding-upon-binding,3 binding without majorstructural transitions, or binding through highly dynamiccomplexes where several conformations are sampled on thesurface of the partner (fuzziness).4,5 The vast majority ofexperimental information about IDP binding modes have beeninferred from crystal structures of proteins in complex withpeptides corresponding to linear motifs of IDPs.6,7 However,only short IDP sequences that remain sufficiently rigid and bindto their partner proteins with moderate-to-high affinity arelikely to crystallize. In addition, crystal structures provide amainly static view of the complexes and do not give informationabout conformational dynamics, or the associated time scales,experienced by the IDP in the complex. Knowledge ofconformational dynamics of IDP complexes is of fundamental

importance to understand the molecular details of IDP bindingmodes and how IDPs engage in highly specific interactionswithout concomitantly high binding affinity.8

Nuclear magnetic resonance (NMR) spectroscopy remainsthe only experimental technique that can access both structureand dynamics of IDP complexes at atomic resolution, even forinteractions displaying low to moderate affinities. However,chemical shift titrations of IDPs with their binding partnersoften lead to excessive line broadening of the NMR resonancesdue to conformational exchange occurring on the micro- tomillisecond (μs-ms) time scale. In order to overcome thisproblem, NMR exchange techniques have been used in a fewcases to visualize IDP interaction trajectories and to determinethe structure of the bound complex by creating a low-populatedbound state of the IDP (detectable by the exchangeexperiments) by adding small substoichiometric amounts ofthe binding partner.9−12 Yet, these studies have not

Received: November 23, 2017Published: December 25, 2017

Article

pubs.acs.org/JACSCite This: J. Am. Chem. Soc. 2018, 140, 1148−1158

© 2017 American Chemical Society 1148 DOI: 10.1021/jacs.7b12407J. Am. Chem. Soc. 2018, 140, 1148−1158

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concentrated directly on the site-specific dynamics of the boundIDP.Here, we study a dynamic signaling complex formed within

the mitogen-activated protein kinase (MAPK) cell signalingpathways.13−15 These pathways feature a kinase moduleconsisting of a MAPK kinase kinase (MKKK) that phosphor-ylates and thereby activates a MAPK kinase (MKK) that in turnactivates a MAPK by phosphorylation. The MKKs specificallyactivate the MAPKs according to the following scheme:MKK1/2/5 activate ERK, MKK3/4/6 activate p38, andMKK4/7 activate JNK. This specificity is mediated byintrinsically disordered N-terminal regulatory domains of theMKKs that recruit their cognate MAPKs. The interaction isfacilitated by docking site motifs composed of two to threebasic residues followed by a submotif containing threehydrophobic residues ΦL, ΦA, ΦB (K/R2−3−X1−6−ΦL−X1−3−ΦA−X−ΦB, where X is any amino acid type).16−18 Thedocking 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 commondocking (CD) groove (Figure 1).19−24 Docking site motifs are

present not only in MKKs, but are also found withinintrinsically disordered regions of a large number of proteinsincluding MAPK substrates, phosphatases, and scaffold proteinsand therefore represent important regulatory motifs in cellsignaling.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 theKIS 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 combinerotating-frame R1ρ,

28−31 Carr−Purcell−Meiboom Gill (CPMG)relaxation dispersion32−34 as well as chemical exchangesaturation transfer (CEST)35,36 to decipher the dynamicinteraction profile of MKK4. Interestingly, our results showhow the docking site motif remains rigid in the complex withp38α and thereby functions as an anchor point, while the KISdomain undergoes fast dynamics and samples several conforma-tional states on the surface of p38α in a fuzzy complex.

■ RESULTSResidual Structure and Dynamics of the Regulatory

Domain of MKK4. We expressed and purified the intrinsicallydisordered regulatory domain of MKK4 (residues 1−86,

MKK41−86) (Figure 2A, B) and obtained the completebackbone assignment using a series of BEST-type triple

resonance experiments37 (Figure S1A of the SupportingInformation, SI). The secondary 13Cα chemical shifts showthat this domain is devoid of transiently populated secondarystructures (Figure 2C). Nuclear relaxation rates can be used toprobe the dynamics of IDPs on the pico- to nanosecond timescale including correlated motions.38−42 We measured 15Ntransverse relaxation rates, R2,

15N transverse chemical shiftanisotropy (CSA)/15N−1H dipole−dipole (DD) cross-corre-

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

Figure 2. Regulatory domain of MKK4 is intrinsically disordered. (A)Domain organization of MKK4 with its intrinsically disorderedregulatory domain (residues 1−86), docking site motif for JNK andp38 (residues 40−48, blue), and the KIS domain (residues 49−62,green). (B) Sequence of the regulatory domain of MKK4 with thedocking site and KIS domain highlighted in blue and green,respectively. (C) Secondary chemical shifts of MKK41−86 calculatedon the basis of the experimental Cα chemical shifts. (D) Experimental15N transverse relaxation rates, R2, and CSA/DD cross-correlatedrelaxation rates, ηxy. (E) Experimental {

1H}−15N nOes of MKK41−86.All relaxation rates were obtained at 5 °C and a 1H frequency of 850MHz.

