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 (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. 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 (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. The regulation of gene expression requires the spatial and temporal orchestration of a complex network of protein-protein and protein-DNA interactions. Central to the control of these interactions are a myriad of post-translational modifications. One such modification involves the reversible, covalent attachment of the ubiquitin-like protein SUMO. 4The abbreviations used are: SUMO, small ubiquitin-like modifier; E1, ubiquitin-like protein-activating enzyme; E2, ubiquitin-like protein-conjugating enzyme; E3, ubiquitin-like protein-isopeptide ligase; GST, glutathione S-transferase; HA, hemagglutinin; HSQC, heteronuclear single quantum correlation; NOE, nuclear Overhauser effect; SUMO-1gg, residues 1–97 of human SUMO-1 plus an N-terminal Gly-Ser-His tripeptide; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy. In addition to serving as an antagonist against ubiquitin-mediated degradation, sumoylation affects a growing number of recognized biological processes, including nuclear transport, signal transduction, transcription, and DNA repair (recent reviews Refs. 1.Muller S. Ledl A. Schmidt D. Oncogene. 2004; 23: 1998-2008Crossref PubMed Scopus (247) Google Scholar, 2.Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 3.Dohmen R.J. Biochim. Biophys. Acta. 2004; 1695: 113-131Crossref PubMed Scopus (210) Google Scholar, 4.Hilgarth R.S. Murphy L.A. Skaggs H.S. Wilkerson D.C. Xing H.Y. Sarge K.D. J. Biol. Chem. 2004; 279: 53899-53902Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 5.Hay R.T. Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). However, despite extensive studies, the molecular mechanisms for this regulation, which likely arise through SUMO-dependent modulation of target protein interactions with other macromolecules, remain largely undefined (2.Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 6.Reverter D. Lima C.D. Nature. 2005; 435: 687-692Crossref PubMed Scopus (383) Google Scholar, 7.Baba D. Maita N. Jee J.G. Uchimura Y. Saitoh H. Sugasawa K. Hanaoka F. Tochio H. Hiroaki H. Shirakawa M. Nature. 2005; 435: 979-982Crossref PubMed Scopus (187) Google Scholar, 8.Pichler A. Knipscheer P. Oberhofer E. van Dijk W.J. Korner R. Olsen J.V. Jentsch S. Melchior F. Sixma T.K. Nature Struct. Mol. Biol. 2005; 12: 264-269Crossref PubMed Scopus (166) Google Scholar). In contrast, the pathways for the sumoylation and desumoylation of target proteins are well established. After proteolytic maturation and ATP-dependent activation, SUMO family members are transferred from the heterodimeric E1-activating enzyme to Cys-93 in the single E2-conjugating enzyme, UBC9. Although E3-ligating enzymes, which facilitate the specificity and efficiency of sumoylation, have been identified, UBC9 is sufficient for conjugating SUMO to many proteins, at least in vitro (9.Gocke C.B. Yu H. Kang J. J. Biol. Chem. 2005; 280: 5004-5012Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The final product is an isopeptide bond joining the C-terminal glycine carboxyl of SUMO with the side chain amino group of a lysine in the target protein. This lysine is generally present in a ψKXE/D consensus motif, where ψ represents a large branched hydrophobic residue (Ile, Val, or and is amino R.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. PubMed Scopus Google Scholar). The molecular for the recognition of this motif by the structure of the C-terminal domain of to UBC9 D. Lima C.D. Nature. 2005; 435: 687-692Crossref PubMed Scopus (383) Google Scholar, Lima C.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the desumoylation of modified proteins from proteolytic of the isopeptide bond by E. Lima C.D. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, F. M. A. Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar). by transcription is of the with sumoylation (9.Gocke C.B. Yu H. Kang J. J. Biol. Chem. 2005; 280: 5004-5012Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, E.S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, A. H. A. M. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). as well as and are SUMO and in their sumoylation to transcriptional (recent reviews Refs. A. J. M. PubMed Scopus Google Scholar, R.T. Cell. Biol. 2004; PubMed Scopus Google Scholar, 2005; Scopus Google Scholar). The regulation of transcription by SUMO conjugation through several Sumoylation may with post-translational such as or of lysine residues in transcription Sumoylation also the or of a protein involved in signal or transcription, as with and M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. M. A. J. Biol. 2004; PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar). sumoylation is involved in the of transcription of the protein with SUMO to the of nuclear nuclear protein or protein to which transcription such as and may as for regulatory or may in activities such as the modification or of transcription Mol. Cell. Biol. PubMed Scopus Google Scholar, Biochem. Biol. PubMed Scopus Google Scholar). sumoylated transcription such as or to and in structure to gene expression D. D. A. L.A. E. R.T. