Michael Sattler

Helmholtz Zentrum München, München, Bavaria, Germany

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Publications (195)1450.15 Total impact

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    ABSTRACT: Canonical membrane protein biogenesis requires co-translational delivery of ribosome-associated proteins to the Sec translocase and depends on the signal recognition particle (SRP) and its receptor (SR). In contrast, high-throughput delivery of abundant light-harvesting chlorophyll a,b-binding proteins (LHCPs) in chloroplasts to the Alb3 insertase occurs post-translationally via a soluble transit complex including the cpSRP43/cpSRP54 heterodimer (cpSRP). Here we describe the molecular mechanisms of tethering cpSRP to the Alb3 insertase by specific interaction of cpSRP43 chromodomain 3 with a linear motif in the Alb3 C-terminal tail. Combining NMR spectroscopy, X-ray crystallography and biochemical analyses, we dissect the structural basis for selectivity of chromodomains 2 and 3 for their respective ligands cpSRP54 and Alb3, respectively. Negative cooperativity in ligand binding can be explained by dynamics in the chromodomain interface. Our study provides a model for membrane recruitment of the transit complex and may serve as a prototype for a functional gain by the tandem arrangement of chromodomains.
    Nature Communications 11/2015; 6:8875. DOI:10.1038/ncomms9875 · 11.47 Impact Factor
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    ABSTRACT: Protein kinases are the most prominent group of Hsp90 (heat shock protein 90) clients and are recruited to the molecular chaperone by the kinase specific cochaperone Cdc37 (cell division cycle 37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of C. elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the MAP-kinase (mitogen activated protein kinase) Erk2 (extracellular-signal regulated kinase 2) we observe that certain features of the interaction with Cdc37-Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.
    Journal of Biological Chemistry 10/2015; DOI:10.1074/jbc.M115.693150 · 4.57 Impact Factor
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    ABSTRACT: The peroxisomal proteins (peroxins) that mediate the import of peroxisomal matrix proteins have been identified. Recently, the purification of a functional peroxisomal translocon has been reported. However, the molecular details of the import pathways and the mechanisms by which the cargo is translocated into the lumen of the organelle are still poorly understood. Structural studies have begun to provide insight into molecular mechanisms of peroxisomal import pathways for cargo proteins that harbor peroxisomal targeting signals, PTS1 and PTS2, at their C- and N-termini, respectively. So far structures have been reported for binary or tertiary protein-protein interfaces, and highlight the role of intrinsically disordered regions for these interactions. Here, we provide an overview of the currently available structural biology of peroxisomal import pathways. Current challenges and future perspectives of the structural biology of peroxisomal protein translocation are discussed.
    Biochimica et Biophysica Acta 10/2015; DOI:10.1016/j.bbamcr.2015.09.034 · 4.66 Impact Factor
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    ABSTRACT: The correct topogenesis of peroxisomal membrane proteins is a crucial step for the formation of functioning peroxisomes. Although this process has been widely studied, the exact mechanism with which it occurs has not yet been fully characterized. Nevertheless, it is generally accepted that peroxisomes employ three proteins - Pex3, Pex19 and Pex16 in mammals - for the insertion of peroxisomal membrane proteins into the peroxisomal membrane. Structural biology approaches have been utilized for the elucidation of the mechanistic questions of peroxisome biogenesis, mainly by providing information on the architecture of the proteins significant for this process. This review aims to summarize, compare and put into perspective the structural knowledge that has been generated mainly for Pex3 and Pex19 and their interaction partners in recent years.
    Biochimica et Biophysica Acta 10/2015; DOI:10.1016/j.bbamcr.2015.09.031 · 4.66 Impact Factor
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    Lee Freiburger · Miriam Sonntag · Janosch Hennig · Jian Li · Peijian Zou · Michael Sattler ·
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    ABSTRACT: NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.
    Journal of Biomolecular NMR 08/2015; 63(1). DOI:10.1007/s10858-015-9981-0 · 3.14 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; 290(36). 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; 37(8). DOI:10.1002/bies.201500033 · 4.73 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; 427(15). 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 · 7.56 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 · 5.45 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 · 2.64 Impact Factor
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    Michael Sattler ·

    RNA 04/2015; 21(4):727-8. DOI:10.1261/rna.050971.115 · 4.94 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; 21(3). DOI:10.1038/nm.3776 · 27.36 Impact Factor
  • Andre Dallmann · Michael Sattler ·
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    ABSTRACT: NMR spectroscopy is a powerful tool to study the structure and dynamics of nucleic acids. In this unit, we give an overview of important experiments to determine and characterize hydrogen bonds in nucleic acids and provide detailed instructions for setting up recently developed sensitivity-improved NMR pulse sequences, i.e., BEST selective long-range HNN-COSY, selective BEST-TROSY-HNNCOSY, and Py H(CC)NN-COSY. The strengths and limitations of these experiments will also be discussed. Detailed step-by-step protocols are provided for each of the three pulse sequences, with special emphasis on adjusting and setting of delays and shaped pulses. The NMR pulse sequences with example datasets and optimized, nonstandard adiabatic pulse shapes used for selective (15) N magnetization transfer are provided. These experiments enable NMR analysis of a broad variety of RNAs ranging from low to high molecular weight and complexity. © 2014 by John Wiley & Sons, Inc. Copyright © 2014 John Wiley & Sons, Inc.
    Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] 12/2014; 59:7.22.1-7.22.19. DOI:10.1002/0471142700.nc0722s59
  • Janosch Hennig · Fatima Gebauer · Michael Sattler ·

    Cell cycle (Georgetown, Tex.) 12/2014; 13(23). DOI:10.4161/15384101.2014.986625 · 4.57 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 · 5.53 Impact Factor
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    Dataset: BUSS

Publication Stats

9k Citations
1,450.15 Total Impact Points


  • 2008-2015
    • Helmholtz Zentrum München
      • Institute of Structural Biology
      München, Bavaria, Germany
  • 2007-2015
    • Technische Universität München
      • Faculty of Chemistry
      München, Bavaria, Germany
  • 1999-2011
    • 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
    • University of Wisconsin–Madison
      Madison, Wisconsin, United States
    • Abbott Laboratories
      North Chicago, Illinois, United States
  • 1994-1996
    • University Hospital Frankfurt
      Frankfurt, Hesse, Germany