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lated relaxation rates, ηxy, as well as {1H}−15N nuclearOverhauser enhancements (nOes) (Figure 2D, E). All ratesfollow the same overall pattern with smaller rates for residues1−20 followed by more elevated rates for the central region ofthe protein and finally the largest rates for the regionencompassing residues 72−78. The profile is consistent withthe local dynamics being dominated by the bulkiness of theamino acids along the chain (Figure S2).43 The similar overallprofile of R2 and ηxy show that contributions from conforma-tional exchange occurring on the micro- to millisecond (μs-ms)time scale are negligible (Figure 2D), except for residue H31that shows a small exchange contribution, potentially due toprotonation/deprotonation of the imidazole side chain.In order to decrease signal overlap, notably within the serine/

glycine rich repeat region (residues 7−19), we also obtainedthe backbone assignment of a smaller subconstruct of theregulatory domain comprising residues 12−86. The spectrumof this construct (MKK412−86) superimposes almost perfectlyon the spectrum of MKK41−86 (Figure S1B), and was,therefore, used for further studies.Ensemble Description of MKK4 from Chemical Shifts

and Residual Dipolar Couplings. In order to precisely mapthe conformational sampling of MKK412−86, we measuredmultiple residual dipolar couplings (RDCs) (1DHN,

1DCαHα,

1DCαC′,2DHNC′, and

4DHNHα). The statistical coil generatorFlexible-Meccano44,45 was used in combination with the geneticalgorithm ASTEROIDS46 to select representative structuralensembles (five ensembles of 200 conformations each) inagreement with experimental 1H, 15N, and 13C chemical shiftsand RDCs.47−49 We used 1DNH,

1DCαHα, and1DCαC′ as active

parameters in the ensemble selections, and 4DHNHα and2DHNC′

were retained for cross validation. The selected ASTEROIDSensembles are in excellent agreement with the experimentaldata and, importantly, are able to reproduce the RDCs thatwere not included in the selections providing quantifiableevidence that the local conformational sampling contained inthe ASTEROIDS ensembles is genuine and accurate (Figure3).50

Analysis of the conformational sampling in terms ofpopulations in different regions of Ramachandran spaceshows an elevated population of poly proline II (PPII) withinthe MKK4 docking site motif and at the C-terminal end of theregulatory domain (Figure 3D). Other regions of MKK4essentially adopt statistical coil conformations and are devoid oftransiently populated secondary structures (Figure 3C, D).

Interaction of the Regulatory Domain of MKK4 withp38α. The interaction between MKK412−86 and p38α wasinitially studied by isothermal titration calorimetry (ITC)

Figure 3. Structural ensemble description of MKK412−86 from chemical shifts (1HN, 15N, 13Cα, 13Cβ and 13C′) 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, and1DCαC′ RDCs were included in the

ASTEROIDS selections (active parameters), while 4DHNHα and 2DHNC′ were retained for cross-validation of the resulting ensembles (passiveparameters). (C, D) Site-specific α-helical (αR) and PPII populations of MKK412−86 derived from five independently selected ASTEROIDSensembles (blue) compared to statistical coil populations (red). Blue and green shadings highlight the location of the docking site motif and the KISdomain, respectively.

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showing the formation of a 1:1 complex with a dissociationconstant of KD = 4.1 μM (Figure S3). Subsequently, wemonitored the interaction by chemical shift titrations. TheNMR signals of MKK412−86 display only small chemical shiftperturbations (CSPs) and undergo extensive line-broadening,even for small substoichiometric amounts of p38α (Figure 4A).

The observed line broadening may arise due to an increase inthe local correlation time due to the larger size of the p38αkinase (41 kDa) and from conformational exchange occurringon the μs−ms time scale between free and p38α-boundMKK412−86. The HSQC intensity profile of MKK412−86 in thepresence of p38α reveals that the interaction is not onlylocalized to the docking site motif, but implicates the KISdomain of MKK4, with the largest line broadening observed forthe residues within the docking site motif (Figure 4B). Thisinteraction profile is also supported by the small 15N CSPsobserved in the initial steps of the chemical shift titration(Figure 4C).To obtain further insight into the interaction profile and

dynamics of the MKK4:p38α complex, we measured 15N

rotating frame relaxation rates, R1ρ, of the free form ofMKK412−86 as well as in the presence of three differentadmixtures of p38α (Figure 5A). The R1ρ rates were converted

into transverse relaxation rates, R2, by taking into account off-resonance effects.31 Large increases in the 15N R2 rates areobserved even for small molar ratios of p38α for all residues ofthe docking site motif and the KIS domain, as well as forresidues immediately adjacent to these regions. These resultsconfirm the extended nature of the MKK4 interaction site withp38α.

