Beads-on-a-string, characterization of Ets-1 sumoylated within its flexible N-terminal sequence

Matthew S. Macauley, Wesley J. Errington, Manuela Schårpf, Cameron D. Mackereth, Adam G. Blaszczak, Barbara J. Graves, Lawrence P. McIntosh

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31 Scopus citations

Abstract

Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide amolecular 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 (Kd ∼400 μM) 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.

Original languageEnglish (US)
Pages (from-to)4164-4172
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number7
DOIs
StatePublished - Feb 17 2006

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Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

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