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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*
  • Gergő Gógl
    Affiliations
    From the Department of Biochemistry,
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  • Anita Alexa
    Affiliations
    the “Momentum” Protein Interaction Group, Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, 1117 Budapest, Hungary, and
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  • Bence Kiss
    Affiliations
    From the Department of Biochemistry,
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  • Gergely Katona
    Affiliations
    the Department of Chemistry and Molecular Biology, University of Gothenburg, 40530 Gothenburg, Sweden
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  • Mihály Kovács
    Affiliations
    ELTE-MTA “Momentum” Motor Enzymology Research Group, Department of Biochemistry, and
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  • Andrea Bodor
    Affiliations
    Institute of Chemistry Eötvös Loránd University, 1117 Budapest, Hungary,
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  • Attila Reményi
    Correspondence
    To whom correspondence may be addressed: Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok Korutja 2, 1117 Budapest, Hungary. Tel.: 36-1-3826613; E-mail:
    Affiliations
    the “Momentum” Protein Interaction Group, Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, 1117 Budapest, Hungary, and
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  • László Nyitray
    Correspondence
    To whom correspondence may be addressed: Dept. of Biochemistry, Eötvös Loránd University, Pázmány Péter Sétány 1/C, 1117 Budapest, Hungary. Tel.: 36-1-3812171; Fax: 36-1-3822172; E-mail:
    Affiliations
    From the Department of Biochemistry,
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  • Author Footnotes
    * 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.
Open AccessPublished:November 02, 2015DOI:https://doi.org/10.1074/jbc.M115.684928
      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.

      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 (
      • Donato R.
      S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles.
      ,
      • Marenholz I.
      • Heizmann C.W.
      • Fritz G.
      S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature).
      ). 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 (
      • Kiss B.
      • Duelli A.
      • Radnai L.
      • Kékesi K.A.
      • Katona G.
      • Nyitray L.
      Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.
      ,
      • Elliott P.R.
      • Irvine A.F.
      • Jung H.S.
      • Tozawa K.
      • Pastok M.W.
      • Picone R.
      • Badyal S.K.
      • Basran J.
      • Rudland P.S.
      • Barraclough R.
      • Lian L.Y.
      • Bagshaw C.R.
      • Kriajevska M.
      • Barsukov I.L.
      Asymmetric mode of Ca2+-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling.
      ). 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 (
      • Bresnick A.R.
      • Weber D.J.
      • Zimmer D.B.
      S100 proteins in cancer.
      ,
      • Riuzzi F.
      • Sorci G.
      • Donato R.
      S100B Stimulated myoblast proliferation and inhibits myoblast differentiation by independently stimulating ERK1/2 and inhibiting p38 MAPK.
      ). 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)
      The 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.
      -type domain of RSK1 by ERK1/2 (
      • Hartman K.G.
      • Vitolo M.I.
      • Pierce A.D.
      • Fox J.M.
      • Shapiro P.
      • Martin S.S.
      • Wilder P.T.
      • Weber D.J.
      Complex formation between s100b protein and the p90 ribosomal S6 kinase (RSK) in malignant melanoma is calcium-dependent and inhibits extracellular signal-regulated kinase (ERK)-mediated phosphorylation of RSK.
      ). In malignant melanomas, S100B expression is highly elevated, which can be used as a prognostic marker for the disease (
      • Gogas H.
      • Eggermont A.M.
      • Hauschild A.
      • Hersey P.
      • Mohr P.
      • Schadendorf D.
      • Spatz A.
      • Dummer R.
      Biomarkers in melanoma.
      ). Moreover, S100B is being explored as a therapeutic target for treating melanomas by inhibiting its protein-protein interactions (
      • Hartman K.G.
      • McKnight L.E.
      • Liriano M.A.
      • Weber D.J.
      The evolution of S100B inhibitors for the treatment of malignant melanoma.
      ).
      RSK1 belongs to the group of MAPK-activated protein kinases (MAPKAPK) (
      • Romeo Y.
      • Zhang X.
      • Roux P.P.
      Regulation and function of the RSK family of protein kinases.
      ). MAPKAPKs are downstream cytoplasmic targets of ERK and/or p38 MAPKs and belong to the CaMK-type protein kinase superfamily (
      • Cargnello M.
      • Roux P.P.
      Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases.
      ). 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 (
      • Garai Á.
      • Zeke A.
      • Gógl G.
      • Törő I.
      • Fördős F.
      • Blankenburg H.
      • Bárkai T.
      • Varga J.
      • Alexa A.
      • Emig D.
      • Albrecht M.
      • Reményi A.
      Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove.
      ,
      • Alexa A.
      • Gógl G.
      • Glatz G.
      • Garai Á.
      • Zeke A.
      • Varga J.
      • Dudás E.
      • Jeszenői N.
      • Bodor A.
      • Hetényi C.
      • Reményi A.
      Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex.
      ). 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 (
      • Malakhova M.
      • Tereshko V.
      • Lee S.-Y.
      • Yao K.
      • Cho Y.-Y.
      • Bode A.
      • Dong Z.
      Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2.
      ). 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 (
      • Rellos P.
      • Pike A.C.
      • Niesen F.H.
      • Salah E.
      • Lee W.H.
      • von Delft F.
      • Knapp S.
      Structure of the CaMKIIδ/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation.
      ). 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 (
      • Pearce L.R.
      • Komander D.
      • Alessi D.R.
      The nuts and bolts of AGC protein kinases.
      ). 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 (
      • Frödin M.
      • Antal T.L.
      • Dümmler B.A.
      • Jensen C.J.
      • Deak M.
      • Gammeltoft S.
      • Biondi R.M.
      A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation.
      ).
      Figure thumbnail gr1
      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 (
      • Rellos P.
      • Pike A.C.
      • Niesen F.H.
      • Salah E.
      • Lee W.H.
      • von Delft F.
      • Knapp S.
      Structure of the CaMKIIδ/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation.
      ). Many CaMK-type kinases share an analogous activation mechanism. B, RSK1 consists of two kinase domains (green) and a flexible C-terminal tail (red) (
      • Alexa A.
      • Gógl G.
      • Glatz G.
      • Garai Á.
      • Zeke A.
      • Varga J.
      • Dudás E.
      • Jeszenői N.
      • Bodor A.
      • Hetényi C.
      • Reményi A.
      Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex.
      ,
      • Ikuta M.
      • Kornienko M.
      • Byrne N.
      • Reid J.C.
      • Mizuarai S.
      • Kotani H.
      • Munshi S.K.
      Crystal structures of the N-terminal kinase domain of human RSK1 bound to three different ligands: Implications for the design of RSK1 specific inhibitors.
      ). ERK2 (orange) binds directly to the C-terminal linear motif and phosphorylates the activation loop of the CTKD of RSK1 (which is a CaMK-type kinase domain). Then the CTKD phosphorylates the HM of the NTKD. The phosphorylated HM can anchor the AGC master kinase PDK1, which will, in turn, activate the NTKD.
      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 (
      • Chrestensen C. A
      • Sturgill T.W.
      Characterization of the p90 ribosomal S6 kinase 2 carboxyl-terminal domain as a protein kinase.
      ). 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 (
      • Frödin M.
      • Antal T.L.
      • Dümmler B.A.
      • Jensen C.J.
      • Deak M.
      • Gammeltoft S.
      • Biondi R.M.
      A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation.
      ).
      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 (
      • Tompa P.
      • Fuxreiter M.
      Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions.
      ). 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 (
      • Kiss B.
      • Duelli A.
      • Radnai L.
      • Kékesi K.A.
      • Katona G.
      • Nyitray L.
      Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.
      ). 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.

      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 (
      • Underwood K.W.
      • Parris K.D.
      • Federico E.
      • Mosyak L.
      • Czerwinski R.M.
      • Shane T.
      • Taylor M.
      • Svenson K.
      • Liu Y.
      • Hsiao C.L.
      • Wolfrom S.
      • Maguire M.
      • Malakian K.
      • Telliez J.B.
      • Lin L.L.
      • et al.
      Catalytically active MAP KAP kinase 2 structures in complex with Staurosporine and ADP reveal differences with the autoinhibited enzyme.
      ). Hartman et al. (
      • Hartman K.G.
      • Vitolo M.I.
      • Pierce A.D.
      • Fox J.M.
      • Shapiro P.
      • Martin S.S.
      • Wilder P.T.
      • Weber D.J.
      Complex formation between s100b protein and the p90 ribosomal S6 kinase (RSK) in malignant melanoma is calcium-dependent and inhibits extracellular signal-regulated kinase (ERK)-mediated phosphorylation of RSK.
      ) 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 (
      • Garai Á.
      • Zeke A.
      • Gógl G.
      • Törő I.
      • Fördős F.
      • Blankenburg H.
      • Bárkai T.
      • Varga J.
      • Alexa A.
      • Emig D.
      • Albrecht M.
      • Reményi A.
      Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove.
      ). 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. (
      • Hartman K.G.
      • Vitolo M.I.
      • Pierce A.D.
      • Fox J.M.