Figure 4. Chemical shift titration of 15N-labeled MKK412−86 withp38α. (A) Regions of the 1H−15N HSQC spectrum of MKK412−86with different admixtures of p38α: 0% (gray), 10% (green), and 15%(red). (B) HSQC intensity profile (I/I0) of isolated MKK412−86 (I

0)compared to MKK412−86 with different admixtures of p38α (I): 6%(blue), 10% (green), and 15% (red). (C) 15N chemical shiftperturbations in MKK412−86 upon addition of different molar ratiosof p38α: 6% (blue), 10% (green), and 15% (red).

Figure 5. Interaction of MKK412−86 with p38α as probed by 15Nnuclear relaxation rates. (A) R2 relaxation rates of isolated MKK412−86(black) and of MKK412−86 in the presence of different admixtures ofp38α: 2.5% (green), 6% (blue), and 10% (red) obtained at a 1Hfrequency of 850 MHz. (B) R2 relaxation rates of MKK412−86 with 6%(molar fraction) of p38α obtained at 600 (gray) and 850 MHz (blue).(C) CPMG RD data showing the difference between the effective R2rates at high (1 kHz) and low (31 Hz) CPMG frequencies obtained at600 (gray) and 850 MHz (blue). (D) Comparison of effective R2 ratesfrom the CPMG RD experiments and R1ρ-derived R2 rates at a 1Hfrequency of 850 MHz. Data are shown of MKK412−86 with 6% p38α.All relaxation rates were obtained at 5 °C in the presence of 5%glycerol.

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To reveal the origin of the increases in relaxation rates, wemeasured R1ρ rates at two different magnetic field strengths ofMKK412−86 containing 6% (molar ratio) of p38α (Figure 5B).The profiles of these rates closely match each other at the twomagnetic field strengths without clear deviations for singleresidues or stretches of residues, demonstrating that the spin-lock field (1.5 kHz) has effectively quenched contributions toR2 arising from exchange between the free and bound state ofMKK412−86. Therefore, the R2 rates of MKK412−86, R2sub,measured at the different admixtures of p38α (Figure 5A)probe an increasing contribution from the R2 rates of thecomplex. The relaxation rates, R2sub, are given by the followingequation:51,52

= +Δ

+ ΔR RR p

1 Rk

2sub 2free2 bound

2

ex

(1)

This expression is valid for exchange between two states ofunequal transverse relaxation rates (ΔR2 = R2bound − R2free) andpbound is the population of MKK412−86 in complex with p38α,while kex is the rate of exchange between the free and boundstate. The dynamic interaction profile (R2bound) of MKK412−86in complex with p38α can therefore be obtained from eq 1provided that the exchange rate, kex, and the population, pbound,can be accurately determined, for example from NMR exchangetechniques as detailed below.Interaction of MKK4 with p38α Detected by CPMG

Relaxation Dispersion and CEST. 15N Carr−Purcell−

Meiboom−Gill (CPMG) relaxation dispersion (RD) inMKK412−86 with three different admixtures of p38α (2.5, 6and 10%) were measured at 1H frequencies of 600 and 850MHz. Conformational exchange contributions are observed forresidues within the docking site motif and the KIS domain(Figure 5C). Significant errors in derived exchange parametersfrom RD can be obtained if the two exchanging species displayunequal intrinsic transverse relaxation rates.53 With this inmind, we complemented our data set with chemical exchangesaturation transfer (CEST) data at 600 MHz (for the sameadmixtures as the RD data) that are sensitive to changes in thetransverse relaxation rate between the exchanging states.Several observations can be made on the basis of theexperimental data (Figure 6): (1) The RD curves ofMKK412−86 are amplified (increasing exchange contributions)with increasing molar ratio of p38α demonstrating that theexcited state detected by these experiments most likelycorrespond to the p38α-bound state. (2) The RD curvesasymptote to the R1ρ-derived R2 rates at high CPMGfrequencies (Figure 5D), and the increase in the plateau valueswith the molar fraction of p38α therefore reflects an increasingcontribution from the relaxation rate, R2bound, in the complex.(3) The 15N CEST data reveal that the chemical shift differencebetween the free and bound state of MKK412−86 is relativelysmall (|Δω| < 1.5 ppm) as all residues within the docking sitemotif and the KIS domain show merged, asymmetric CESTpeaks, with the exception of residue K45 that display two nicelyseparated peaks (|Δω| ≈ 4 ppm).

