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Structural and dynamic insights into the mechanism of allosteric signal transmission in ERK2-mediated MKP3 activation Chang Lu, Xin Liu, Chen-Song Zhang, Haipeng Gong, Jia-Wei Wu, and Zhi-Xin Wang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00827 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 28, 2017
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Biochemistry
Structural and dynamic insights into the mechanism of allosteric
signal
transmission
in
ERK2-mediated
MKP3
activation
Chang Lu1, #, Xin Liu1, #, Chen-Song Zhang2, Haipeng Gong1, Jia-Wei Wu1,* and Zhi-Xin Wang1,*
1
Key Laboratory of Ministry of Education for Protein Science, School of Life Sciences,
Tsinghua University, Beijing 100084, P.R. China 2
State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University,
Xiamen, Fujian 361005, P.R. China.
#
These authors contributed equally to this work.
*
Corresponding author.
E-mail:
[email protected] or
[email protected] Phone: 86-10-62785505, FAX: 86-10-62792826
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ABSTRACT The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the 334FNFM337 motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process, in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the timescale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2 induced MKP3 activation.
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INTRODUCTION The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are tightly regulated through dephosphorylation of both threonine and tyrosine in the activation loop by a family of dual-specificity MAPK phosphatases (MKPs).1-4 MKPs belong to the protein-tyrosine phosphatases (PTPs) superfamily, which is defined by the PTP signature motif, HC(X)5R, in their active sites (also called P-loop).5-6 In addition to the P-loop, the active site of the classical PTP also include a flexible “WPD (Trp-Pro-Asp) loop” that is important in the PTP-mediated catalytic process. PTPs catalyze the hydrolysis of tyrosine phosphate esters by the two-step mechanism, in which the aspartic acid in the WPD loop functions as a general acid in the first step and as a general base catalyst in the second step.7 The opening/closing transition of the WPD loop has implicated a role for PTP substrate recognition and catalytic efficiency and is also of paramount importance for the development of selective PTP inhibitors that may recognize a certain loop conformation.8-9 Therefore, the conformational changes and dynamic properties of the WPD loop have been the subject of many experimental and computational studies.7, 10 In mammalian cells, there are 10 distinct catalytically active MKPs, which can be classified into three groups based on their subcellular location and substrate specificity.1, 11 The first group are inducible nuclear phosphatases and can dephosphorylate all three major MAPKs, which comprises MKP1, MKP2, PAC1 and hVH3. The second group are cytoplasmic ERK-specific MKPs, including MKP3, MKP4 and MKPX. The third group are located in both nucleus and cytoplasm and selectively inactivate JNK and p38, including MKP5, MKP7 and hVH5. These MKPs share two common structural features: an N-terminal non-catalytic domain of MKP, which is critical for the selective binding of MAPKs (kinase binding domain, KBD), and a C-terminal catalytic domain (CD), which is responsible for the hydrolysis of the phosphate group and exhibits between 40% and 90% sequence identity among different MKPs (Table S1). A striking feature of MAPK and MKP interaction is that, several MKPs of the first and second groups are shown to be catalytically activated by direct binding of their corresponding MAPK
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substrates,12-16 suggesting that the substrate-induced activation may contribute to the highly specific molecular recognition. MKP3 is widely expressed and functions as a highly specific negative regulator of ERK1/2 activation.17-19 Full-length MKP3 has little activity towards the p-nitrophenol phosphate (pNPP) model substrate, but in the presence of ERK, the ability of MKP3 to dephosphorylate pNPP increases by ~20-30-fold.20-21 However, the structural basis of substrate recognition and the mechanism of substrate-induced catalytic activation of MKPs are still poorly understood. In this study, the allosteric activation of MKP3 by ERK2 is studied by using a combination of experimental and computational methods. We first provide detailed biochemical evidence that binding of ERK2 to a hydrophobic motif 334FNFM337 in the MKP3 catalytic domain (MKP3-CD) is critical for catalytic activation of MKP3. To elucidate the mechanistic basis for the dramatic catalytic enhancement, we further perform long-time, all-atom MD simulations for both the apo and ERK2-bound MKP3-CD. The simulation studies provide direct observation that ERK binding induces conformational change in the MKP3-CD, and therefore unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site of MKP3. The network of allosteric interactions taking place in the interface between the MKP3-CD and ERK2 also provides an explanation for the specificity of the MKP3-ERK2 recognition.
