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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 January 1; 291(1): 11–27.
Published online 2015 November 2. doi:  10.1074/jbc.M115.684928
PMCID: PMC4697148

Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein

MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY*

Abstract

Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca2+-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca2+-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca2+-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a “fuzzy” complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.

Keywords: calcium, crystal structure, extracellular-signal-regulated kinase (ERK), kinetics, melanoma, mitogen-activated protein kinase (MAPK), nuclear magnetic resonance (NMR), RSK, S100 proteins, small-angle X-ray scattering (SAXS)

Introduction

The vertebrate-specific S100 proteins belong to the EF-hand-containing, Ca2+-binding superfamily of proteins with more than 20 paralogs in the human proteome (1, 2). They are small (~100 amino acids) and mostly homodimeric proteins where each monomer can bind two calcium ions. In their Ca2+-bound form, each monomer exposes a hydrophobic surface and becomes capable of binding to target proteins. In most cases, two partner proteins bind to one S100 dimer symmetrically; however, there are a few examples where an elongated motif interacts with the two identical hydrophobic grooves simultaneously and asymmetrically (3, 4). S100 proteins can be found both intra- and extracellularly. On the cell surface, they can bind to receptors (such as receptor for advanced glycosylation end product) and activate ERK/p38 mitogen-activated protein kinase (MAPK) pathways indirectly (5, 6). Despite the fact that these small proteins have been extensively studied for decades, the precise and specific intracellular role of most S100 proteins still remains to be determined.

It has recently been shown that S100B can form a complex with ribosomal S6 kinase 1 (RSK1) in malignant melanoma cell lines, and this interaction negatively affects phosphorylation of the C-terminal Ca2+/calmodulin-dependent kinase (CaMK)3-type domain of RSK1 by ERK1/2 (7). In malignant melanomas, S100B expression is highly elevated, which can be used as a prognostic marker for the disease (8). Moreover, S100B is being explored as a therapeutic target for treating melanomas by inhibiting its protein-protein interactions (9).

RSK1 belongs to the group of MAPK-activated protein kinases (MAPKAPK) (10). MAPKAPKs are downstream cytoplasmic targets of ERK and/or p38 MAPKs and belong to the CaMK-type protein kinase superfamily (11). Inactive MAPKAPKs are in an autoinhibited form where the C-terminal inhibitory helix (αL) blocks substrate as well as ATP binding. This inhibitory helix is followed by a short linker and an MAPK-binding linear motif, where the latter determines MAPK binding specificity (12, 13). The first step of MAPKAPK activation is activation loop (AL) phosphorylation by its cognate MAPK. Next, the autoinhibitory helix is extruded by the phosphorylated AL via an unknown mechanism (14). Although most MAPKAPK proteins contain a single catalytic domain, the RSK subfamilies are tandem kinases; in addition to their C-terminal CaMK-type domain (CTKD), they have an N-terminal AGC-type kinase domain (NTKD).

The regulatory mechanism of CaMK-type kinases usually involves intracellular Ca2+ signals. These kinases have a regulatory C-terminal extension, and in the inactive form the first segment of this tail forms a helical inhibitory helix that blocks substrate or cofactor binding, whereas the second segment is disordered. Upon Ca2+ binding, calmodulin (CaM) opens up and binds to the unstructured tail and to the terminal part of the inhibitory helix (15). This interaction remodels the inhibitory segment and brings about an active kinase state (Fig. 1A). In contrast, AGC-type kinases have a more complicated regulation mechanism that includes multiple phosphorylation steps. They also have a C-terminal regulatory element that includes two short sequence motifs as follows: the turn motif and the hydrophobic motif (HM). Upstream kinases phosphorylate the HM and turn motif, which will bind to the N-lobe of the kinase domain. Phospho-HM binding remodels the allosteric αC helix of the kinase domain, which in turn activates the kinase, although phospho-turn motif will increase HM binding (16). Fully active kinase requires another phosphorylation on its AL. In some cases phospho-HM also has a binding partner in trans; it can bind to an AGC master kinase, for example phosphoinositide-dependent kinase 1 (PDK1), which will then phosphorylate the AL (17).

FIGURE 1.
Regulation of CaMK domain containing protein kinase activity. A, inactive CaMKII has an autoinhibitory C-terminal fragment (blue) that is released by calmodulin binding (15). Many CaMK-type kinases share an analogous activation mechanism. B, RSK1 consists ...

Phosphorylation on the CaMK-type domain of RSK1 by activated ERK2 sets off the full multistep activation of RSK1 (Fig. 1B). In RSK1, the only known role of the C-terminal CaMK-type kinase domain (CTKD) is the phosphorylation of the regulatory hydrophobic motif of the NTKD (18). The phospho-HM of NTKD serves as an anchor motif for PDK1, which will eventually phosphorylate the activation loop of NTKD, resulting in an active kinase that can now phosphorylate a diverse set of substrates downstream of RSK1 (17).

S100B binding to the CTKD of RSK1 may be intuitively similar to typical CaMK activation. Inactive CaMKII has a very similar structure to RSK1; moreover, both the structure and function of S100B are analogous to that of calmodulin. In this study, we have performed biochemical and structural characterization of S100B binding to RSK1. We show that S100B binds to a C-terminal RSK1 segment that is required not only for ERK2 recruitment but also for the autoinhibition of the RSK1 CaMK-type domain. Interestingly, S100B not only directly interferes with the assembly of the ERK2-RSK1 heterodimeric complex, it also negatively affects the activity of the CaMK-type domain of RSK1. The structural basis for this unusual dual-inhibitory mechanism was revealed by combining high resolution x-ray crystallographic analysis with lower resolution solution small angle x-ray scattering and NMR spectroscopy studies on a minimal S100B-RSK1 complex. This analysis revealed a highly dynamic, fuzzy complex (19). We also found that a single RSK1 fragment binds to an S100B dimer, which is rather unusual in the symmetric homodimer-forming S100 protein family (3). Kinetic studies indicated that S100B binding to the CaMK-type domain involves both a conformational selection and an induced fit step. Based on the results of our structural analysis, it was possible to assign a structural state to all observed kinetic steps. Overall, our study gives a detailed biochemical insight into the S100B-RSK1 interaction and could facilitate future drug design studies to treat malignant melanomas.