Figure 6. Interaction between MKK412−86 and p38α 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 p38α: 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 CESTexperiments were recorded at 600 MHz using a B1 saturating field of 26 Hz. Experimental data are displayed as points, while full drawn linescorrespond 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 inthe presence of 5% glycerol.

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We selected a subset of residues displaying large exchangecontributions in the RD data (residues R41, K45, A49, F53, andK54, Figure 5C), and we analyzed the RD and CEST datasimultaneously across all admixtures according to a two-stateexchange model using ChemEx (Figure 6).12,36 For eachadmixture, global values (across all residues) for kex and pboundwere optimized, while the chemical shift difference, Δω,between free and bound MKK412−86 as well as the rates R2freeand R2bound were obtained on a per-residue basis with commonvalues across all admixtures (Table S1).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 theexpected values according to the measured dissociationconstant of the complex (Figure S3). The fitted exchangerates were 231 ± 5, 249 ± 5, and 269 ± 8 s−1 for the 2.6, 6.1,and 9.6% admixtures, respectively, showing that the exchangerates are almost entirely dominated by the off-rate of thecomplex. We note that the fitted values for R2free (Table S1)correspond within experimental error to those measuredindependently in isolated MKK412−86 (Figure 5A, black data)showing that the NMR exchange data indeed detect conforma-tional exchange departing from the free state of MKK412−86.Importantly, the fitted values of R2bound (Table S1) range from70 s−1 up to 155 s−1 depending on the residue and the magneticfield strength showing that the excited state corresponds to thep38α-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 tworegions. The pbound value derived for residues within thedocking site motif is identical within experimental error to thevalue derived for residues within the KIS domain (Table S2).The analysis of the experimental data does therefore not showevidence for a different exchange behavior of the KIS domaincompared to the docking site motif.Dynamic Interaction Profile of the MKK4-p38α

Signaling Complex. The analysis of the RD and CESTdata shows that the formation of the MKK4:p38α signalingcomplex can be described by a simple two-site exchangebetween the free state of MKK412−86 and the p38α-bound state.The dynamic interaction profile (R2bound) of MKK412−86 cantherefore be calculated for all residues from the R2 values(Figure 5A) measured for the different admixtures of p38αusing eq 1. The R2bound values calculated for the individualadmixtures closely resemble each other showing consistencybetween the different data sets (Figure 7).The R2bound values decay to the experimentally measured

values of R2free as we approach the N-terminus of MKK412−86,showing that these residues remain dynamically unaffected bythe interaction with p38α. The largest R2bound values areobserved for residues within the docking site motif, whileresidues of the KIS domain show lower rates. This observationis consistent with the docking site motif mediating a specificinteraction with p38α while the KIS domain experiences fastdynamics (nanosecond motions) in the complex by sampling anumber of different conformations on the surface of p38α. Wenote that as the NMR exchange experiments show nearlyidentical populations of bound MKK4 for residues within thedocking site motif and the KIS domain (Table S2), theexperimental data support a model, where the KIS domain ispersistently in contact with the surface of p38 rather thanundergoing exchange between a single ordered (on-surface)

and multiple disordered (off-surface) conformations. Theresidues located C-terminally of the KIS domain also showslightly elevated R2bound rates. Interestingly, this region showsincreased rigidity in free MKK412−86 (Figure 2D, E), andsubsequently experiences further restriction of motion uponinteraction with p38α according to the calculated R2boundrelaxation rates.

Structure and Dynamics of the MKK4-p38α SignalingComplex. The RD and CEST experiments of MKK412−86 withdifferent admixtures of p38α provide clear evidence that theKIS domain of MKK4 is directly involved in complex formation(Figure 5C, D). In order to map the binding site of this domainon p38α, we recorded TROSY spectra of perdeuterated p38αwith saturating amounts of MKK4 peptides of different lengths(Figure 8). Addition of the canonical docking site peptide ofMKK4 (residues 39−50) leads to CSPs primarily in threedifferent regions of p38α involving residues 110−129, 160−164, and 304−320 that are all located at or in close proximity tothe docking groove. Upon addition of the MKK4 peptideencompassing both the docking site motif and the KIS domain(residues 33−62), additional CSPs are observed in p38α for theresidues 216−221 and 269−280. No additional CSPs wereobserved upon addition of the entire regulatory domain (FigureS4) showing that the residues located C-terminally of the KISdomain of MKK412−86 do not make specific contacts with p38α.On the basis of this information, we propose a model of the