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MATERIALS AND EXPERIMENTAL DETAILS Constructs, mutagenesis, protein purification and characterization. The rat MKP3, MKP3∆N151 (residues 152-381), MKP3-KBD (1-154), MKP3-CD (204-350) and mouse ERK2 were subcloned into pET21b, pET15b, pET-Duet or pGEX4T-2 vectors for generation of His-tagged or GST-tagged proteins. The active pERK2 was obtained by coexpressing ERK2 and the constitutively active MEK1EE∆N4. Mutations were generated by overlap PCR procedure. All constructs were verified by DNA sequencing. All proteins were purified over Ni-NTA (Qiagen) or GS4B (GE Healthcare) columns and ion exchange and gel filtration chromatography (Source-15Q/15S and Superdex-200, GE Healthcare). The appropriate folding of various ERK2 and MKP3 proteins were examined by circular dichroism analysis, and the ERK2-MKP3 interactions were examined by gel filtration analysis.22 Proteins were stored at -80°C, and stocks for phosphatase assays were supplemented with glycerol to final concentration of 20% (v/v). Phosphatase assays. The activity of MKP3 was assayed using pERK2 as substrate in the coupled enzyme system containing 50 mM MOPS, pH 7.0, 100 mM NaCl, 0.1 mM EDTA, 0.1 mg/ml purine nucleoside phsphorylase (PNPase), and 50 µM 7-methyl-6-thioguanosine (MESG). The reactions were initiated by addition of the phosphatase and monitored as reported.18 The kinetic data were analyzed using the general initial velocity equation, taking substrate depletion into account: v0 =
{
kcat [ E ]0 + [S]0 + K m − 2
([ E ]0 + [S]0 + K m )
2
− 4[ E ]0 [S]0
}
(1)
where Km is the dissociation constant involved in the pERK2 dephosphorylation by MKP3. Activation of MKP3 by ERK2 was measured using the p-nitrophenyl phosphate (pNPP) assay in the reaction mixture containing 50 mM MOPS, pH 7.0, 100 mM NaCl, 0.1 mM EDTA, and 20 mM pNPP.18 At the fixed pNPP concentration, the rate equation for ERK2-induced MKP3 activation is given by v = v0 +
( vmax − v0 ) 2[ E ]0
{[E ] + [ERK] + K 0
0
d
−
([ E ]0 + [ERK]0 + K d )
2
− 4[ E ]0 [ERK]0
where Kd is the dissociation constant for binding of ERK2 to MKP3.
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}
(2)
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Simulation procedures. The apo catalytic domains was obtained by deleting the sulfate from the crystal structures MKP5 (PDB 1ZZW, chain A) and chloride ion and MPD from MKP3 (PDB 1MKP). The program CHARMM22 was then used to add hydrogen atoms, N- and Cterminal patches to the structures 23. The generated structures were solvated and neutralized in a box with TIP3P water at a minimum of 13 Å between the model and the wall of the box. All simulations were run using NAMD 2.9 with periodic boundary conditions (PBC) applied 24. The temperature was held at 300 K while the pressure was controlled at 1 atm. The time step was set to 2 fs and the particle mesh Ewald method was applied to model the electrostatics and the van der Waals interactions cutoff was set at 12 Å. Both apo MKP-CDs followed a 3-step pre-equilibration totaling 600 ps, the last snapshots of which were chosen as the starting structures for 1000 ns productive simulations without constraints. The ERK2-MKP3-CD complex was constructed by superimposition of the structures of MKP3-CD (PDB 1MKP) and D-motif-bound ERK2 (PDB 2FYS) to the corresponding domains in the crystal structure of JNK1-MKP7-CD (PDB 4YR8). A 3-step pre-equilibration of (1) 200 ps fixing the protein while allowing water molecules to move, (2) 400 ps constraining the protein while doing equilibration and energy minimization, and (3) a final 200 ps of unrestrained equilibration and energy minimization were performed. The steric clashes from superimposition were removed after the second step, and the final snapshot of the third step was used as a starting model for subsequent 1500 ns MD simulation. The same MD program and parameters were used for simulation of the complex as in the apo MKPs. Analysis of all the simulations was implemented using GROMACS 5.2 and VMD 1.9.1.25-26
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RESULTS AND DISCUSSION Identification of the binding interface between ERK2 and MKP3 catalytic domain Like all MKPs, MKP3 consists of an N-terminal kinase binding domain and a C-terminal catalytic domain (Figure 1A). Previous studies showed that recognition and activation of MKP3 by ERK2 involves three regions of MKP3: residues 61-75 in the N-terminal domain (also called kinase interaction motif, KIM or D-motif), residues 161–177 localized in the linker region, and residues 364–367 in the C-terminal tail of MKP3 (residues 348–381).27 The D-motif mediated interaction between ERK2 and N-terminal domain of MKP3 was believed to play an important role in the ERK2-induced MKP3 activation. The structure of ERK2 in complex with the D-motif peptide of MKP3 revealed that the D-motif binding site (also called D-site) on ERK2 is located in a noncatalytic region opposite of the kinase catalytic pocket. However, no structural information is available for the linker region and the C-terminal tail of MKP3. A widely accepted model
for
the
ERK2-induced
MKP3
activation
is
the
so-called
“self-inactivation
mechanism”.28-29 In this model, the MKP3-CD interacts directly with its N-terminal KBD and the interdomain binding stabilizes the inactive conformation of the catalytic site. Binding of ERK to the MKP3-KBD alters its interactions with the MKP3-CD such that this interaction allosterically triggers the active site residues to reconfigure to a conformation optimal for the efficient catalysis. To quantitatively evaluate the contributions of the three regions of MKP3, we first determined the kinetic parameters for the full-length MKP3, a C-terminal fragment of MKP3 (MKP3∆N151, residues 152-381) and the MKP3-CD (residues 204-350), using dually phosphorylated ERK2 (pERK2) as a substrate. As shown in Figures 1B and 1C, the kcat of MKP3∆N151-catalyzed reaction is 10-fold lower than that of wild-type MKP3, whereas the Km value for MKP3∆N151is about 10-fold higher than that for MKP3, indicating that the interaction between the D-site of ERK2 and the MKP3-KBD contributes directly to efficient ERK2 dephosphorylation by MKP3. Unexpectedly, the kcat and Km values for MKP3-CD are very similar to those of MKP3∆N151, suggesting that both the linker and C-terminal tail have no
