Steric mechanism of auto-inhibitory regulation of specific and non-specific DNA binding by the ETS transcriptional repressor ETV6.

DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus (5')GGAA(3') recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing (5')GGAA(3')versus(5')GGAT(3'). These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.

[1]  Albert Jeltsch,et al.  Transition from Nonspecific to Specific DNA Interactions along the Substrate-Recognition Pathway of Dam Methyltransferase , 2005, Cell.

[2]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[3]  G. Poon,et al.  Base coupling in sequence-specific site recognition by the ETS domain of murine PU.1. , 2003, Journal of molecular biology.

[4]  R. Kaptein,et al.  Toward an Integrated Model of Protein‐DNA Recognition as Inferred from NMR Studies on the Lac Repressor System , 2004 .

[5]  L. Kay,et al.  An NMR Experiment for Measuring Methyl−Methyl NOEs in 13C-Labeled Proteins with High Resolution , 1998 .

[6]  J. Chaires.  Analysis and interpretation of ligand-DNA binding isotherms. , 2001, Methods in enzymology.

[7]  A. Szabó,et al.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity , 1982 .

[8]  K. Gaston,et al.  Protein flexibility directs DNA recognition by the papillomavirus E2 proteins , 2010, Nucleic acids research.

[9]  J. Mackay,et al.  New Insights into DNA Recognition by Zinc Fingers Revealed by Structural Analysis of the Oncoprotein ZNF217* , 2013, The Journal of Biological Chemistry.

[10]  Christian Griesinger,et al.  Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients , 1999 .

[11]  T. Pawson,et al.  Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. , 1994, Biochemistry.

[12]  Huan‐Xiang Zhou.  Rapid search for specific sites on DNA through conformational switch of nonspecifically bound proteins , 2011, Proceedings of the National Academy of Sciences.

[13]  C. Wolberger,et al.  Insights into nonspecific binding of homeodomains from a structure of MATα2 bound to DNA , 2003, Proteins.

[14]  G. Marius Clore,et al.  Detecting transient intermediates in macromolecular binding by paramagnetic NMR , 2006, Nature.

[15]  P. Güntert.  Automated NMR structure calculation with CYANA. , 2004, Methods in molecular biology.

[16]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[17]  G. Lipari.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules , 1982 .

[18]  H. Viadiu,et al.  Structure of BamHI bound to nonspecific DNA: a model for DNA sliding. , 2000, Molecular cell.

[19]  Shuang-yong Xu,et al.  BstYI bound to noncognate DNA reveals a "hemispecific" complex: implications for DNA scanning. , 2007, Structure.

[20]  John H. Bushweller,et al.  Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited , 2013, Proceedings of the National Academy of Sciences.

[21]  Sequence Discrimination by DNA-binding Domain of ETS Family Transcription Factor PU.1 Is Linked to Specific Hydration of Protein-DNA Interface* , 2012, The Journal of Biological Chemistry.

[22]  P. Sigler,et al.  The basis for half-site specificity explored through a non-cognate steroid receptor-DNA complex , 1995, Nature Structural Biology.

[23]  Gregory M Lee,et al.  The affinity of Ets-1 for DNA is modulated by phosphorylation through transient interactions of an unstructured region. , 2008, Journal of molecular biology.

[24]  Ron D. Appel,et al.  ExPASy: the proteomics server for in-depth protein knowledge and analysis , 2003, Nucleic Acids Res..

[25]  L. Kay,et al.  Variable Control of Ets-1 DNA Binding by Multiple Phosphates in an Unstructured Region , 2005, Science.

[26]  Peter C. Hollenhorst,et al.  Genomic and biochemical insights into the specificity of ETS transcription factors. , 2011, Annual review of biochemistry.

[27]  Mark Okon,et al.  Autoinhibition of ETV6 (TEL) DNA binding: appended helices sterically block the ETS domain. , 2012, Journal of molecular biology.

[28]  G. Clore,et al.  Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR , 2011, Proceedings of the National Academy of Sciences.

[29]  K. Mayo,et al.  Motional Model Analyses of Protein and Peptide Dynamics Using 13C and 15N NMR Relaxation , 1997 .

[30]  K. Yamamoto,et al.  Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA , 2003, Nature.

[31]  Andrew R. Gehrke,et al.  Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo , 2010, The EMBO journal.

[32]  Rajeevan Selvaratnam,et al.  The Auto-Inhibitory Role of the EPAC Hinge Helix as Mapped by NMR , 2012, PloS one.

[33]  Diego U Ferreiro,et al.  Experimental snapshots of a protein-DNA binding landscape , 2010, Proceedings of the National Academy of Sciences.

[34]  Y. Levy,et al.  Asymmetrical roles of zinc fingers in dynamic DNA-scanning process by the inducible transcription factor Egr-1 , 2012, Proceedings of the National Academy of Sciences.

[35]  T. Alber,et al.  Modulation of transcription factor Ets-1 DNA binding: DNA-induced unfolding of an alpha helix. , 1995, Science.

[36]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[37]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[38]  Monika Fuxreiter,et al.  Fuzziness: linking regulation to protein dynamics. , 2012, Molecular bioSystems.

[39]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[40]  D. Rau,et al.  Differences in water release for the binding of EcoRI to specific and nonspecific DNA sequences. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[41]  M. Pufall,et al.  Autoinhibitory domains: modular effectors of cellular regulation. , 2002, Annual review of cell and developmental biology.

[42]  C. Arrowsmith,et al.  DNA Binding Specificity Studies of Four ETS Proteins Support an Indirect Read-out Mechanism of Protein-DNA Recognition* 210 , 2000, The Journal of Biological Chemistry.

