title: Beads-on-a-string, characterization of ETS-1 sumoylated within its flexible N-terminal sequence.
abstract: Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide a molecular framework for understanding this regulation, we have characterized the conjugation of Ets-1 with SUMO-1. Ets-1 is modified in vivo predominantly at a consensus sumoylation motif containing Lys-15. This lysine is located within the unstructured N-terminal segment of Ets-1 preceding its PNT domain. Using NMR spectroscopy, we demonstrate that the Ets-1 sumoylation motif associates with the substrate binding site on the SUMO-conjugating enzyme UBC9 (K(d) approximately 400 microm) and that the PNT domain is not involved in this interaction. Ets-1 with Lys-15 mutated to an arginine still binds UBC9 with an affinity similar to the wild type protein, but is no longer sumoylated. NMR chemical shift and relaxation measurements reveal that the covalent attachment of mature SUMO-1, via its flexible C-terminal Gly-97, to Lys-15 of Ets-1 does not perturb the structure or dynamic properties of either protein. Therefore sumoylated Ets-1 behaves as "beads-on-a-string" with the two proteins tethered by flexible polypeptide segments containing the isopeptide linkage. Accordingly, SUMO-1 may mediate interactions of Ets-1 with signaling or transcriptional regulatory macromolecules by acting as a structurally independent docking module, rather than through the induction of a conformational change in either protein upon their covalent linkage. We also hypothesize that the flexibility of the linking polypeptide sequence may be a general feature contributing to the recognition of SUMO-modified proteins by their downstream effectors.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Macauley
authors.forename: Wesley J
authors.initials: WJ
authors.lastname: Errington
authors.forename: Manuela
authors.initials: M
authors.lastname: Schärpf
authors.forename: Cameron D
authors.initials: CD
authors.lastname: Mackereth
authors.forename: Adam G
authors.initials: AG
authors.lastname: Blaszczak
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Graves
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D041681:NIH 3T3 Cells; D050784:Proto-Oncogene Protein c-ets-1 / Q000737:chemistry* / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: C494042:Ets1 protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D025842:SUMO-1 Protein; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9