Experimental Procedures

Protein/Peptide Expression and Purification

The cDNA of S100 proteins, the RSK1CTKD construct containing the C-terminal RSK1 region between residues 411 and 735, as well as different peptides from RSK1 were cloned into modified pET expression vectors. All protein constructs were expressed in Escherichia coli Rosetta (DE3) pLysS (Novagen) cells with standard techniques. RSK1CTKD was expressed as N-terminal GST fusion protein with a C-terminal non-cleavable hexahistidine tag. S100B/A4/A2/P/A10 was cloned with an N-terminal TEV-cleavable hexahistidine tag. All peptides were cloned with an N-terminal GST fusion tag. For labeling, the peptide 689–735 contained an N-terminal Cys residue. The shorter reporter peptide (residues 712–735) was chemically synthesized and was N-terminally labeled with carboxyfluorescein. The RSK1 HM construct (AGAHQLFRGFSFVATG) was expressed as an N-terminal GST fusion protein with a C-terminal non-cleavable hexahistidine tag. S100B/A4/A2/P was purified with a nickel-affinity column and cleaved by TEV protease. After complete cleavage, S100B/A4/A2/P was supplemented with Ca2+/Zn2+ and injected into a phenyl-Sepharose column. Elution was done with 5 mm EDTA, and the eluted fractions were dialyzed against a Ca2+/Zn2+-containing buffer. After cleavage, S100A10 was further purified by cation exchange chromatography. For biochemical measurements, double affinity-purified RSK1 was used. For SAXS measurement, this double-purified RSK1 was cleaved by the TEV protease, and the sample was further purified on a HiTrap Blue-Sepharose column (GE Healthcare), and then it was complexed with S100B. Peptides were captured by GST affinity purification. After TEV cleavage, GST and TEV were precipitated by boiling the sample. The protein precipitation was spun down, and the supernatant was further purified by reverse-phase HPLC on a Jupiter 300 C5 column (Phenomenex). After lyophilization, the peptides were dissolved in water. For NMR experiments, the 15N-labeled peptide was produced similarly, but before induction, cells were transferred to a 15NH4Cl-containing minimal medium. Labeling of cysteine-containing peptides was carried out using 5-(iodoacetamido)fluorescein (Sigma), and the reaction mixture was also purified by HPLC. HM peptide was prepared by double affinity purification.

Crystallization, Data Collection, and Structural Determination

Crystallization samples contained 1 mm S100B monomer and 2-fold excess (~1 mm) of RSK1 peptide in each case. Crystallization was done in a standard sitting drop vapor-diffusion setup at 23 °C. The crystallization solution consisted of 0.1 m HEPES, pH 7, 150 mm NaCl, 20% PEG6000 in all four cases. Crystals were supplemented with 10% glycerol before flash-cooling in liquid nitrogen. Data were collected on the PXIII beamline of the Swiss Light Source (Villigen) and on the ID23 and ID30 beamlines of ESRF (Grenoble, France) at 100 K (Table 1). Data were processed with XDS. Matthews probability calculations indicate the highest probability of an asymmetric complex forming between a single dimer of S100B and a single RSK1 peptide in all crystal forms (20). This was later confirmed after the phase problem was solved by molecular replacement in PHASER with a high resolution structure of S100B as a searching model (21,23). The molecular replacement search identified a single S100B dimer in the asymmetric unit in all cases. Structure refinement was carried out in PHENIX, and structure remodeling/building was done in Coot (24, 25). The final model was refined to 2.4 Å resolution for crystal structure A (with peptide 683–735), to 2.13 Å resolution for crystal structure A′ (with peptide 689–735), to 2.7 Å resolution for the crystal structure B (with peptide 696–735), and to 2.95 Å resolution for the crystal structure C (with peptide 683–720) (Table 1).

TABLE 1
Data collection and refinement statistics

Kinase Assays

Activated MAPKs were produced by co-expressing them with constitutively active forms of GST-tagged MAPK kinases in E. coli with bicistronic modified pET vectors as described before (13). MAPKs were purified by affinity purification using a HiTrap nickel column (GE Healthcare) followed by ion exchange chromatography on a 1-ml Mono Q column (GE Healthcare). 20 nm activated MAPK was incubated with 1 μm GST-eluted MAPKAPK substrates at room temperature with increasing amounts of S100B. Kinase reactions were carried out in 50 mm HEPES, pH 7.5, 100 mm NaCl, 5 mm MgCl2, 0.05% IGEPAL, 5% glycerol, 2 mm DTT, 2.5 mm CaCl2, 0.5 mm EGTA in the presence of 250 μm ATP and ~5 μCi of [γ-32P]ATP. For RSK1 HM phosphorylation experiments, first 100 nm activated MAPK was incubated with 0.5 μm GST-eluted MAPKAPK substrates at room temperature for 30 min with 250 μm ATP to fully pre-activate MAPKAPK. Then this in situ phosphorylated RSK1 was added to a mixture of 20 μm GST-HM and ~5 μCi of [γ-32P]ATP with increasing amounts of S100B. Reactions were stopped with protein loading sample buffer complemented with 20 mm EDTA, boiled, and then subjected to SDS-PAGE. The gel was dried before analysis by phosphorimaging on a Typhoon Trio+ scanner (GE Healthcare). Rates of 32P incorporation into MAPKAPK substrate bands were obtained by the least squares method using three to five data points from the linear range of kinetic phosphorylation curves. Inhibitory constants were determined by fitting a quadratic equation to the measured initial reaction rates. Note that the presented constants are calculated for a symmetrical binding mode, and therefore they have to be divided by a factor of 2 to get the asymmetrical binding constant.