[43]  A. Grishin,et al.  Conserved structural features of ETS domain-DNA complexes , 2009, Molecular Biology.

[44]  A. Szabó,et al.  Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 2. Analysis of experimental results , 1982 .

[45]  M. Record,et al.  Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer. , 2001, Biophysical journal.

[46]  R. Bryan,et al.  The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. , 1993 .

[47]  A. Bax,et al.  TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts , 2009, Journal of biomolecular NMR.

[48]  X. Xie,et al.  Nonspecifically bound proteins spin while diffusing along DNA , 2009, Nature Structural &Molecular Biology.

[49]  T. Gu,et al.  Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2 , 2000, Molecular and Cellular Biology.

[50]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[51]  K S Wilson,et al.  The crystal structure of EcoRV endonuclease and of its complexes with cognate and non‐cognate DNA fragments. , 1993, The EMBO journal.

[52]  S. Bohlander.  ETV6: a versatile player in leukemogenesis. , 2005, Seminars in cancer biology.

[53]  R. Kaptein,et al.  Structure and Flexibility Adaptation in Nonspecific and Specific Protein-DNA Complexes , 2004, Science.

[54]  I. Rebay,et al.  Sterile Alpha Motif Domain-Mediated Self-Association Plays an Essential Role in Modulating the Activity of the Drosophila ETS Family Transcriptional Repressor Yan , 2010, Molecular and Cellular Biology.

[55]  C. Garvie,et al.  Structural Analysis of the Autoinhibition of Ets-1 and Its Role in Protein Partnerships* , 2002, The Journal of Biological Chemistry.

[56]  Yaakov Levy,et al.  Frustration in protein–DNA binding influences conformational switching and target search kinetics , 2011, Proceedings of the National Academy of Sciences.

[57]  R. Marmorstein,et al.  Structures of SAP-1 bound to DNA targets from the E74 and c-fos promoters: insights into DNA sequence discrimination by Ets proteins. , 1998, Molecular cell.

[58]  R. Lutz,et al.  Highly cooperative recruitment of Ets-1 and release of autoinhibition by Pax5. , 2009, Journal of molecular biology.

[59]  H. Rüterjans,et al.  Nuclear magnetic resonance investigation of 15N-labeled histidine in aqueous solution. , 1977, Journal of the American Chemical Society.

[60]  B. Matthews,et al.  Crystal structure of an engineered cro monomer bound nonspecifically to DNA: Possible implications for nonspecific binding by the wild‐type protein , 1998, Protein science : a publication of the Protein Society.

[61]  G Marius Clore,et al.  NMR structural and kinetic characterization of a homeodomain diffusing and hopping on nonspecific DNA , 2006, Proceedings of the National Academy of Sciences.

[62]  Yangzhou Wang,et al.  Analysis of the 2.0 A crystal structure of the protein-DNA complex of the human PDEF Ets domain bound to the prostate specific antigen regulatory site. , 2005, Biochemistry.

[63]  K. Loth,et al.  Sliding and target location of DNA-binding proteins:an NMR view of the lac repressor system , 2013, Journal of biomolecular NMR.

[64]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[65]  Yaakov Levy,et al.  Protein sliding along DNA: dynamics and structural characterization. , 2009, Journal of molecular biology.

[66]  James U Bowie,et al.  Derepression by Depolymerization Structural Insights into the Regulation of Yan by Mae , 2004, Cell.

[67]  M. Kovács,et al.  From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins. , 2013, Trends in biochemical sciences.

[68]  L. Kay,et al.  Simultaneous Acquisition of 15N- and 13C-Edited NOE Spectra of Proteins Dissolved in H2O , 1994 .

[69]  R. Kodandapani,et al.  A new pattern for helix–turn–helix recognition revealed by the PU.1 ETS-domain–DNA complex , 1998, Nature.

[70]  J. Hus,et al.  Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data , 2000, Journal of biomolecular NMR.

[71]  P. V. von Hippel,et al.  Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. , 1974, Journal of molecular biology.

[72]  David S Wishart,et al.  A simple method to predict protein flexibility using secondary chemical shifts. , 2005, Journal of the American Chemical Society.

[73]  G. Clore,et al.  Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement. , 2004, Journal of the American Chemical Society.

[74]  J U Bowie,et al.  Polymerization of the SAM domain of TEL in leukemogenesis and transcriptional repression , 2001, The EMBO journal.

[75]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[76]  Barbara J. Graves,et al.  The Structural and Dynamic Basis of Ets-1 DNA Binding Autoinhibition* , 2005, Journal of Biological Chemistry.

[77]  Academician D. V. Skobel’tsyn.  Analysis of Experimental Results , 1969 .

[78]  R. Mann,et al.  Origins of specificity in protein-DNA recognition. , 2010, Annual review of biochemistry.

[79]  S. Tzeng,et al.  Allosteric inhibition through suppression of transient conformational states. , 2013, Nature chemical biology.

[80]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[81]  Yang Shen,et al.  Identification of helix capping and β-turn motifs from NMR chemical shifts , 2012, Journal of biomolecular NMR.

[82]  L. Kay,et al.  Improved 1HN-detected triple resonance TROSY-based experiments , 1999, Journal of biomolecular NMR.

[83]  L. McIntosh,et al.  DNA Binding by the ETS Protein TEL (ETV6) Is Regulated by Autoinhibition and Self-association* , 2010, The Journal of Biological Chemistry.

[84]  P. V. von Hippel,et al.  Facilitated Target Location in Biological Systems* , 2022 .