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effects on the MKP3 phosphatase activity (Figure 1D). Note that although the catalytic efficiency (kcat/Km) for the pERK2 dephosphorylation by MKP3-CD is 100-fold lower than that of the full-length MKP3, it is about 4 orders of magnitude higher than that for dephosphorylating the biphosphorylated peptide derived from the activation loop of ERK2 (5 M-1 s-1) 30. The superiority of the pERK2 to the ERK2-derived phosphopeptide suggests that substrate recognition involves other regions of MKP3-CD in addition to its active site. In addition to the D-site, ERK2 has a second binding site utilized by transcription factor substrates and phosphatases, the FXF-motif binding site (also called F-site or DEF-site).31-35 The specific region in MKP3 that interacts with the F-site on ERK2 has been assigned to the sequence
364
FTAP367 localized in the C-terminus of MKP3.27, 36 Recently, we have determined
the crystal structure of JNK1 in complex with the MKP7-CD, which reveals an FXF-docking interaction mode between MAPK and MKP.22 Examination of the sequence alignment of the MKPs indicated that interacting residues FNFL in MKP7 are highly specific to the MKP family (334FNFM337 in MKP3-CD) (Figure S1). Therefore, it is tempting to speculate that the MKP3-CD may bind to the F-site of ERK2 in a manner analogous to the way by which MKP7-CD bind to JNK1. To test our hypothesis, we generated a series of point mutations on MKP3∆N151 and examined their effect on catalytic function (Figure 1E). As shown in Figure 1E, when the key hydrophobic residues Phe334 and Phe336 on the MKP3-CD interface was replaced by a charged residue (F334D or F336D), their catalytic efficiencies were decreased approximately tenfold. In contrast, mutating the four residues 364FTAP367 in the C-terminus of MKP3 simultaneously to Ala or Asp had no effect on phosphatase activity. Interestingly, mutation of Lys324 in helix α4 to an alanine residue results in significant increase of the MKP3-CD activity, suggesting that helix α4 may not be directly involved in the MKP3-ERK2 interaction, probably due to the role of electrostatic repelling between Lys324 of MKP3 and the positively charged surface on ERK2. Gel filtration analysis further confirmed that the FXF-motif mediated-docking interaction does not directly involve the
364
FTAP367 sequence in MKP3, and the sequence
MKP3-CD is essential for ERK2 substrate binding (Figure S2).
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334
FNFM337 in the
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The allosteric activation of MKP3 has been well documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate
27
. Using the activation-based
assay, we are able to elucidate the molecular basis of the specific ERK2 recognition by MKP3. Figure 1F shows the concentration dependence of the MKP3-catalyzed pNPP hydrolysis on the ERK2 concentration. For wild-type MKP3, the values of Kd and maximum activation fold (vmax/v0) were determined to be 0.13 ± 0.01 µM and 25.8 ± 0.2, respectively. When the residues 364
FTAP367 were replaced by Ala residues, the mutant MKP3/A4 exhibits a similar affinity
towards ERK2 (Kd = 0.11 ± 0.01 µM) and can still be activated to ~40% (vmax//v0 = 10.0 ± 0.1) of wild-type MKP3 activity by ERK2. In comparison, mutant MKP3/F334D completely impairs its ability to be activated by ERK2. To determine the contribution of the KBD of MKP3 to ERK2-induced activation, we also measured the activation of MKP3∆N151 by ERK2 (Figure 1G). The MKP3∆N151 fragment alone has very low phosphatase activity, comparable to that of the full-length MKP3 in the absence of ERK2, but it can still be activated by ERK2 to a similar degree. The Kd value of MKP3∆N151 for ERK2 was determined to be 20 ± 2 µM, which is 153-fold higher than that of the full length MKP3. As in the case of wild-type MKP3, the pNPP phosphatase activity of MKP3∆N151 with the F334D mutant is unable to be activated by ERK2, indicating again that the
334
FNFM337 motif, but not the sequence
364
FTAP367 located in the C
terminus of MKP3, is essential for ERK2-induced MKP3 activation (Table 1). In order to further confirm that the F-site in ERK2 is indeed important for the ERK2-induced MKP3 activation, we made several substitutions in this region of ERK2 and quantitatively analyzed their ability to activate MKP3 (Figure 1H). As shown in this figure, mutation of hydrophobic residues in the F-site of ERK2 shows decreased ability to activate MKP3 to the maximum activity even at saturating concentrations. In summary, our results clearly demonstrate that (1) the N-terminal domain of MKP3 is conducive to but not responsible for the ERK2-induced MKP3 activation, (2) the ERK2-induced allosteric activation is an intrinsic property of the MKP3-CD, and (3) the FXF-motif mediated docking interaction is a critical determinant for both enzyme activity and allosteric activation.
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Figure 1. FXF-motif (334FNFM337) is essential for ERK2 binding and allosteric activation of MKP3. (A) Domain organization of MKP3 and structures of MKP3-KBD (PDB entry 1HZM), MKP3-CD (PDB entry 1MKP) and ERK2 (PDB entry 2ERK). (B-D) Plots of initial velocity of the full-length or different truncations of MKP3 catalyzed reaction versus pERK2 concentration. The solid lines are best-fitting results to eqn. (1). (E) Effects of mutations in MKP3∆N151 on the ERK2 dephosphorylation (mean ± SEM, n = 3). Residues involved in hydrophobic and hydrophilic contacts are colored in red and blue, respectively. (F) ERK2 induced activation of full-length MKP3 and its mutants. (G) ERK2 induced activation of MKP3∆N151 and its mutants. The solid lines are best-fitting results according to eqn. (2). (H) Relative activity of MKP3-catalyzed pNPP hydrolysis in the presence of ERK2 mutants as compared to wildtype ERK2 (mean ± SEM, n = 3).