Binding Assays and Kinetic Measurements

Steady-state tryptophan fluorescence was measured with a Synergy H4 (BioTek Instruments) plate reader in 384-well plates. The excitation wavelength was 295 nm, and the emission was detected at 330 nm. 1 μm GST-RSK1CTKD was mixed with increasing amounts of S100B or CaM. As a negative control, 1 μm GST was also mixed with increasing amounts of S100B (data not shown). Fluorescence intensity change was only observable in the case of RSK1-S100B. The experiment was carried out in triplicate. The measured data were fitted by a quadratic binding equation.

Fast kinetic measurements were performed with the stopped-flow instrument SFM-300 (Bio-Logic) with excitation at 297 nm. Fluorescence emission from Trp residues was observed through a 340-nm bandpass filter (Comar Optics). All reactions were performed at 25 °C in 20 mm Tris, pH 7.5, 100 mm NaCl, 0.5 mm TCEP, 2 mm CaCl2, and 1 mm Ca-EDTA. Post-mixing GST-RSK1CTKD concentration was fixed to 1 μm. Amplitudes and the observed rate constants of double exponential fits were determined using Origin 7 (OriginLab Corp.). To determine the off-rate constants, complex samples were mixed with the RSK1(683–735) peptide.

In direct FP measurements, 50 nm fluorescein-conjugated reporter peptide was mixed with increasing amounts of S100, and the FP signal was measured with a Synergy H4 (BioTek Instruments) plate reader in 384-well plates. In competitive FP measurements 50 nm labeled reporter peptide was mixed with S100 in a concentration to achieve ~60–80% complex formation. Subsequently, increasing amounts of unlabeled peptide or GST-RSK1 constructs were added. In all simple competitive measurements, 20 μm S100B was mixed with labeled peptide. The Kd value for each experiment was determined by fitting the data to a quadratic or a competition binding equation. Titration experiments were carried out in triplicate, and the average FP signal was used for fitting the data with Origin 7.

Gel filtration experiments were carried out using an in-house packed 10/300 Superdex 75 column and GF buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm CaCl2, and 25 μm TCEP. Each sample was injected at least two times into the column. S100B concentration was 1 mm in all cases, which is much higher than the observed dissociation constant.

Secondary and Solution Structure Determination

CD spectroscopy measurements were carried out on a Jasco J-810 spectropolarimeter using a 0.1-mm wide cuvette in a buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 2 mm CaCl2, 2 mm Ca-EDTA, and 1 mm TCEP. The free RSK1(683–735) peptide concentration was 50 μm. To obtain the CD spectrum of the S100B-bound peptide, the spectrum of free S100B (120 μm S100B monomer) was subtracted from the spectrum of the protein complex (50 μm peptide and 120 μm S100B monomer) assuming that the secondary structure of S100B does not change upon RSK1(683–735) binding. Each spectrum was measured in triplicate, and the averaged spectra were used for analysis. The subtracted spectra of the free and the bound peptide was fitted with BeStSel (26).

All SAXS measurements were performed at the BM29 beamline at ESRF. Data were analyzed using the ATSAS program package (27). A dilution series of a concentrated sample of the gel filtrated RSK1(683–735)-S100B complex was measured from 8 to 0.4 mg/ml. No concentration effect was observed during the measurement. The primary data analysis was performed in PRIMUS (Fig. 7). The scattering curve was interpreted with CORAL (27). In the CORAL modeling of structure A, 16-residue long N-terminal, 25-residue long linker, and 4-residue long C-terminal regions were modeled to the peptide fragments obtained in the crystal structure (Figs. 6 and and7).7). Fragments from the crystal structure were treated as rigid bodies, and only the “invisible” flexible elements were modeled. Solution scattering from the RSK1 CTKD construct was determined with on-line size-exclusion chromatography-SAXS using a Superdex 75 column. Fit of the crystal structure was determined by CRYSOL, and an ab initio model was generated by 15 independent run of DAMMIN (27). The best fit was achieved by ensemble modeling with EOM, including both ordered and disordered C-terminal tails (Fig. 10) (28). Ensemble modeling with solely ordered or disordered C terminus resulted in worse fit and not reliable models (data not shown).

FIGURE 6.
Solution structure of the S100B-RSK1 complex. A, CD spectra of free (red) and RSK1(683–735)-bound (black) S100B is shown on the left side and the CD spectra of free (red) and S100B-bound (black) RSK1(683–735) peptide is shown on the right ...
FIGURE 7.
Detailed analysis of the solution structure of the fuzzy S100B-RSK1 complex. A, Guinier fit (red lines) to the obtained scattering curve in the concentration series. The measured concentrations of the isolated complex were 8.3, 3.8, 2.0, 0.8, and 0.5 ...
FIGURE 10.
Assembly of the S100B-RSK1 complex. A, stopped-flow based kinetic analysis (presented in Fig. 8) by global fitting based on the model shown in B (black lines) (see also Fig. 9). (The S100B concentration decreases from red to green.) B, proposed kinetic ...

