title: DNA binding by the ETS protein TEL (ETV6) is regulated by autoinhibition and self-association.
abstract: The ETS protein TEL, a transcriptional repressor, contains a PNT domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT domain and DNA-binding ETS domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.
authors.affiliation: Department of Oncological Sciences, University of Utah School of Medicine, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112-5550, USA.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Green
authors.forename: H Jerome
authors.initials: HJ
authors.lastname: Coyne
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Graves
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D050783:Proto-Oncogene Proteins c-ets / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D014158:Transcription, Genetic; D014357:Trypsin / Q000737:chemistry; D000097764:ETS Translocation Variant 6 Protein
chemical_list: D000409:Alanine; D004247:DNA; D018698:Glutamic Acid; D011108:Polymers; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D014357:Trypsin; D000097764:ETS Translocation Variant 6 Protein
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