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Table 1. Kinetic parameters for ERK2-induced MKP3 activation. MKP3
mutation
vmax / v0
Kd (µ µM)
Wild type
25.8 ± 0.2
0.13 ± 0.01
FTAP/AAAA
10.0 ± 0.1
0.11 ± 0.01
F334D
1.3 ± 0.1
N.D.
Wild type
21.8 ± 0.2
20 ± 2
FTAP/AAAA
7.8 ± 0.1
22 ± 2
F334D
1.1 ± 0.1
N.D.
CD
Wild type
9.2 ± 0.1
33 ± 7
∆N151 + KBD*
Wild type
20.6 ± 0.6
18 ± 1
Full length
∆N151
N.D. Not determined. *ERK2 induced MKP3∆N151 activation in the presence of 11 µM MKP3-KBD.
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Molecular dynamics simulations of apo MKP5-CD and apo MKP3-CD. Catalytic domains of human MKPs exhibit good structural alignment in the core region, but there are deviations in the loops and secondary structures outside the core (Figure S3).4 MKP5, the human MKP family member with the highest resolution structure generated to date, is widely expressed and plays an important role in the control of the immune response.37 MKP5-CD possesses the intrinsic enzyme activity and its catalytic efficiency (kcat/Km) is ~10-fold higher than that of MKP3-CD towards small molecule substrates.37 The structure of MKP5-CD adopts a typical active conformation of PTPases as found in PTP1B and VHR (Figure 2A). In contrast to MKP5-CD, the MKP3-CD structure is resolved in the inactive state of PTPase loop (Figure 2B).38-39 The Cα positions of the P-loop residues in MKP3-CD are shifted on average 3.5 Å relative to MKP5-CD. The side chain of Arg299 in MKP3-CD is oriented toward the negatively charged chloride ion, departing from the canonical phosphate-coordinating conformation. In particular, the conserved aspartate Asp262 in the D-loop is remote from the active site arginine residue and makes a hydrogen bond interaction with Asn242 from a hairpin loop connecting strands β3 and β3’ (Figure 2B). Besides the key features of the active site, the three loops that constitute a catalytic cleft (loop β2–α1, loop β3–β4, and loop α4–α5) are seriously deviated from those of MKP5-CD, which may result in disengagement of important residues for enzyme activity. The largest difference between MKP3-CD and MKP5-CD is the presence of an extra β-strand (β3’,
246
LFENA250) between strand β3 and strand β4 in MKP3-CD. The residues of the
β3’ strand in MKP3-CD are ~11 Å, on average, away from the corresponding residues of the β3-β4 loop in MKP5-CD. The pNPPase activity of MKP3-CD can be significantly increased by ERK2 binding, suggesting that MKP3-CD alone is able to undergo catalytic activation in the presence of ERK2 (Figure S4). These results indicate that MKP3-CD retains structural features necessary and sufficient for the substrate-induced activation, and the structural differences between MKP5-CD and MKP3-CD may reflect the well-known substrate-induced activation properties of MKPs. In order to understand the dynamic features of MKPs with distinct intrinsic catalytic
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activities, 1 µs MD simulations for the isolated (ligand-free) MKP5-CD and MKP3-CD were performed in explicit solvent starting with their crystal structures (Movie S1 and Movie S2). For apo MKP3-CD, the residues with higher fluctuation values are observed in several loop regions, including the β2-α1 loop (residues 219-223), the β3’ strand (residues 245-249), the D-loop (residues 261-267), the α4-α5 loop, and α5 helix (residues 334-347), whereas for apo MKP5-CD, only the D-loop residues are seen to have large fluctuation (Figure 2D). The β3’ region of MKP3-CD exhibits the most prominent change and high flexibility. The single β3’ strand is spontaneously disrupted in apo MKP3, indicating that strand-to-loop conformation switch is likely to be the inherent property of MKP3-CD (Figure 2E). In comparison, the β3-β4 region in MKP5-CD (residues 360-365, corresponding residues of β3’ in MKP3-CD) is quite stable, making numerous contacts with the flanking helix α1 and β-sheet throughout the simulation. Surprisingly, although the superposition of the MKP5-CD structure with that of MKP3-CD shows that helix α5 could be well aligned, large fluctuations are also found in a broad region at the α4-α5 loop and the following helix α5 in MKP3-CD. The average B-factors for the helix α5 are much higher than those of the entire MKP3-CD, also suggesting that the conformation of helix α5 is flexible. In most previously reported PTP structures, residues of the P-loop have an identical conformation,5, 8 while the WPD loop (corresponding to the D-loop in MKPs) has two distinct stable conformations (open and closed). Recently, unrestrained MD simulations of the apo classical PTP1B showed only marginal changes in the conformations of both loops over the timescale of ~2 µs.40 Our MD simulations show, however, that the P-loop of MKP5-CD undergoes conformational rearrangements when sulfate ion was moved from the active site, where a distinctive P-loop conformation is formed after ~100 ns of simulation (Figures 2E, 2F). In comparison, the P-loop of MKP3-CD is quite stable across the entire trajectories with an average RMSD of 0.3Å, indicating that the determined inactive structure of MKP3-CD reflects a stable P-loop structure in the apo state (Figure 2F). It is also shown that the D-loop of both MKPs are flexible throughout the simulations (Figure 2F). Since the conformational space of the
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P-loops and D-loops in both simulations are very similar to what has been observed for the MKP3-CD crystal structure, our simulation studies suggest that the conformations of both loops are inducible and dependent on the presence of a negative charge, underscoring the importance of the proper structural context of these two loops during substrate binding. Thus, our MD simulation studies present the model structures for both intrinsically and inducibly active MKPs in the apo states, allowing a detailed analysis of the active-site flexibility of MKPs in comparison with other PTP structures. It should be noted that the transition between the open and closed forms of the D-loop, which is important for catalysis of PTPases, has been shown to occur on a slow timescale (microseconds to milliseconds).7 This transition, if relevant in MKP3, is unlikely to be observed by the simulations (1.5 µs) presented here.