NMR spectra were recorded on a Bruker Avance III 700 spectrometer operating at 700.17 MHz for 1H, equipped with 5-mm triple-resonance probe head with a z axis gradient. The optimal spectral dispersion was achieved at 303 K, and this temperature was selected from a series of HSQC spectra recorded in the 277–320 K range (for both the free and complexed peptide). 1H chemical shifts were referenced to the internal 4,4-dimethyl-4-silapentane-1-sulfonic acid standard, whereas 15N chemical shifts were referenced indirectly via the gyromagnetic ratios. Both samples had the following compositions: ~0.9 mm 15N-labeled peptide, 20 mm MES buffer, pH 6.00, 20 mm NaCl, 3 mm NaN3, 2 mm TCEP, 5 mm CaCl2 and 10% D2O. S100B concentration was ~2 mm in the complex. Peak assignment and sequential connectivities were determined from the analysis of standard three-dimensional HSQC-TOCSY and three-dimensional HSQC-NOESY measurements. Spectra were processed with TOPSPIN and analyzed using the CARA (29) software program. Chemical shifts were deposited to the BMRB database under accession number 25873.

Results

S100B Exerts Dual-level Inhibition on ERK2 → RSK1 Activation

To quantitatively characterize the binding of S100B to RSK1CTKD, we measured the intrinsic tryptophan fluorescence change of RSK1 upon S100B binding. Because none of the MAPKAPKs can bind to CaM, we used it as a negative control in the experiment. Because neither CaM nor S100B contains a tryptophan residue, the fluorescent signal originates from RSK1. A relatively large intensity decrease (~25%) was found in the presence of large excess of S100B but not in the case of CaM. The measured dissociation constant of the S100B-RSK1CTKD complex was found to be about 5 μm (Fig. 2A).

FIGURE 2.
S100B binding inhibits RSK1 activation. A, S100B binding induces a conformational change in GST-RSK1CTKD, which can be monitored by the change of intrinsic tryptophan fluorescence. The dissociation constant calculated based on this change is 4.8 μ ...

Activated ERK2 can phosphorylate the CTKD of RSK1 on Thr-573 in in vitro kinase assays. In turn, activated CTKD can efficiently phosphorylate an HM containing GST fusion protein in trans (18). An inhibiting role of S100B on RSK1CTKD phosphorylation has been recently reported (7). We also find that S100B clearly impedes RSK1CTKD phosphorylation by ERK2 in an in vitro kinase assay using recombinantly expressed and purified components (Fig. 2B). In addition, S100B also blocks the activity of activated RSK1CTKD on an HM-containing NTKD substrate fragment in trans (Fig. 2C). The calculated inhibitory constants were found to be around 6 μm for ERK2 → CTKD signaling, which showed a good agreement with the binding affinity of the S100B-CTKD complex (see above). It is noteworthy that S100B binding did not completely abolish CTKD kinase activity on the NTKD HM fragment but only lowered the reaction rate to ~40% of the original activity under saturating conditions. From these observations, we could rule out that S100B has a CaM-like effect, which is known to exert activation on CaMKII (30). In conclusion, S100B was shown here to be a dual-level specific inhibitor of RSK1 activation, acting both on the ERK2 → CTKD and also on the CTKD → NTKD level.

C-terminal Region of CTKD Mediates High Affinity Binding to S100B

It was proposed that the S100B-RSK1 interaction requires the flexible C terminus of RSK1 (7). Interestingly, ERK2-RSK1 binding also depends on this C-terminal tail (31). These findings suggest that S100B and ERK2 binding to RSK1 is mutually exclusive. To show that the two RSK1-binding partners indeed compete for the same RSK1 region, we used FP-based binding assays with a fluorescently labeled C-terminal fragment of RSK1. Titrating this labeled reporter peptide RSK1(712–735) with S100B in the presence of calcium ions indeed increased its fluorescence anisotropy, indicating direct interaction (Fig. 3A). In contrast, in the presence of EDTA (or in the absence of Ca2+) no changes in FP were observed (Fig. 3B). Thus, the interaction seemed to be specific; however, the binding affinity was found to be low (Kd = 30 μm). We next extended the length of the reporter peptide toward the N terminus (residues 689–735). The binding affinity of this extended peptide, including the inhibitory helix region (αL) as well, was found to be about 6-fold higher compared with the shorter RSK1 peptide (Fig. 3C).

FIGURE 3.
Characterization of RSK1 C-terminal region as the interaction hot spot for S100B binding. A, MAPK binding C-terminal peptide (RSK1(712–735)) binds weakly to S100B (Kd ≈30 μm). B, in the absence of Ca2+, this interaction is abolished ...

FP-based binding measurements can also be used to determine the binding affinities of unlabeled constructs. The dissociation constant of the RSK1CTKD protein as well as different RSK1 C-terminal fragments were measured in competition assays. The binding affinity of RSK1CTKD determined in this assay (4 μm) was similarly in the low micromolar range as the affinity measured by the assay based on monitoring the tryptophan fluorescence change upon S100B binding (Fig. 3D). Extending the peptide length to the end of the core kinase domain of CTKD(683–735) greatly increased the strength of complex formation (Kd = 40 nm) (Fig. 3, E and F). For this high affinity complex, the binding stoichiometry can be determined from the competition curve. In the presence of 20 μm S100B, maximal signal decrease was observed around 10 μm unlabeled RSK1(683–735) peptide, indicating an asymmetrical binding mode. This asymmetry means that a single RSK1(683–735) peptide binds to one S100B dimer. A similar 1:2 binding stoichiometry was observed in the complex of non-muscle myosin IIA and S100A4, but it has not been previously observed in any S100B complex (3). To further confirm the 1:2 binding ratio, we examined the complex formation in gel-filtration experiments. Free S100B dimer and free RSK1(683–735) peptides have different elution volumes, and a slight upward shift in the elution volume is apparent when both components are added, which is suggestive for complex formation. Mixing the partners in a 1:2 molar ratio gives a homogeneous fast eluting peak, whereas adding equimolar RSK1(683–735) peptide to S100B chains (2:2 ratio) results in a mixture of the previously identified complex and free RSK1 peptide (Fig. 3G).