      • Shapiro P.
      • Martin S.S.
      • Wilder P.T.
      • Weber D.J.
      Complex formation between s100b protein and the p90 ribosomal S6 kinase (RSK) in malignant melanoma is calcium-dependent and inhibits extracellular signal-regulated kinase (ERK)-mediated phosphorylation of RSK.
      ), 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 (
      • Zaru R.
      • Ronkina N.
      • Gaestel M.
      • Arthur J.S.
      • Watts C.
      The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways.
      ), 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 (
      • Dunker A.K.
      • Lawson J.D.
      • Brown C.J.
      • Williams R.M.
      • Romero P.
      • Oh J.S.
      • Oldfield C.J.
      • Campen A.M.
      • Ratliff C.M.
      • Hipps K.W.
      • Ausio J.
      • Nissen M.S.
      • Reeves R.
      • Kang C.
      • Kissinger C.R.
      • et al.
      Intrinsically disordered protein.
      ). In contrast, fuzzy complexes retain a degree of flexibility even in their bound form (
      • Tompa P.
      • Fuxreiter M.
      Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions.
      ). 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 (
      • Kiss B.
      • Duelli A.
      • Radnai L.
      • Kékesi K.A.
      • Katona G.
      • Nyitray L.
      Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.
      ,
      • Elliott P.R.
      • Irvine A.F.
      • Jung H.S.
      • Tozawa K.
      • Pastok M.W.
      • Picone R.
      • Badyal S.K.
      • Basran J.
      • Rudland P.S.
      • Barraclough R.
      • Lian L.Y.
      • Bagshaw C.R.
      • Kriajevska M.
      • Barsukov I.L.
      Asymmetric mode of Ca2+-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling.
      ).
      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. (
      • Hartman K.G.
      • Vitolo M.I.
      • Pierce A.D.
      • Fox J.M.
      • Shapiro P.
      • Martin S.S.
      • Wilder P.T.
      • Weber D.J.
      Complex formation between s100b protein and the p90 ribosomal S6 kinase (RSK) in malignant melanoma is calcium-dependent and inhibits extracellular signal-regulated kinase (ERK)-mediated phosphorylation of RSK.
      ) 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 (
      • Romeo Y.
      • Zhang X.
      • Roux P.P.
      Regulation and function of the RSK family of protein kinases.
      ,
      • Anjum R.
      • Roux P.P.
      • Ballif B.A.
      • Gygi S.P.
      • Blenis J.
      The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling.
      ,
      • Romeo Y.
      • Moreau J.
      • Zindy P.-J.
      • Saba-El-Leil M.
      • Lavoie G.
      • Dandachi F.
      • Baptissart M.
      • Borden K.L.B.
      • Meloche S.
      • Roux P.P.
      RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth.
      ). 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 (
      • Gao X.
      • Chaturvedi D.
      • Patel T.B.
      Localization and retention of p90 ribosomal S6 kinase 1 in the nucleus: implications for its function.
      ). 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) (
      • Cavalier M.C.
      • Pierce A.D.
      • Wilder P.T.
      • Alasady M.J.
      • Hartman K.G.
      • Neau D.B.
      • Foley T.L.
      • Jadhav A.
      • Maloney D.J.
      • Simeonov A.
      • Toth E.A.
      • Weber D.J.
      Covalent small molecule inhibitors of Ca2+-bound S100B.
      ). Selective targeting of these sites is already a promising opportunity for the treatment of malignant melanoma (
      • Hartman K.G.
      • McKnight L.E.
      • Liriano M.A.
      • Weber D.J.
      The evolution of S100B inhibitors for the treatment of malignant melanoma.
      ). 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 thumbnail gr11
      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 S100B partner protein surfaces (
      • Cavalier M.C.
      • Pierce A.D.
      • Wilder P.T.
      • Alasady M.J.
      • Hartman K.G.
      • Neau D.B.
      • Foley T.L.
      • Jadhav A.
      • Maloney D.J.
      • Simeonov A.
      • Toth E.A.
      • Weber D.J.
      Covalent small molecule inhibitors of Ca2+-bound S100B.
      ). However, site 3 is unoccupied in all S100B-RSK1 structures, whereas the novel site 4 is occupied in crystal structure C; therefore, site 4 may be uniquely used in the “S100B-inhibited” complex of RSK1. B, side view of the fuzzy and the S100B inhibited complex. In the fuzzy complex, site 4 remains free. Targeting this binding interface with small molecule inhibitors may tentatively interfere with only the S100B-inhibited complex but leaves fuzzy complex formation intact.

      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.

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