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Figure 2. MD simulations of apo MKP5-CD and apo MKP3-CD. (A-B) Comparison between crystal structures of the intrinsically active (A, MKP5-CD) and inducibly active (B, MKP3-CD) MKP catalytic domain. The P-loop and the D-loop are highlighted in marine and magenta, respectively. The key residues are shown in sticks, and the blue dashed lines represent polar interactions. (C) RMSD (Cα) of MKP5-CD and MKP3-CD are seen to reach plateau values of 1.9 ± 0.2 Å and 2.2 ± 0.3 Å for the last 900 ns of each simulation. (D) RMSF of the two proteins calculated from last 900 ns of each simulation. (E) Residues that are highly flexible or essential for catalysis are selected for regional RMSD plot. (F) Superposition of 15 structural snapshots from 1 µs simulations of apo MKP5-CD (left) and apo MKP3-CD (right). The backbone of the structures is shown as gray tube, with the first and final snapshots of each simulation highlighted in red and blue respectively.
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Molecular dynamics simulation of ERK2-bound MKP3-CD. To further understand the substrate-induced activation in the context of the MKP3-CD, the ERK2-MKP3-CD interaction model was constructed based on existing three-dimensional structures, which was then subjected to a long time-scale MD simulation. Briefly, MKP3-CD was docked onto the ERK2 structure with molecular interactions analogous to those found in the experimentally determined JNK1-MKP7-CD structure (see Materials and Experimental Details). In this section, the conformational features of ERK2-bound MKP3-CD during the 1500 ns MD simulation are examined and compared in detail to those of apo MKP5-CD and apo MKP3-CD, which are representatives of the active-state and inactive-state MKP catalytic domains, respectively. Compared to the MD simulations of the apo MKP3-CD, the network of cross-correlation is increased substantially throughout the entire catalytic domain for the ERK2-bound MKP3-CD, suggesting that the enzyme becomes more rigid when ERK2 is bound and that concerted motion has replaced random fluctuations (Figure 3A). The network of correlated fluctuations of the apo MKP3-CD shows that the fluctuations of the β3’ residues are negatively correlated with the flanking β3, β4, and β5 residues. In ERK2-bound MKP3, additional elevated levels of correlation between β3’ and multiple distal regions are clearly observed, particularly for the D-loop and helix α5 (including
334
FNF336 motif). These results
suggest that α5, the β3’ region and the D-loop residues together form a coupled network of interactions through which the propagation of the allosteric effect following ERK2 binding is transmitted to the active site (Figure 3B). These observations are also consistent with the HDX results of Zhou et. al. which showed that binding of ERK2 reduces the hydrogen/deuterium exchange rates throughout the MKP3 catalytic domain, including all of the above-mentioned regions.36 In the crystal structure of MKP3-CD, α5 is shortened by several residues compared with that in MKP5, and the conserved residues
334
FNF336 of MKP3 forms an irregular turn. As previously
indicated, the α4-α5 loop and the following helix α5 are very flexible in apo MKP3-CD, and that the residues
334
FNF336 visit both the helical and the bend conformations at equilibrium (Figure
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3C). Flexibility of this region of MKP3-CD could cause a serious deviation of Phe336 from its optimum position. As shown in Figure 3E, there is a large change in the distance plot between Phe336 and Arg299 at ~600 ns simulation of apo MKP3-CD, indicating that, in this system, Phe336 can move away from the binding pocket formed by residues from the P-loop and D-loop. During the MD simulations for the ERK2-MKP3-CD complex, however, the RMSD values of α5 region increased very rapidly within the first nanoseconds of the simulation and reached a plateau value after about 10 ns. The helical conformation of Phe334 and Asn335 of MKP3 began to form at 2 ns and quickly reached a rather stable state over the time course from 10 ns to 1500 ns (Figure 3C). These two residues (Phe334 and Asn335) anchor the MKP3-CD to the F-site of ERK2, thus significantly reducing the movements of the α5 in the ERK2-bound complex. These results indicate that FXF-motif mediated interaction plays an important role that keeps Phe336 the in the correct orientation for catalysis. The family-wide superposition of MKP-CDs reveals that the β3-β4 region shows greater variability than other regions of the structure among the members of MKPs (Figure S3). As previously shown, the β3-β4 region in MKP5-CD is quite stable and makes numerous contacts with the flanking helix α1 and β-sheet throughout the simulation, whereas the corresponding β3’ strand of MKP3-CD exhibits the most prominent change and high flexibility: these residues undergo a reversible strand-to-loop conformational switch during the MD (Figure 3D). The ERK2-MKP3-CD complex simulation reveals that, although the β3’ strand of the ERK2-bound MKP3-CD shows a similar strand-to-loop dynamic behavior to the apo MKP3-CD initially (~100 ns), noticeable differences are observed when the simulation time is extended to 1500 ns (Figure 3D). Firstly, the residues 246-249 in the ERK2-bound MKP3-CD remains in the loop conformation longer than the apo MKP3-CD, indicating that the loop conformation is more favorable in the bound protein. More importantly, a major conformational change in the β3-β4 region is observed at ~800 ns of the ERK2-bound MKP3-CD simulation, resulting in a large jump in RMSD values and formation of a new helical turn (Figure 3D). Such conformational change is likely to be driven by the balance of two factors, the net attractive interaction between
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two loop regions and the chain’s conformational entropy, which pushes loop β3–β4 towards the P-loop to form extensive interactions with the hydrophobic residues from the α1 helix and β3, β5 strands (Figure S5). As a result, Pro241 from the β3-β4 region, which has been suggested as the residue hindering optimal positioning of substrate-binding residue Arg299 in MKP3-CD,38 is displaceable to permit torsion of the Arg299 side chain, which would render catalytically important residues to be situated in the proper positions and lead to formation of active MKP3-CD conformation (Figure 3F). The time evolution of the distance between Pro241 and Arg299 indicates that Pro241 move away from its initial position observed in crystal structure after 800 ns (Figure 3F).
