Michael Sattler

Helmholtz Zentrum München, München, Bavaria, Germany

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Publications (188)1428.05 Total impact

  • Journal of Biomolecular NMR 08/2015; DOI:10.1007/s10858-015-9981-0 · 3.31 Impact Factor
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    ABSTRACT: IPSE/alpha-1, the major secretory product of eggs from the parasitic worm Schistosoma mansoni, efficiently triggers basophils to release the immunomodulatory key cytokine interleukin-4. Activation by IPSE/alpha-1 requires the presence of IgE on the basophils, but the detailed molecular mechanism underlying activation is unknown. NMR and crystallographic analysis of IPSEδNLS, a monomeric IPSE/alpha-1 mutant, revealed that IPSE/alpha-1 is a new member of the β&]gamma]-crystallin superfamily. We demonstrate that this molecule is a general immunoglobulin-binding factor with highest affinity for IgE. NMR binding studies of IPSEδNLS with the 180-kDa molecule IgE identified a large positively charged binding surface that includes a flexible loop, which is unique to the IPSE/alpha-1 crystallin fold. Mutational analysis of amino acids in the binding interface showed that residues contributing to IgE binding are important for IgE-dependent activation of basophils. As IPSE/alpha-1 is unable to cross-link IgE, we propose that this molecule, by taking advantage of its unique IgE-binding crystallin fold, activates basophils by a novel, cross-linking-independent mechanism. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 07/2015; DOI:10.1074/jbc.M115.675066 · 4.57 Impact Factor
  • Janosch Hennig · Michael Sattler
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    ABSTRACT: RNA binding proteins (RBPs) are key factors for the regulation of gene expression by binding to cis elements, i.e. short sequence motifs in RNAs. Recent studies demonstrate that cooperative binding of multiple RBPs is important for the sequence-specific recognition of RNA and thereby enables the regulation of diverse biological activities by a limited set of RBPs. Cross-linking immuno-precipitation (CLIP) and other recently developed high-throughput methods provide comprehensive, genome-wide maps of protein-RNA interactions in the cell. Structural biology gives detailed insights into molecular mechanisms and principles of RNA recognition by RBPs, but has so far focused on single RNA binding proteins and often on single RNA binding domains. The combination of high-throughput methods and detailed structural biology studies is expected to greatly advance our understanding of the code for protein-RNA recognition in gene regulation, as we review in this article. © 2015 WILEY Periodicals, Inc.
    BioEssays 06/2015; DOI:10.1002/bies.201500033 · 4.84 Impact Factor
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    ABSTRACT: The pH-responsive one component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity, α-helix-forming propensity), is robust to extension, but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH<6.8. Strikingly, a variant with reversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement which facilitates dimerization of the cytoplasmic CadC DNA-binding domain. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular Biology 05/2015; DOI:10.1016/j.jmb.2015.05.001 · 4.33 Impact Factor
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    ABSTRACT: Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.
    PLoS Pathogens 05/2015; 11(5):e1004910. DOI:10.1371/journal.ppat.1004910 · 8.06 Impact Factor
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    ABSTRACT: The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 04/2015; 43(10). DOI:10.1093/nar/gkv365 · 9.11 Impact Factor
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    ABSTRACT: Kinase inhibition is considered to be an important therapeutic target for LRRK2 mediated Parkinson's disease (PD). Many LRRK2 kinase inhibitors have been reported, but have yet to be optimized in order to qualify as drug candidates for the treatment of the disease. In order to start a structure-function analysis of such inhibitors we mutated the active site of Dictyostelium Roco4 kinase to resemble LRRK2. Here, we show Saturation Transfer Difference (STD) NMR and the first co-crystal structures of two potent in vitro inhibitors, LRRK2-IN-1 and Compound19, with mutated Roco4. Our data demonstrate that this system can serve as excellent tool for the structural characterization and optimization of LRRK2 inhibitors using X-ray crystallography and NMR spectroscopy.
    Journal of Medicinal Chemistry 04/2015; 58(9). DOI:10.1021/jm5018779 · 5.48 Impact Factor
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    ABSTRACT: Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds. © 2015 Authors.
    Bioscience Reports 04/2015; 35(3). DOI:10.1042/BSR20150042 · 2.85 Impact Factor
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    Michael Sattler
    RNA 04/2015; 21(4):727-8. DOI:10.1261/rna.050971.115 · 4.62 Impact Factor