S100 proteins are structurally highly homologous, and therefore it is important to determine whether the S100B-RSK1 interaction is specific within the S100 family. Direct binding between the RSK1(683–735) peptide and S100B, S100A4, S100A2, and S100P was observed by FP-based binding experiments. Nevertheless, it was found that only S100B was able to mediate high affinity binding with the RSK1CTKD (Figs. 3H and and4).4). This result suggests that after anchoring itself to the C-terminal region of RSK1, S100B may form additional specific interactions with the kinase domain itself. In summary, the RSK1-S100B interaction seems to be specific among S100 protein family members.

FIGURE 4.
Fits of the fluorescence polarization binding measurements. A, direct (left) and competitive (middle, right) FP measurements for different S100 proteins. In competitive experiments, unlabeled RSK1(683–735) (middle) or RSK1CTKD (right) was used ...

The short labeled RSK1(712–735) peptide has a marginal affinity (30 μm) toward S100B; however, it can form a high affinity complex with ERK2 (0.5–1 μm). This phenomenon can be exploited to investigate co-binding of ERK2 and S100B to RSK1. At high S100B concentration, ERK2-RSK1(712–735) complex formation could still be measured both in the presence or the absence of calcium ions in direct FP measurements (Fig. 3I). In competitive titration experiments using the RSK1(689–735) peptide fragment, which is a much better S100B binder, we could measure the effect of active/inactive S100B on ERK2-RSK1(689–735) complex formation. If co-binding can occur then almost identical competitive titration curves are expected. However, if there is a competition between ERK2 and S100B then one can measure an increase in the observed dissociation constant for the ERK2-RSK1 binding event. The measured affinity indeed decreased by about 1 order of magnitude (Fig. 3I). This result indicates that there is a direct competition for the RSK1 tailpiece between the two binding partners.

Crystal and Solution Structure of the Minimal RSK1-S100B Complex

For structural studies, a set of RSK1 peptides with various deletions both from the C- and N-terminal ends of RSK1(683–735) were made (Figs. 4 and and55A). We found that if either the C- or the N-terminal part was truncated, the binding affinity decreased. To visualize the atomic details of the minimized RSK1-S100B interaction, we determined the crystal structure of the high affinity RSK1(683–735)-S100B complex (crystal structure A). Unfortunately, only a small N- and C-terminal portion of RSK1 could be built in the crystallographic model; nonetheless, the structure verified the asymmetric nature of the complex (Fig. 5, B and C). To improve the quality of the crystal structure, we attempted protein crystallization with truncated peptide constructs to decrease the conformational flexibility. An N-terminally truncated peptide (RSK1(689–735)) gave a very similar crystal structure but with better resolution and a more interpretable electron density map (crystal structure A′) (Fig. 5D). A non-isomorphous crystal formed with a yet shorter peptide (696–735; crystal structure B), whereas a nearly isomorphous crystal was obtained with the peptide 683–720 (crystal structure C) (Fig. 5C). Comparison of these four structures suggests that the crystallographic models likely captured different conformations of the interaction (Fig. 5D). In crystal structure B, the middle part of the RSK1 peptide was also missing, but the C-terminal part showed helical structure. Similarly to structure A, only the two classical S100 binding pockets were occupied by the terminal regions of the RSK1 peptide. Structure C displayed a very different structure for the bound RSK1 peptide. For this peptide (683–720), in agreement with its lower affinity compared with the full-length peptide (residues 683–735) or the N-terminally truncated peptide (residues 696–735), one of the two canonical pockets was unoccupied. However, a helical segment from the peptide was found in the non-conventional binding site of S100B, which is a shallow groove connecting the two canonical sites as described in Ref. 3.

FIGURE 5.
Structural properties of the S100B-RSK1 complex. A, summary of binding affinities between S100B and various truncated RSK1 peptide constructs. (See Fig. 3 and and44 for details.) Removal of small regions at both sides greatly influences the binding ...

In structure B, the second canonical S100 binding interface interacts with a C-terminal helical element of RSK1. This region of RSK1 has helical propensity in solution and also it forms a helix when it is bound in the ERK2 docking groove (13). Moreover, the helical element from structure C was found to be analogous to the surface-oriented side of the bound inhibitory helix of the previously solved RSK1 structures (Fig. 5C) (13, 14, 32). We propose that structure C captured the S100B-RSK1 complex in a state where the interaction between the S100B and the RSK1 C-terminal segment may be such as in an S100 inhibited complex with full-length RSK1. Superimposing the S100B complex with an inhibited RSK1 structure via this helical element (between residues 700 and 707 with a root mean square deviation of 1.1 Å for 51 atoms) suggests that the S100B dimer could help locking the inhibitory helix in its position, and thus promote its interaction with the core kinase domain while it simultaneously blocks MAPK binding through the secondary classical S100-binding interface (Fig. 5E). However, it should be noted that atomic clashes are apparent between S100B and the αG helix of RSK1 in a model generated by this simple superimposition. Thus, the αG helix must undergo some kind of structural rearrangement upon S100B binding to be compatible with the binding mode that was observed in the crystallographic model C.