Figure 3. Mobility of α5 and β3-β β4 region in MKP3-CD. (A) Covariance map of atomic fluctuations of the ERK2-MKP3-CD (upper-left triangle) and apo MKP3-CD (lower-right triangle). Corresponding fields with distinct levels of atomic correlations are outlined in black and magenta. (B) Residues involved in allosteric transition pathway are shown as colored surfaces on MKP3-CD. Arrows indicate time-dependent sequence of observed change during MD. The active site is circled out with dashed line. The catalytic residues and key residues influencing active site are labeled and
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shown in stick. (C-D) Upper panels: RMSD values of α5 (C) and β3’ region (D) for the three MD systems. Lower panels: secondary structural components of corresponding region as a function of time. (E) Upper: distances between the second Phe in FXF motif (Phe336 in MKP3, Phe451 in MKP5) and the Arg in the P-loop for three MDs. Lower: 10 snapshots from simulation of apo and ERK2-bound MKP3-CD, with Phe336 and Arg299 shown in sticks. (F) Upper: distances between the Arg and Pro241 (in MKP3) or the equivalent Thr356 (in MKP5) for three MDs. Lower: 10 snapshots from simulation of apo and ERK2-bound MKP3-CD, with Arg299 and Pro241 shown in sticks.
Besides the P-loop, which is primarily responsible for substrate binding, the D-loop is also an essential part of the active site of MKPs, as well as the larger superfamily of the PTPases. The mobility of the important general acid (Asp) has implicated a role for PTP substrate recognition and catalytic efficiency of classical PTPases.7, 10 To provide a quantitative analysis regarding the mobility of the general acid and its neighboring residues, the time evolution of distances between the Asp and selected residues are shown in Figure 4 for the three systems, apo MKP5-CD, apo MKP3-CD and ERK2-bound MKP3-CD. A very interesting picture evolves for the mobility of the general acid residue. For apo-MKP5-CD, the general acid Asp377 frequently forms a salt bridge with the sidechain nitrogen of Arg414, where a high probability is observed at short distances (i.e., < 3 Å) (Figure 4A1). For apo-MKP3-CD, however, the distance between Asp262 and Arg299 shows a bipartite distribution, probably due to the presence of a hydrogen bond between Asp262 and Asn242 that may impair the Asp262-Arg299 electrostatic interactions (Figure 4B1 and 4B2). In contrast, the general acid Asp377 in apo-MKP5-CD is always distant from His357 (that is equivalent to Asn242 in MKP3-CD) throughout the simulation (Figure 4A1 and 4A2). For the ERK2-bound MKP3-CD, no short distances are monitored between Asp262 and Arg299 during the first 300 ns simulation and Asp262 forms a stable hydrogen bond with Asn242. Following the change in the interaction surface between ERK2 and MKP3-CD, particularly the flip of sidechain of Trp264 in D-loop at ~300 ns, the sidechain of Asp262 turned approximately 90° and orients towards the active site (Figure 4C1 and S6). As a result, there is a
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higher probability of encountering interactions between Asp262 and Arg299 during the 300-800 ns simulations. Finally, accompanying further conformational change of β3’ region at ~800 ns, Asn242 is dragged away from the D-loop, allowing Asp262 to adopt a favorable sidechain orientation over the active site. Clearly, these results demonstrate that the general acid of apo MKP3-CD fluctuates between multiple conformations and has an intrinsic ability to sample conformations that meet functional requirements. Binding of ERK2 to the allosteric site plays a stabilizing role for this loop region, thus the general acid behaves in a manner analogous to that of MKP5-CD (Figures 4A1 and 4C1).
Figure 4. Mobility of the general acid. (A-C) Time evolution of distances between the general acid (Asp) and the substrate binding residue (Arg) or His/Asn from the β3-β4 region from the simulations of apo MKP5-CD (A, blue), apo MKP3-CD (B, gray) and ERK2-bound MKP3-CD (C, red). (A1-C1)
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The normalized distribution of distances between Asp and Arg or His/Asn calculated from the indicated time periods, during the simulations of the apo MKP5 (A1), the apo MKP3 (B1) and the ERK2-bound MKP3 (C1). The bins in the histograms are obtained by rounding off to the integer part of the distances. (A2-C2) The typical orientations of these residues are highlighted in sticks for the three systems, namely the apo MKP5 (A2), the apo MKP3 (B2) and the ERK2-bound MKP3 (C2).
