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

Gógl, Gergő; Alexa, Anita; Kiss, Bence; Katona, Gergely; Kovács, Mihály; Bodor, Andrea; Reményi, Attila; Nyitray, László

doi:10.1074/jbc.m115.684928

Cited by 48 publications

(59 citation statements)

References 40 publications

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“…Since both the fluorescent labeling at position 516 and truncation at position 560 affected the binding affinity of the C-ERMAD, we can conclude that an extended region of the C-ERMAD binds to S100A4 likely forming an asymmetric complex, similar to the N-ERMAD and to other recently characterized S100 protein-protein interactions [18, 41, 42]. Finally, we carried out binding experiments with Fl-C-ERMAD T567D variant to evaluate the potential effect of Thr567 phosphorylation on S100A4 binding.…”

Section: Resultsmentioning

confidence: 71%

Ezrin interacts with S100A4 via both its N- and C-terminal domains

et al. 2017

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Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process.

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Section: Resultsmentioning

confidence: 71%

Ezrin interacts with S100A4 via both its N- and C-terminal domains

et al. 2017

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“…Usually, the tested S100 proteins only covered the closest relatives (e.g. S100A2, S100A4, S100A6, S100B, S100P), and the results often showed redundant bindings [19,27,[36][37][38]. Based on functional clustering, we have revealed here that the S100ome can be separated into two groups.…”

Section: Competitive Fp As a Potent Tool To Measure High-throughput Mmentioning

confidence: 77%

“…Earlier studies showed that a symmetric S100 dimer can recognize two identical binding motifs, symmetrically [17,25,26]. In recent studies however, several asymmetric complexes were also described [18,27,28]. In those cases, an S100 dimer captures a single straddling the two binding sites.…”

Section: Validation With Itc Measurementsmentioning

confidence: 99%

“…Peptide synthesis. The CapZ (265-276), NCX1 (254-265), SIP (188-202), TRPM4 (129-147) and MDM4(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) peptides were chemically synthesized using solid phase peptide synthesis (PS3 peptide synthesizer, Protein Technologies) with Fmoc/tBu strategy in the case of (5(6)-carboxyfluorescein) labeled and unlabeled version. Peptides were purified by RP-HPLC using a Jupiter 300 Å C 18 column (Phenomenex).…”

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confidence: 99%

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High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

Simon

Gógl

Ecsédi

et al. 2019

Preprint

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The calcium-binding, vertebrate-specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To assess their interactome, high-throughput, systematic analysis is indispensable, which allows one to get not only qualitative but quantitative insight into their protein-protein interactions (PPIs). We have chosen an unbiased assay, fluorescence polarization (FP) that revealed a partial functional redundancy when the complete S100ome (n=20) was tested against numerous model partners (n=13). Based on their specificity, the S100ome can be grouped into two distinct classes: promiscuous and orphan. In the first group, members bound to several ligands (>4-5) with comparable high affinity, while in the second one, the paralogs bound only one partner weakly, or no ligand was identified (orphan). Our results demonstrate that in vitro FP assays are highly suitable for quantitative ligand binding studies of selected protein families. Moreover, we provide evidence that PPI-based phenotypic characterization can complement the information obtained from the sequence-based phylogenetic analysis of the S100ome, an evolutionary young protein family. Author summaryFunctional similarity among a protein family can be essential in order to understand proteomic data, to find biomarkers, or in inhibitor design. Proteins with similar functions can compensate the loss-offunction of the others, their expression can co-vary under pathological conditions, and simultaneous targeting can lead to better results in the clinics. To investigate this property one can use sequencebased approaches. However, this path can be difficult. In the case of the vertebrate specific, evolutionary young, S100 family, phylogenetic approaches lead to ambiguous results. To overcome this problem, we applied a high-throughput biochemical approach to experimentally measure the binding affinities of a large number of S100 interactions. We performed unbiased fluorescence polarization assay, involving the complete human S100ome (20 paralogs) and 13 known interaction partners. We used this measured 20x13 (260) protein-protein interaction array to reveal the functional relationships within the family. Our work provide a general framework for studies focusing on phenotype-based domain classification.

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“…A well‐conserved series of hydrophobic residues at the dimer interface contributes to a strong noncovalent interaction between the S100 protomers, resulting in dissociation (dimerization) constants in the low to sub‐micromolar affinity range . Thus, nearly all homodimeric S100 proteins possess a pair of symmetrical target binding sites that recognize two copies of target protein , although some examples of asymmetric binding have been reported . Despite the very high sequence similarities among S100 family members , only a few examples of heterodimeric S100 proteins have been explored.…”

Section: Introductionmentioning

confidence: 99%

A subset of calcium‐binding S100 proteins show preferential heterodimerization

et al. 2019

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The assembly of proteins into dimers and oligomers is a necessary step for the proper function of transcription factors, muscle proteins, and proteases. In uncontrolled states, oligomerization can also contribute to illnesses such as Alzheimer's disease. The S100 protein family is a group of dimeric proteins that have important roles in enzyme regulation, cell membrane repair, and cell growth. Most S100 proteins have been examined in their homodimeric state, yet some of these important proteins are found in similar tissues implying that heterodimeric molecules can also be formed from the combination of two different S100 members. In this work, we have established co‐expression methods in order to identify and quantify the distribution of homo‐ and heterodimers for four specific pairs of S100 proteins in their calcium‐free states. The split GFP trap methodology was used in combination with other GFP variants to simultaneously quantify homo‐ and heterodimeric S100 proteins in vitro and in living cells. For the specific S100 proteins examined, NMR, mass spectrometry, and GFP trap experiments consistently show that S100A1:S100B, S100A1:S100P, and S100A11:S100B heterodimers are the predominant species formed compared to their corresponding homodimers. We expect the tools developed here will help establish the roles of S100 heterodimeric proteins and identify how heterodimerization might alter the specificity for S100 protein action in cells.

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

Cited by 48 publications

References 40 publications

Ezrin interacts with S100A4 via both its N- and C-terminal domains

Ezrin interacts with S100A4 via both its N- and C-terminal domains

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

A subset of calcium‐binding S100 proteins show preferential heterodimerization

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