All crystal structures included a segment of the αL helix (between residues 697 and 701) on the N-terminal binding side that is in coil conformation. This apparently key interaction however does not contain any directed bonds toward S100B. In structure A, A′, and B, the C-terminal binding side is occupied by the MAPK-binding basic motif with different conformations. Interestingly, in structure B this segment was helical just as in the case of the MAPK-bound conformation. This interaction is responsible for the direct competition with ERK2 binding. In structure C, where this basic motif was truncated, a helical element appeared next to the N-terminal binding side. Because different regions showed helical propensity in our crystal structures, we wanted to investigate the secondary structure of the bound RSK1(683–735) peptide. We measured the solution structure using CD spectroscopy (Fig. 6A). The free peptide is highly flexible and lacks any secondary structural element, but the bound form contains a single ~12-amino acid-long helical element. For the other characterized asymmetric S100-binding partner (NMIIA), a much larger helical content was calculated (>60%, ~20 residues long) by an identical method, which was later also confirmed by high resolution crystal and solution structures (3, 4). This suggests that the RSK1 binding mode to S100B is different from the NMIIA binding to S100A4. In addition, these data also suggest that either of the two observed helical element is compatible with the observed total helical content, but both cannot exist at the same time.

Based on our multiple crystallographic models, it appears that the S100B-RSK1 complex can acquire many possible structural states. The first model is based on crystal structures A and A′; both canonical binding pockets are filled with the N- and C-terminal part of the linear motif adopting a coil structure. The second model is based on crystal structure B; the N-terminal binding region is in coil conformation, but the C-terminal is helical. The last two possible models are based on structure C. In these complexes, the N-terminal part is in coil, but it is extended by a helical intervening part, and the C-terminal binding region may bind as it is seen in either the A (or A′) or the B crystal structure. Because we wanted to further analyze this structurally diverse, apparently fuzzy complex, as revealed by x-ray crystallographic analysis, we used SAXS to obtain at least low resolution structural information about the shape of the complex in solution. We were able to measure high quality SAXS from an isolated complex with the RSK1(683–735) peptide (Fig. 7). In all crystal structures, only small peptide fragments could be located in the electron density. Using SAXS data, however, we could simulate the whole complex using the above described four templates (Fig. 6B). In these simulations the “crystallographically” invisible parts were ab initio modeled using a program called CORAL (27). Out of the four structural models, only the first one (based on structure A and A′) fit the obtained SAXS curve, and therefore this describes the solution structure of the RSK1-S100B complex the best (Figs. 6 and and7).7). The simulated models have high flexibility between the two anchor positions; therefore, it can be considered as a clamp-type fuzzy complex (19). From 10 independent simulations, two types of clusters were observed as follows: one with a free and one with an S100-bound N-terminal part (Fig. 7). This N-terminal flanking segment seems to be responsible for a large increase in the binding affinity (Figs. 4 and and55A). Unfortunately, this site on S100B is close to a crystal packing interaction surface; therefore, this additional interaction remains hidden in our crystal structures. To further characterize this flanking element, we performed mutational scanning on the first six residues (Figs. 4 and and55A). We found that the first two residues do not directly contribute to the binding, but if either residues 685–686 or 687–688 were mutated to Ala, the binding affinity decreased with an order of magnitude. We assume that the hydrophobic Leu-686 and basic His-688 are most likely the key residues determining the high affinity binding of RSK1(683–735) to S100B.

As an additional analysis, we measured the changes in the solution structure of this minimal RSK1 peptide by NMR spectroscopy. We characterized the RSK1(683–735) peptide in free form and performed peak assignment at 303 K from the three-dimensional TOCSY-HSQC and NOESY-HSQC measurements. Already the narrow signal dispersion of the HSQC spectrum informed us about the disordered nature of this free RSK1 tail fragment. Addition of excess S100B under identical conditions resulted in changes in the spectrum (Fig. 7E). The complex had an increased molecular mass and as a consequence a different tumbling time in solution. As changes were monitored from the 15N RSK1 peptide side (S100B protein was unlabeled), the consequence of complex formation would be observable as shifted and broadened peaks and also peaks broadened below the detection limit in the HSQC spectrum.

Unfortunately, we were unable to assign the complex spectrum under these experimental conditions. Nevertheless, if we assume that close peaks in the HSQC spectra correspond to the same amino acid, we could gain residue level information of the changes in the chemical environment of the RSK1 peptide upon complex formation. Based on the overlay of the spectra, it appears that many peaks showed no or little chemical shift changes. These are the residues that belong to the N-terminal flanking and the intervening parts (for example Val-694, Gly-696, Ser-707, and Lys-709 remained mostly unchanged, see Fig. 5). Although 54 peaks (including minor peaks) were detectable for the free peptide, only 47 peaks were present in the spectrum of the complex (Fig. 7E). Disappearing peaks correspond to residues involved in direct binding interaction with intermediate exchange. However, these resonances can also belong to the shifted, newly appearing broad peaks. These changes clearly affect the C-terminal binding side (residues 724–730) and residues from the N-terminal side (like Ala-697 or Ala-704); they are in full accordance with the crystallographic model (Fig. 5) and indicating that these regions could provide the “clamps” of the fuzzy complex. In addition, the N-terminal flanking part was also changed upon binding (e.g. Leu-686). The lack of dramatic changes in signal dispersion upon complex formation suggests that there are no new secondary structural elements forming in the RSK1 peptide. This suggests that the observed helical content by CD spectroscopy is due to transient helix formation. Overall, these observations corroborate the existence of a fuzzy complex and are in agreement with the crystallographic analysis of the RSK1-S100B complex.

