title: Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix.
abstract: The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge-dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Soumya
authors.initials: S
authors.lastname: De
authors.affiliation: Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Okon
authors.affiliation: Department of Oncological Sciences, School of Medicine, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112-5550, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815-6789, USA.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Graves
authors.affiliation: Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address: mcintosh@chem.ubc.ca.
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
mesh_terms: D000577:Amides / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D004247:DNA / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050783:Proto-Oncogene Proteins c-ets / Q000737:chemistry* / Q000502:physiology*; D012097:Repressor Proteins / Q000737:chemistry* / Q000502:physiology*; D013816:Thermodynamics; D000097764:ETS Translocation Variant 6 Protein
chemical_list: D000577:Amides; D000596:Amino Acids; D004247:DNA; D004220:Disulfides; D006859:Hydrogen; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D000097764:ETS Translocation Variant 6 Protein
keywords: ETS family; NMR spectroscopy; charge–dipole interaction; hydrogen exchange; protein folding