Based on the results presented in this study and the previous knowledge on the MKP structures, the detailed processes in substrate-induced MKP3 activation can be illustrated by a model in the structural aspect of Figure 5. In the absence of ERK2, MKP3-CD is intrinsically dynamic in its apo state, with several important loop regions uncommitted in their flexibilities and conformations. The enzyme can exist in a low-activity state because of disengagement of its active site residues, such as Asp264 in the D-loop, Arg299 in the P-loop, and Phe336 in the helix α5. The MKP3-CD-ERK2 interaction triggers a network of allosteric changes in the F-site ERK2, leading to the rearrangement of the contacting residues from the FXF-motif (334FNFM337) and the D-loop of MKP3-CD. As a result, the Phe336 is stabilized in the ERK2-bound MKP3-CD, and the sidechain of the residue Asp264 is faced towards the active site. As the motion of the β3-β4 region is coupled to α5 and the D-loop, ERK2-binding induced displacement of these residues from their original positions, allowing the active site Arg299 of P-loop to orient optimal for efficient catalysis. The final model of MKP3-CD shows orientations of the active site residues with stunning similar characteristics to those of the intrinsically active MKP5-CD (Figure S5). Finally, it should be noted that we have performed simulation in the absence of N-terminal KBD of MKP3. As aforementioned, the KBD makes a significant contribution to the interactions with ERK2 and also to its catalytic efficiency, the presence of the MKP3-KBD may affect the allosteric network obtained through the present MD simulations. However, we think that the current simulation has captured the primary characteristics of allosteric activation network of
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MKP3 by ERK2, as the activation of MKP3∆N151 is not influenced by the presence of excess isolated MKP3-KBD (Table 1 and Figure S4).
Figure 5. Dynamic process underlying allosteric signal transmission in ERK2-mediated MKP3-CD activation. Snapshots at indicated time points during the MD are illustrated, with MKP3-CD shown in cartoon and ERK2 in surface representation colored according to electrostatic potential (positive, blue; negative, red). 0 ns: initial ERK2 and MKP3-CD docking model. 10 ns: Phe334 of MKP3-CD interact directly with the F-site of ERK2, and the FXF motif of MKP3 to is folded into a stable 1-turn helix. 150 ns: the original β3’ strand of MKP3-CD (shown in gray cartoon) is able to switch between ‘strand’ and ‘loop’ conformations. 320 ns: Trp264 from the D-loop of MKP3-CD flipped through which the general acid Asp262 is brought in close proximity to the active site Arg299. 850 ns: the residues of β3’ strand region begin to form extensive interactions with the residues from the α1 helix and β3, β5 strand. 1500 ns: the
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complex structure has been relative stable since 850 ns, where residues from the original β3’ strand have assumed a new stable conformation. A dynamic illustration of MKP3-CD conformational change is provided in Movies S3 and S4.
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CONCLUSION The dynamic conformational changes of enzymes are essential for enzyme function and regulation.41 Allosteric transition, defined as conformational changes induced by ligand binding, is one of the fundamental properties of proteins.42 Fifty years ago, a simple, two-state allosteric model was presented by Monod, Wyman, and Changeux (MWC),43 which has been widely applied to characterize signal transduction in biology.44-45 One of the key concepts in the MWC model is that signaling molecules undergo reversible transitions between discrete conformations, which are accessible in the absence of ligand. Such spontaneous “conformational switches”, whose states are selectively stabilized by the ligands to which they preferentially bind, contrast with
the
sequential,
induced-fit
mechanism
initially
suggested
for
the
enzyme-substrate interaction.46 Recent advances in theoretical and experimental methods have provided increasing evidence that under native state conditions proteins have an intrinsic ability to sample conformations that meet functional requirements, and shifts of pre-existing equilibrium may be a fundamental paradigm of ligand binding.47-50 However, due to the complex nature of the protein dynamics, the in-depth investigation of how this allosteric conformational change is transmitted still remains a challenge. In most cases, the time length of classical MD simulation is not long enough to sample the different conformational states in an allosteric regulation, and restrained or accelerated simulations have been employed to probe allosteric conformational change.51-52 In this study, we perform unconstrained, all-atom MD simulations for the ERK2-MKP3-CD complex. Unlike the conventional MD simulations of PTP1B, which showed only marginal changes in the WPD loop conformations (closed or open) over the timescale of the simulation (~2 µs),40 our MD simulations showed the allosteric signal transduction over the timescale of the simulation (1.5 µs), and provide a direct observation for the dynamic process of inactive-active conformation transition. This dynamic structural model is completely different from those proposed previously and unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site of MKP3.
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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.
The extended experimental methods and 6 supporting figures providing the gel-filtration results, analysis of all available MKP-CD structures, comparison between MKP3-CD crystal structure and final model (PDF) 4 movies of the MD simulations (MPG) AUTHOR INFORMATION Corresponding Author *
Corresponding author. E-mail:
[email protected] or
[email protected] ORCID Chang Lu: 0000-0002-3272-0120 Haipeng Gong: 0000-0002-5532-1640
Author Contributions #
These authors contributed equally.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT The MD simulations were performed on the “Explorer 100” cluster system of Tsinghua National Laboratory for Information Science and Technology. This work was supported in part by grant 2016YFA0502004 from the Ministry of Science and Technology of the People’s Republic of China and grant 31770841 from the National Natural Science Foundation of China.