MKK4:p38α signaling complex that encodes the dynamicinteraction profile of MKK412−86 obtained from the NMRrelaxation experiments (Figure 9). The docking site motif ofMKK4 binds at the docking groove of p38α, while the KISdomain makes contacts with a region located at the bottom ofthe C-lobe of p38α, 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 motifadopts a specific, rigid conformation within the complex andanchors the regulatory domain to p38α, the KIS domainexperiences fast dynamics in the complex and samples a

Figure 7. Calculated transverse relaxation rates, R2bound, of MKK412−86in complex with p38α at a 1H frequency of 850 MHz. The rates wereobtained from the relaxation data presented in Figure 5A for eachadmixture separately using eq 1. The values for pbound and kex wereextracted for each admixture from the RD and CEST experiments(Figure 6 and Table S1). The dashed line corresponds to the expectedrelaxation rate of free p38α: Global analysis of 15N relaxation rates inp38α at 25 °C resulted in a rotational correlation time of 21.8 ns.54

Under our experimental conditions (5 °C and 5% glycerol), weestimate a correlation time of 47.1 ns by taking into account thechange in viscosity55 using the Stokes−Einstein equation. Using thisvalue for the correlation time, we estimate an average R2 rate of 85 s

−1

(850 MHz) for p38α taking into account dipolar and CSAcontributions.

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number of conformations in a dynamic equilibrium consistentwith the relaxation rates, R2bound, within the complex (Figure 9).

■ DISCUSSIONIn the MAPK pathways, signaling specificity is controlled byintrinsically disordered domains of kinases, phosphatases, andscaffold proteins that mediate the assembly and colocalizationof multiple enzymes into large signaling complexes. The sevenhuman MKKs, all have disordered regulatory domains thatspecifically recruit their cognate MAPKs. While numerousstructures of folded, catalytic domains of MAPKs and MKKshave been solved by X-ray crystallography, we have very littleinformation about the structural propensities and dynamics ofthe regulatory domains of the MKKs and their interactionmechanisms with MAPKs.Here we have obtained an atomic resolution description of

the disordered domain of MKK4 showing that this regionbehaves as a statistical coil with the exception of slightlyelevated populations of PPII conformations within its dockingsite motif and at the C-terminus (Figure 3). While elevatedPPII conformations were also observed within the seconddocking site motif of MKK7, the statistical coil behavior of

MKK4 is in contrast to the regulatory domain of MKK7 thatcontains a 30-residue helical molecular recognition element atits N-terminus.23 The disordered domains of the MKKstherefore display a large structural diversity encoded in theirprimary sequences.In general docking site motifs are thought to determine

signaling specificity by varying the number and position ofhydrophobic and basic residues. However, it has beenimpossible to establish general rules for pathway discriminationby docking site motifs. Analysis of crystal structures and affinitymeasurements pointed toward an essential role for theintervening region between the consensus positions of thedocking site as the key determinants for specificity,22 while arecent study, using substrate competition assays, suggested thatthe nature of the hydrophobic residues within the docking sitegoverns specificity.56

Here, we provide evidence that KIS domains are key playersin the recognition of MAPKs by MKKs and should beconsidered, in addition to docking site motifs, as mediators ofspecificity. The role of KIS domains in molecular recognitionhas previously been noted in the binding of p38α to theregulatory domains of different phosphatases. It was found thatthe docking site motif is essential for binding to p38α, while thespecificity toward different phosphatases is encoded in the KISdomain.25 In addition, Peti, Page, and co-workers directlyproved the implication of the KIS domain in binding to p38α

Figure 8. Titration of p38α (1H, 15N, 2H) with different unlabeledMKK4 peptides. (A) Two different regions of the 1H−15N TROSYspectra of p38α (red) with five times excess of the canonical dockingsite peptide of MKK4 (residues 39−50, blue) and a peptideencompassing both the docking site motif and the KIS domain(residues 33−62, green). (B) Combined 1H and 15N CSPs (15N shiftsdivided by a factor of 8 compared to 1H shifts) in p38α upon additionof the docking site peptide (blue) and the peptide encompassing boththe docking site motif and the KIS domain (green). All spectra wererecorded at 25 °C in the absence of glycerol.

Figure 9. Structural model of the MKK4:p38α signaling complexencoding the dynamic interaction profile of MKK412−86 derived fromNMR relaxation. The structure of p38α is shown in cartoonrepresentation (beige) with Cα atoms shown as spheres for theresidues displaying CSPs upon interaction with the canonical dockingsite peptide of MKK4 (residues 39−50, blue) and the peptideencompassing both the docking site motif and the KIS domain(residues 33−62, green). The regulatory domain of MKK4 isrepresented by a single structure of the ensemble of theMKK4:p38α complex. MKK4 is color coded according to thetransverse relaxation rate, R2bound, in the complex (Figure 7). Grayresidues indicate proline residues for which no data could be obtained.For clarity, the first 30 N-terminal amino acids of the regulatorydomain of MKK4 are not displayed as they remain dynamicallyunaffected by the interaction with p38α.