Rapid Kinetic Experiments Reveal That S100B Binding to RSK1 Involves Both Conformational Selection and Induced Fit

The inherent tryptophan fluorescence change of RSK1 upon S100B binding (Fig. 2A) could be used to decipher the kinetic mechanism of the interaction. In stopped-flow experiments, we were able to follow RSK1-S100B complex formation in real time by mixing RSK1 with S100B as well as to monitor its dissociation by mixing the complex with a “chaser” peptide from RSK1 (Fig. 8). Both the observed association and dissociation transients could be fitted by double exponential functions. The observed rate constants of the first and second phases of the association transients showed a decreasing and an increasing tendency, respectively, with increasing S100B concentration. The rate constants of both phases reached saturation at a relatively low S100B concentration (Fig. 8, C and D). These profiles are indicative of the presence of both conformational selection (first phase) and induced fit (second phase) processes. As dissociation was dependent on chaser peptide concentration, dissociation rate constants were determined by linear extrapolation to zero chaser concentration (Fig. 8, G and H). Based on the exponential approximations (Fig. 8), we constructed the kinetic model shown in Fig. 9, which we used for global fitting kinetic analysis (Fig. 10). The results of this analysis (Fig. 10, A and B) were in good agreement with those resulting from experimental fits (Fig. 9). In the proposed model RSK1 displays conformational heterogeneity, termed autoinhibited and released state, indicating the position of αL helix. The interaction of the released species with S100B is followed by an isomerization step. Note that S100B binding by the predominant autoinhibited species is not favorable. S100B binding therefore can be described as a combination of conformational selection and induced fit model (Fig. 10B). First, S100B can only bind to the free C-terminal tail. After the formation of the fuzzy complex, the anchored S100B can mediate further interactions with the kinase domain. The population of this “S100B-inhibited” state is ~44%, and it can share structural relationship with our crystal structure C. We speculate that this latter complex is responsible for the inactivation of the activated CTKD domain of RSK1.

FIGURE 8.
Tryptophan fluorescence-based kinetic experiments, exponential analysis. A, binding traces of GST-RSK1CTKD and S100B fitted with a double exponential function. (The S100B concentration decreases from red to blue.) GST-RSK1CTKD Trp fluorescence transients ...
FIGURE 9.
Comparison of the kinetic parameters based on the exponential analysis and the global fit. The proposed model consists of two consecutive steps involving conformational selection or induced fit based steps. Free RSK1 is in a mixture of S100B binding competent ...

To validate the apparent conformational heterogeneity of RSK1, we attempted to analyze the RSK1CTKD construct by SAXS (Fig. 10C). Unfortunately, all attempts to measure scattering of the S100B-RSK1CTKD complex failed, because the complex dissociated under gel filtration. Moreover, obtaining good SAXS data on the RSK1CTKD construct alone turned out to be technically challenging due to the protein's aggregation tendency. Fortunately, on-line gel filtration of the S100B-RSK1CTKD complex without Ca2+ finally gave useful scattering data on the RSK1CTKD construct. The measured pattern showed an acceptable fit with an RSK1 model obtained from the ERK2-RSK1CTKD heterodimeric crystal structure (χ = 1.00) (Fig. 10C) (13). In contrast, a more reasonable model (χ = 0.88) could be described as an ensemble of both “autoinhibited” and “released” states of the CTKD (Fig. 10D). Although this may not be regarded as unambiguous validation of all the structural models proposed, it lends support to some aspects of the complex kinetic model.

Discussion

CaMK domains share similar architecture and therefore similar regulation. Their C-terminal extensions usually have an inhibitory effect on the kinase domain, and this autoinhibition needs to be released before the kinase can become enzymatically active. In many cases, this is achieved through CaM binding. In MAPKAPK-type kinases, the activation loop needs to be phosphorylated by a MAPK. Then the phosphorylated activation loop and the autoinhibitory element must go through substantial remolding at the active site (33). Hartman et al. (7) have recently reported that S100B can bind to RSK1 and suggested that this interaction is positioned to the same RSK1 C-terminal region where ERK2 also binds (12). However, the possibility that S100B may bind to the autoinhibitory RSK1 C-terminal region has not been postulated before. Based on our results, we conclude that CaM and S100B binding is structurally similar. However, their binding to cognate CaMK domains can have dramatically different functional readouts. CaM binding promotes CaMKII activity by stabilizing an open and active conformation of the kinase (see Fig. 1A), whereas S100B binding inhibits RSK1 activity, possibly by stabilizing a closed and autoinhibited form of RSK1, in addition to also directly blocking ERK2 recruitment. In the work of Hartman et al. (7), it was stated that S100B binding had no effect on the phosphorylation of the RSK hydrophobic motif. In the light of our in vitro kinase assay data and considering that MAPKAPK HM phosphorylation may occur reportedly in trans as well (34), this discrepancy may be explained by the insensitivity and/or nonspecific nature of the used antibody in the former report.

X-ray crystallographic, SAXS, and NMR-based structural analysis indicate that S100B forms a fuzzy complex with the C-terminal tail of RSK1. Disordered binding regions (IDRs) of proteins usually show flexibility only in their unbound state, but upon partner binding they fold (35). In contrast, fuzzy complexes retain a degree of flexibility even in their bound form (19). They could be prevalent in interactomes, but they currently stay mostly uncharacterized due to limitations of our experimental approaches. Capturing static interactions from a fuzzy complex is challenging, and determining the in-solution structural ensemble is almost impossible. We managed to crystallize the minimal S100B-RSK1 complex in three different binding modes regarding the structure of the RSK1 C-terminal region. This was necessary to obtain a reliable structural ensemble by SAXS as experimentally determined crystallographic structural states could be used to model in-solution scattering. Interestingly, it was the fuzziest crystallographic model with only two small anchoring contacts between the flexible RSK1 C-terminal region and the dimeric S100B protein that matched the determined in-solution molecular ensemble the best. In this complex, a large part of the bound peptide is not involved in making contacts and is highly unstructured, which we also proved by solution NMR measurements. The fuzziness of the complex and the observed increase in helical content upon binding raise the question as to where and how a helix may form, albeit likely only transiently. Our diverse crystallographic models suggest that the shallow binding interface of S100B is able to interact with helical partners. Also, the distance between the two observed anchored regions is ~26 Å, which is connected by a 25-residue-long flexible linker. It is possible that the propensity for helix formation within this intervening region is highly increased because the motion of this relatively large linker is sterically limited by a clamping mechanism. It is noteworthy that a similar shallow groove of S100A4 mediates interaction with a helical peptide segment of NMIIA (3, 4).