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28. Farooq, A.; Zhou, M. M., Structure and regulation of MAPK phosphatases. Cell. Signal. 2004, 16 (7), 769-779. 29. Mark, J. K.; Aubin, R. A.; Smith, S.; Hefford, M. A., Inhibition of mitogen-activated protein kinase phosphatase 3 activity by interdomain binding. J. Biol. Chem. 2008, 283 (42), 28574-28583. 30. Wiland, A. M.; Denu, J. M.; Mourey, R. J.; Dixon, J. E., Purification and kinetic characterization of the mitogen-activated protein kinase phosphatase rVH6. J. Biol. Chem. 1996, 271 (52), 33486-33492. 31. Sharrocks, A. D.; Yang, S. H.; Galanis, A., Docking domains and substrate-specificity determination for MAP kinases. Trends Biochem. Sci. 2000, 25 (9), 448-453. 32. Zhang, J.; Zhou, B.; Zheng, C. F.; Zhang, Z. Y., A bipartite mechanism for ERK2 recognition by its cognate regulators and substrates. J. Biol. Chem. 2003, 278 (32), 29901-29912. 33. Fantz, D. A.; Jacobs, D.; Glossip, D.; Kornfeld, K., Docking sites on substrate proteins direct extracellular signal-regulated kinase to phosphorylate specific residues. J. Biol. Chem. 2001, 276 (29), 27256-27265. 34. Canagarajah, B. J.; Khokhlatchev, A.; Cobb, M. H.; Goldsmith, E. J., Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 1997, 90 (5), 859-869. 35. Jacobs, D.; Glossip, D.; Xing, H.; Muslin, A. J.; Kornfeld, K., Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. Genes Dev. 1999, 13 (2), 163-175. 36. Zhou, B.; Zhang, J. L.; Liu, S. J.; Reddy, S.; Wang, F.; Zhang, Z. Y., Mapping ERK2-MKP3 binding interfaces by hydrogen/deuterium exchange mass spectrometry. J. Biol. Chem. 2006, 281 (50), 38834-38844. 37. Jeong, D. G.; Yoon, T. S.; Kim, J. H.; Shim, M. Y.; Jung, S. K.; Son, J. H.; Ryu, S. E.; Kim, S. J., Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase. J. Mol. Biol. 2006, 360 (5), 946-955. 38. Stewart, A. E.; Dowd, S.; Keyse, S. M.; McDonald, N. Q., Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation. Nat. Struct. Biol. 1999, 6 (2), 174-181. 39. Bakan, A.; Lazo, J. S.; Wipf, P.; Brummond, K. M.; Bahar, I., Toward a molecular understanding of the interaction of dual specificity phosphatases with substrates: insights from structure-based modeling and high throughput screening. Curr. Med. Chem. 2008, 15 (25), 2536-44. 40. Choy, M. S.; Li, Y.; Machado, L. E. S. F.; Kunze, M. B. A.; Connors, C. R.; Wei, X.; Lindorff-Larsen, K.; Page, R.; Peti, W., Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery. Mol. Cell 2017, 65 (4), 644-658. 41. Berezovsky, I. N.; Guarnera, E.; Zheng, Z.; Eisenhaber, B.; Eisenhaber, F., Protein function machinery: from basic structural units to modulation of activity. Curr. Opin. Struct. Biol. 2017, 42, 67-74. 42. Liu, J.; Nussinov, R., Allostery: an overview of its history, concepts, methods, and applications. PLoS Comput. Biol. 2016, 12 (6), e1004966.
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43. Monod, J.; Wyman, J.; Changeux, J.-P., On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 1965, 12 (1), 88-118. 44. Changeux, J.-P.; Edelstein, S. J., Allosteric mechanisms of signal transduction. Science 2005, 308 (5727), 1424-1428. 45. Nussinov, R.; Tsai, C.-J.; Liu, J., Principles of allosteric interactions in cell signaling. J. Am. Chem. Soc. 2014, 136 (51), 17692-17701. 46. Koshland, D., Application of a theory of enzyme specificity to protein synthesis. Proc. Natl. Acad. Sci. U. S. A. 1958, 44 (2), 98-104. 47. Wang, Z.-X.; Kihara, H., Some applications of statistical mechanics in enzymology 2. Statistical mechanical explanation on allosteric enzyme models. J. Theor. Biol. 1990, 143 (4), 455-464. 48. Wang, Z.-X., Some applications of statistical mechanics in enzymology 1. Elementary principle. J. Theor. Biol. 1990, 143 (4), 445-453. 49. Freire, E., The propagation of binding interactions to remote sites in proteins: analysis of the binding of the monoclonal antibody D1. 3 to lysozyme. Proc. Natl. Acad. Sci. U. S. A. 1999, 96 (18), 10118-10122. 50. D'Abramo, M.; Rabal, O.; Oyarzabal, J.; Gervasio, F. L., Conformational selection versus induced fit in kinases: the case of PI3K-gamma. Angew. Chem. Int. Ed. Engl. 2012, 51 (3), 642-646. 51. Perez, A.; Morrone, J. A.; Simmerling, C.; Dill, K. A., Advances in free-energy-based simulations of protein folding and ligand binding. Curr. Opin. Struct. Biol. 2016, 36, 25-31. 52. Aci-Seche, S.; Ziada, S.; Braka, A.; Arora, R.; Bonnet, P., Advanced molecular dynamics simulation methods for kinase drug discovery. Future Med. Chem. 2016, 8 (5), 545-566.
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Biochemistry
Figure 1. FXF-motif (334FNFM337) is essential for ERK2 binding and allosteric activation of MKP3. 176x138mm (300 x 300 DPI)
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Biochemistry
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Figure 2. MD simulations of apo MKP5-CD and apo MKP3-CD. 166x157mm (300 x 300 DPI)
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Biochemistry
Figure 3. Mobility of α5 and β3-β4 region in MKP3-CD. 176x110mm (300 x 300 DPI)
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Figure 4. Mobility of the general acid (Asp). 156x131mm (300 x 300 DPI)
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Biochemistry
Figure 5. Dynamic process underlying allosteric signal transmission in ERK2-mediated MKP3-CD activation. 170x117mm (300 x 300 DPI)
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