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on the basis of NMR titrations of labeled p38α with unlabeledconstructs of the regulatory domain of hematopoietic tyrosinephosphatase (HePTP).26 Our structural model of theMKK4:p38α complex show striking similarity to theHePTP:p38α complex (Figure S5) suggesting that p38α ingeneral relies on KIS domains for molecular recognition inorder to enhance affinity and specificity. Interestingly, despitesuch high similarity between the complexes, the KIS domains ofMKK4 and HePTP show essentially no sequence identity(Figure S5). This observation is remarkable from a structuralpoint of view and may only be understood by accounting forthe dynamics of the KIS domain on the surface of p38α asdescribed below.In this study, we have deciphered the dynamic interaction

profile of the disordered domain of MKK4 in complex withp38α using a combination of R1ρ, CPMG RD, and CEST. TheRD and CEST experiments show that the docking site motifand the KIS domain experience the same exchange process,revealing identical parameters for the exchange rate and thepopulation of bound MKK4 (Table S2). This demonstrates aconcerted binding of these two regions upon formation of thecomplex and that the KIS domain makes persistent contactswith the surface of p38α. The experimental R1ρ rates measuredfor different admixtures allowed us to derive the dynamicprofile, R2bound, of MKK4 in the complex. The docking sitemotif shows values that are elevated above those estimated forfree p38α under the same experimental conditions (Figure 7).This is in agreement with a significant contribution to the totalcorrelation time of the complex from the N- and C-terminalregions of the regulatory domain that remain dynamicallyunaffected by the interaction with p38α. Disordered segmentsare known to significantly increase the correlation time offolded domains by exerting a so-called “drag” effect.57 Inaddition, we note that the R2bound values are significantly lowerfor the basic cluster (K40-R41-K42) of the docking site motifsuggesting that these residues do not make stable contacts withp38α, in contrast to the hydrophobic cluster.Surprisingly, the KIS domain shows R2bound values that are

significantly lower than the docking site motif. This can beexplained by fast dynamics of the KIS domain on the surface ofp38α, presumably controlled by fluctuating hydrophobiccontacts between the KIS domain and p38α. This interactionmode would also explain how the KIS domains of both MKK4and HePTP retain the same binding mode to p38α (Figures 9and S5) despite low or absent sequence identity in the KISdomains. The KIS domains of both MKK4 and HePTP containa number of hydrophobic residues that could be responsible formediating this dynamic interaction (in particular F53 and F59in MKK4 that show high R2bound values).In general, our study demonstrates that IDPs can employ a

combination of interaction modes leading to a complexdisplaying significantly different dynamics across the boundregions. This feature may ensure that interactions becomehighly specific due to the use of an extended linear motif forbinding, while at the same time retaining the dynamic behaviornecessary for rapid dissociation characteristic of signalingpathways, where components are linked by a multitude oflinear motifs. In addition, the possibility of describing theconformational dynamics of IDP complexes at atomicresolution will be of fundamental importance in the rationalconception of drug design for IDPs.

■ EXPERIMENTAL SECTIONExpression 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-28avector with an N-terminal thioredoxin and a 6xHis tag followed by atobacco etch virus (TEV) cleavage site. Escherichia coli (E. coli)BL21(DE3) cells were transformed with one of the MKK4 constructsand grown in lysogeny broth (LB) medium at 37 °C until the opticaldensity (OD) at 600 nm reached 0.6. Protein expression was inducedby addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a finalconcentration of 1 mM. The cultures were grown while shaking at 37°C for an additional 3 h or overnight at 18 °C, and the cells wereharvested by centrifugation. Isotopically 15N/13C and 15N labeledsamples were produced by growing transformed E. coli BL21(DE3)cells according to the protocol described previously.58

The proteins were purified using Ni affinity chromatographyfollowed by size-exclusion chromatography. For cell lysis, inhibitorcocktail (Roche) was added to the purification buffer (50 mM HEPESpH 8.0, 150 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol(BME)). The elution buffer was the same as the purification buffer, butwith 250 mM imidazole. After removing thioredoxin and the 6xHis tagwith 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 pH7.0, 150 mM NaCl, 2 mM dithiothreitol (DTT)).