The described mode of S100B binding directly to RSK1 is possible only if the inhibitory αL helix is released from the core kinase domain. If this is not the case, then S100B needs to pull out the helix from the active site. The first case would be a classical conformational selection mechanism, and the second one is an induced fit scenario. In theory, they can both be present, but our measured kinetic data suggest that the primary binding of S100B to RSK1CTKD can only be described with the conformational selection model. However, we also observed an induced fit step where the bound S100B further interacts with the kinase domain. This latter interaction could explain that S100B allosterically inhibits the activity of phosphorylated RSK1. Based on one of the crystallographic models (crystal structure C), it is likely that S100B can interact with the inhibitory αL helix in situ, and thus it may increase its stability in the autoinhibited versus the released state of RSK1 CTKD. Nevertheless, this complex is rather unlikely to form because the αG helix is in steric clash in the superimposed models. Releasing the αL inhibitory helix can cause a high degree of structural rearrangement, which can be ideal for the S100B bound inhibitory complex. From a kinetic perspective, the proposed mechanistic model can also be considered as a thermodynamic box (Fig. 10B). S100B binding to the autoinhibited state of the kinase can then directly result in the S100B inhibited complex, with a theoretically calculated Kd of <3 μm, which cannot be neglected compared with the observed binding constants. However, it is clear that that this path is kinetically blocked. Could the rearrangement of the αG helix be the reason for this kinetic block? Tentatively, the answer is yes; however, to describe this presumed S100B-RSK1 inhibitory complex in more detail, an atomic resolution structure of S100B in complex with the RSK1 CTKD is needed.

In malignant melanoma, the MAPK/ERK pathway is usually up-regulated by activating mutations of upstream kinases. This could result in hyperphosphorylation and hyperactivation of RSK1. However, overexpression of S100B in melanoma protects RSK1 against active ERK2 and in turn from autophosphorylation. The mechanistic explanation why S100B inhibition of RSK1 activation is a hallmark feature in melanoma is not understood. Hartman et al. (7) also showed that the subcellular localization of RSK1 is altered by S100B, which could shift remaining RSK1 activity into the cytoplasm. These effects may be beneficial to melanoma cells by negatively affecting important tumor suppressor proteins such as DAPK1, LKB1, or TSC2 (10, 36, 37). Although little is known about the precise mechanism behind RSK1 localization, cellular studies showed that activation of the NTKD involving both AL and HM phosphorylation is essential to nuclear accumulation (38). CTKD inhibition by S100B can directly affect HM phosphorylation and therefore the NTKD AL phosphorylation, too.

Before this study, three binding pockets were identified on S100B (Fig. 11A) (39). Selective targeting of these sites is already a promising opportunity for the treatment of malignant melanoma (9). Here, we showed that the fuzzy complex uses site 1 and site 2 simultaneously, whereas the inhibitory complex involves an asymmetric fourth binding site, which is placed between the classical symmetric binding pockets (Fig. 11). Targeting the classical binding sites can inhibit interactions of S100B indiscriminately, but targeting of this middle binding pocket may be a highly selective inhibitor for the assembly of the S100B-RSK1 inhibitory complex. Based on the observation that primary complex formation only yields the fuzzy complex where this fourth binding site remains free, this novel site may be a potent switch that can freeze the S100B-RSK1CTKD complex into a CaMK-CaM type complex. Stabilization of this latter conformation would presumably promote activation of the kinase, thus turning S100B into an activator of the ERK pathway rather than an inhibitor. Overall, our data and structural models potentially may open up avenues for selective modulation, inhibition and activation alike, of RSK1 activity. In turn, this could enable mapping out the physiological consequences of the apparent signaling cross-talk between a Ca2+-dependent protein and an important kinase regulator of cell growth.

FIGURE 11.
Protein interaction surfaces of S100B and their relevance on differential S100B-RSK1 complex formation. A, schematic view of RSK1-binding sites on S100B as projected on crystal structure A and C. Sites 1–3 had been previously identified as canonical ...

Author Contributions

G. G. and A. A. designed and performed the experiments and analyzed the data. B. K., G. K., and M. K. contributed in designing the experiments and analyzing data. G. G., A. R., and L. N. oversaw the research and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgments

We thank the staff members at beamlines PXIII of Swiss Light Source (SLS) and ID23, ID30, and BM29 of European Synchrotron Radiation Facility (ESRF) for assistance in data collection.

*This work was supported in part by Hungarian National Research Fund (OTKA) Grants K108437 (to L. N.) and NK101072 (to A. B.), the Momentum Program of the Hungarian Academy of Sciences Grants LP2013–57 (to A. R.) and LP-006/2011 (to M. K.), the Swedish Research Council (to G. K.), the MedInProt Program of the Hungarian Academy of Sciences, the European Union, and the European Social Fund Grant (TÁMOP 4.2.1./B-09/KMR-2010-0003). The authors declare that they have no conflicts of interest with the contents of this article.

This article was selected as a Paper of the Week.

The atomic coordinates and structure factors (codes 5CSF, 5CSI, 5CSJ, and 5CSN) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Submitted to the BMRB Database under code 25873.

3The abbreviations used are:

CaMK
calcium/calmodulin-dependent protein kinase
CaM
calmodulin
SAXS
small angle x-ray scattering
MAPKAPK
MAPK-activated protein kinase
CTKD
C-terminal CaMK-type domain
NTKD
N-terminal AGC-type kinase domain
HM
hydrophobic motif
AL
activation loop
TEV
tobacco etch virus
TCEP
tris(2-carboxyethyl)phosphine
FP
fluorescence polarization
HSQC
heteronuclear single quantum coherence.

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