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    ABSTRACT: Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30kDa as data are noisy and sparse. Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20Å) for structural analysis of large complexes and also report on conformational dynamics in solution. Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes. © 2015 Elsevier Inc. All rights reserved.
    Structures of Large RNA Molecules and Their Complexes, 558 edited by Sarah A. Woodson and Frédéric H.T. Allain, 03/2015: chapter 11: pages 333-362; Elsevier., ISBN: 978-0-12-801934-4
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    ABSTRACT: One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.
    Nature Medicine 02/2015; DOI:10.1038/nm.3776 · 28.05 Impact Factor
  • Janosch Hennig · Fatima Gebauer · Michael Sattler
    Cell cycle (Georgetown, Tex.) 12/2014; 13(23). DOI:10.4161/15384101.2014.986625 · 5.01 Impact Factor
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    ABSTRACT: Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that-amongst the 10 Tyr residues present in MSD1-Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.
    Journal of Experimental Botany 11/2014; 66(3). DOI:10.1093/jxb/eru458 · 5.79 Impact Factor
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    Dataset: BUSS
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    ABSTRACT: Der Reaktionszyklus des molekularen Chaperons Hsp90 ist durch ATP-getriebene Konformationsänderungen charakterisiert, die der ATP-Hydrolyse vorgeschaltet sind. Es gibt bereits niedermolekulare Inhibitoren von Hsp90, die mit der ATP-Bindung konkurrieren. Wir fragten uns, ob Verbindungen existieren, die den konformationellen Zyklus beschleunigen können. In einem FRET-basierten Screen, der Konformationsänderungen in Hsp90 detektiert, identifizierten wir zwei Verbindungen und analysierten ihre Wirkungsweise. Wir können zeigen, dass die intrinsische Inhibition von Hsp90, welche konformationelle Umlagerungen verhindert, durch diese Substanzen überwunden wird. Diese Wirkungsweise ähnelt der des Cochaperons Aha1 von Hsp90, das die ATPase-Aktivität beschleunigt. Während beide Verbindungen Konformationsänderungen beeinflussen, so steuern sie jeweils verschiedene Aspekte der Strukturübergänge. Zudem unterscheiden sich ihre durch NMR-Spektroskopie bestimmten Bindestellen. Unsere Studie zeigt, dass kleine Moleküle in der Lage sind, spezifische geschwindigkeitsbestimmende Übergänge in Hsp90 in ähnlicher Weise zu beschleunigen wie dies durch Protein-Cofaktoren geschieht.
    Angewandte Chemie 11/2014; 126(45). DOI:10.1002/ange.201406578
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    ABSTRACT: The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET-based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co-chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate-limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors.
    Angewandte Chemie International Edition 11/2014; 53(45). DOI:10.1002/anie.201406578 · 11.26 Impact Factor
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    ABSTRACT: Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex1, 2. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3′ untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit3. Here we report the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl–Unr–msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. Our results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression.
    Nature 09/2014; 515(7526). DOI:10.1038/nature13693 · 42.35 Impact Factor
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    ABSTRACT: The REtention and Splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and to promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total eighteen residues of Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp232 in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that the Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RNA recognition motif (RRM)-protein interactions, analogous to the one seen in for nucleic acid interactions.
    Journal of Biological Chemistry 08/2014; 289(41). DOI:10.1074/jbc.M114.592311 · 4.57 Impact Factor

Publication Stats

8k Citations
1,428.05 Total Impact Points

Institutions

  • 2008–2015
    • Helmholtz Zentrum München
      • Institute of Structural Biology
      München, Bavaria, Germany
  • 2007–2014
    • Technische Universität München
      • Faculty of Chemistry
      München, Bavaria, Germany
  • 1999–2008
    • European Molecular Biology Laboratory
      • Structural and Computational Biology Unit (Heidelberg)
      Heidelburg, Baden-Württemberg, Germany
  • 2004–2006
    • Institute of Molecular Biology
      Mayence, Rheinland-Pfalz, Germany
  • 1997
    • Goethe-Universität Frankfurt am Main
      • Institut für Organische Chemie und Chemische Biologie
      Frankfurt am Main, Hesse, Germany
  • 1996
    • Abbott Laboratories
      North Chicago, Illinois, United States
  • 1994–1996
    • University Hospital Frankfurt
      Frankfurt, Hesse, Germany