Expression and Purification of p38α. Synthetic codon-optimized p38α corresponding to residues 1−360 (UniprotQ16539) 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 wasinduced at 18 °C with addition of IPTG at OD600 of 0.6. Cells wereharvested 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% (v/v) glycerol, 10 mM imidazole, 2 mM BME,protease inhibitor mixture (Roche)), sonicated on ice, applied to Niaffinity chromatography column, and washed with lysis buffer withoutprotease inhibitor mixture. Protein was eluted in elution buffer (50mM Tris pH 8.0, 150 mM NaCl, 250 mM imidazole, 2 mM BME).The eluted protein was dialyzed against 50 mM HEPES pH 7.0, 150mM NaCl, 5% (v/v) glycerol, and 2 mM BME and digested usingTEV protease. The protein was loaded on a size exclusionchromatography column equilibrated with 50 mM HEPES pH 7.0,150 mM NaCl, 5% (v/v) glycerol, 2 mM DTT. All purification stepswere performed at 4 °C. 13C, 15N, 2D-labeled p38α was obtained bygrowing the cells in D2O and supplementing the growth medium withdeuterated glucose.

NMR Spectroscopy. Spectral assignments of the regulatorydomain 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 andthe program MARS61 was used for automatic assignment of spinsystems followed by manual verification. Random coil values for thecalculation of secondary chemical shifts were obtained from theneighbor corrected intrinsically disordered protein library.62

Residual dipolar couplings (RDCs) were obtained at 5 °C byaligning 15N, 13C-labeled MKK412−86 in a liquid crystal composed ofpoly ethylene glycol (PEG C8E5, Sigma) and 1-octanol giving rise to a2H quadrupole splitting of 23 Hz.63 The RDCs were obtained usingBEST-type three-dimensional HNCO- and HN(CO)CA experimentsas described previously.64

The 15N relaxation rates R1, R1ρ, and {1H}-15N heteronuclearOverhauser effects (nOes) of MKK41−86 were measured in NMRbuffer at 5 °C and a 1H frequency of 850 MHz using HSQC-detectedpulse sequences.65 In addition, CSA-DD transverse cross-correctedcross-relaxation rates (ηxy) were measured under the sameconditions.66 The measured R1ρ and R1 rates were converted intotransverse relaxation rates, R2, by taking into account off-resonanceeffects.31

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Chemical shift titrations of 15N labeled MKK412−86 with unlabeledp38α were carried out at 5 °C in NMR buffer supplemented with 5%glycerol (v/v). The concentration of MKK412−86 was kept constant(247 μM), while varying the concentration of p38α (0, 2.5, 6, 10 and15% molar ratio). A 1H−15N HSQC spectrum was recorded at a 1Hfrequency of 950 MHz at each step of the titration. In addition, 15N R1

and R1ρ were recorded at a1H frequency of 850 MHz and 5 °C for the

samples containing 0, 2.5, 6 and 10% p38α. In addition, 15N R1 and R1ρ

was also measured for the 6% sample at a 1H frequency of 600 MHz at5 °C.The 15N relaxation dispersion CPMG experiments34 were carried

out at 5 °C at 1H frequencies of 600 and 850 MHz on the samples of15N-labeled MKK412−86 containing 2.5, 6 and 10% of unlabeled p38α.A constant-time relaxation delay of 32 ms was employed with CPMGfrequencies 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 relaxationdispersion. An 15N B1 field strength of 26 Hz was applied for allsamples during a constant period of 300 ms. All NMR exchange datawere fitted simultaneously as described in the main text using theprogram ChemEx.36

Interaction of perdeuterated p38α with different unlabeledconstructs 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 conditionsused for the spectral assignment of p38α.26 Peptides (>98% purity)corresponding to the docking site of MKK4 (residues 39−50) or thedocking site and the KIS domain (residues 33−62) were obtainedfrom 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 bufferbefore interaction studies, whereas the canonical peptide was dissolveddirectly in the p38α buffer followed by a verification of the pH.1H−15N TROSY spectra of perdeuterated p38α (200 μM) with fivetimes excess of the two peptides were recorded and compared to thespectrum of free p38α.Isothermal Titration Calorimetry. ITC measurements were

performed on a MicroCal iTC200 (GE Healthcare, PA) at 20 °C.Injections of 3 μL were carried out every 180 s, 13 in total at a stirringspeed of 800 rpm. Prior to the experiments, MKK412−86 and p38αwere dialyzed into ITC buffer (50 mM HEPES pH 7.0, 150 mM NaCl,5% glycerol (v/v), 2 mM BME). MKK412−86 (1.0 mM) was titratedinto a solution of p38α with a concentration of 50 μM. The resultingbinding isotherms were analyzed by a nonlinear least-square fit of theexperimental data to a single site model using the Microcal Origin 7.0software.67,68 Experiments were done in triplicates, and the variabilityestimated to be 5% in the binding enthalpy and 10% in both thebinding 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 basisof the statistical coil library implemented in the program Flexible-Meccano.44,45 This library closely resembles alternative statistical coillibraries bearing the same local sampling features.69,70 Side chains wereadded using SCCOMP71 and chemical shifts were predicted usingSPARTA.72 Five subensembles of 200 conformers each were selectedfrom the pool using ASTEROIDS46,47,73 on the basis of theexperimental chemical shifts using the following estimated errors:1HN (0.04 ppm), 15N (0.2 ppm), 13Cα (0.1 ppm), 13C′ (0.1 ppm), and13Cβ (0.1 ppm). As the experimental chemical shifts were obtained at5 °C, a correction was applied using previously determinedtemperature coefficients in order to match the temperature (25 °C)at which most proteins were assigned in the SPARTA database.74

The backbone dihedral angles were subsequently extracted from thefive representative ensembles giving 1000 ϕ/ψ pairs for each residue.These dihedral angle distributions were used to generate a new pool ofconformers from which a new round of selections of five ensembleswas carried out. This procedure was repeated five times (iterations)until convergence with respect to the experimental chemical shifts wasachieved.

In a final step, experimental 1DNH,1DCαHα, and

1DCαC′ RDCs wereincluded in the target function to select subensembles in agreementwith both experimental chemical shifts and RDCs starting from a poolof structures (25 000 conformers) created using the 1000 ϕ/ψ pairsextracted from the fifth chemical shift iteration (20 000 conformers)and a standard statistical coil ensemble (5000 conformers). RDCswere calculated for each member of the ensemble using PALES75 usinga local alignment window of 15 amino acids in length76 combined witha baseline taking into account the chain-like nature of the protein.46

Two types of RDCs (4DHαHN and 2DHNC′) were retained for cross-validation of the resulting ensembles. The final ensembles wereanalyzed in terms of site-specific populations in four distinct regions ofRamachandran space defined as αL {ϕ > 0°}; αR {ϕ < 0, −120° < ψ <50°}; βP {−100° < ϕ < 0°, ψ > 50° or ψ < −120°}; βS {−180° < ϕ <−100°, ψ > 50° or ψ < −120°}.

Modeling the Complex of p38α:MKK4. A model of the dockingsite peptide of MKK4 in complex with p38α was obtained on the basisof the X-ray structure of the p38α:MEF2A complex (PDB 1LEW).The sequences of the docking sites of MKK4 and MEF2A werealigned, and PyMol was used to change the residues of the MEF2Apeptide into the corresponding residues of the MKK4 peptide.Appropriate side chain rotamers devoid of steric clashes with p38αwere selected. The missing activation loop in the X-ray structures ofp38α were modeled using the ARCH_Pred server.77 The chains C-and N-terminally of the docking site motif of MKK4 weresubsequently built using Flexible-Meccano45 on the basis of theamino acid specific conformational sampling derived from theexperimental NMR chemical shifts and RDCs. Steric clashes of thedisordered parts of MKK4 with p38α were avoided. Structures of thecomplex were sorted by minimizing the distance from any residue inthe KIS domain to any residue experiencing CSPs in p38α. Thesestructures are then expected to fulfill the CSPs observed both withinthe KIS domain (Figure 4) and within p38α (Figure 8). Figure 9presents one of these structures color coded according to the dynamicsin the complex.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/jacs.7b12407.

Assigned HSQC spectra of MKK4; the bulkiness profileof MKK4; ITC data of the p38α:MKK4 complex; CSPsin p38α upon interaction with MKK412−86; comparisonof the p38α:MKK4 complex with p38α:HePTP; andresults of the analysis of NMR exchange data (PDF)

■ AUTHOR INFORMATIONCorresponding Author*malene.ringkjobing-jensen@ibs.frORCIDAndres Palencia: 0000-0002-1805-319XMartin Blackledge: 0000-0003-0935-721XMalene Ringkjøbing Jensen: 0000-0003-0419-2196Author Contributions⊥These authors contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank Prof. Wolfgang Peti for providing theensemble of the p38α:HePTP complex. This work used theplatforms of the Grenoble Instruct-ERIC Center (ISBG; UMS3518 CNRS-CEA-UGA-EMBL) with support from FRISBI(ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01)within the Grenoble Partnership for Structural Biology

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(PSB). Financial support is acknowledged from the FrenchAgence Nationale de la Recherche through ANR JCJCNMRSignal (to M.R.J.). J.K. acknowledges support from theIDPbyNMR Marie Curie action of the European commission(Contract no 264257). We thank Caroline Mas for assistanceand access to the biophysical platform.

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