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). Although generally with sumoylation also in transcriptional activation, as with the and Y. R. M. Sarge K.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). sumoylation of transcription may be or by regulatory such as E. R.T. Mol. Cell. 12: Full Text Full Text PDF PubMed Scopus Google Scholar, R. E. R. Schmidt D. S. M. Mol. Biol. Cell. 2005; Scopus Google Scholar). members of the ETS transcription factor family are by for this regulation through such as UBC9 as an with ETS family members including Ets-1 P. Oncogene. PubMed Scopus Google R. S. S. S. A. PubMed Scopus Google D. K. 2005; PubMed Scopus Google P. Oncogene. 2005; PubMed Scopus Google and D. E. R.T. Y. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). of with SUMO-1 its as a transcriptional by to nuclear from the S. A. PubMed Scopus Google Scholar, R. S. S. A. PubMed Scopus Google Scholar). Sumoylation to predominantly at a lysine residue within the PNT domain of and to be by that the of this domain S. A. PubMed Scopus Google Scholar, R. S. S. A. PubMed Scopus Google Scholar). In to sumoylation of other ETS family members to in addition of SUMO-1 to the motif of with to transcriptional Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). This dynamic regulatory is by the of which the of its domain and the of SUMO-1 conjugation E. R.T. Mol. Cell. 12: Full Text Full Text PDF PubMed Scopus Google Scholar). sumoylation of to in transcriptional of for by binding to a protein with the and complex D. K. 2005; PubMed Scopus Google Scholar). In this we have the sumoylation of the ETS protein Ets-1 with a on its interactions with the conjugating enzyme UBC9 and on the and dynamic of this Ets-1 proteins consensus for sumoylation we have characterized sumoylation at Lys-15 in vivo and in vitro within the of a are by residues to residues of the amino protein. that is to NMR C.D. S. A. PubMed Scopus Google Scholar). NMR we that binds the site of UBC9 via the consensus sumoylation motif Lys-15. covalent attachment of SUMO-1 to does not perturb the structure or dynamic properties of either protein, that as “beads-on-a-string” tethered by a flexible isopeptide linkage. This site of sumoylation a protein site and the PNT domain of Ets-1 The PNT domain as an docking site PubMed Scopus Google with of the transcriptional Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). the sumoylation of Lys-15 may have biological to the of the N-terminal of DNA expression for and mature human SUMO-1gg, and human human and have been or as C.D. S. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The gene the with the of to to by the Ets-1 PubMed Scopus Google Scholar). The with a and from a Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). The expression for been S. S. A. P. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google and a of and E. in or in containing and proteins or W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). by C.D. S. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The or and by to the the and with affinity in a to the and protein. The an N-terminal Gly-Ser-His proteolytic The final by E. A. A. PubMed Scopus Google Scholar). and used to the and of protein. been Mol. Cell. Biol. PubMed Scopus Google Scholar). and from from of and Ets-1 in and the with of or expression of mature and of After as Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). with and in of and with as Mol. Cell. Biol. 2004; PubMed Scopus Google and at for at with of a of protein at for at least To the of a of or affinity and at for in to the and proteins by and against or the as in the In at or containing target protein, SUMO-1gg, or and at W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). To the and the of the the and the of the or The by and with a in The and the this plus to the as The final protein by to and to an and used to the of the NMR for and the at on a for and for and its on a in or with at The protein The and for and NMR in with the for C.D. S. A. PubMed Scopus Google Scholar). for residues 1–97 of P. A. S. R. Melchior F. R. J. J. Mol. Biol. 280: PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google and UBC9 Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google on and for and including of the isopeptide W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google and and F. S. J. A. J. PubMed Scopus Google and of Scholar). relaxation measurements to R. PubMed Scopus Google Scholar). of Ets-1 and UBC9 with of protein, to which of protein in the in to a final of by least of the chemical of the protein the of protein to the the of a complex in the P. PubMed Scopus Google Scholar). are on the and for the Ets-1 in and in with consensus Ets-1 to SUMO-1 at in However, this sumoylation by the of Lys-15 to an that this lysine is the site of modification this consensus sequence been in Ets-1 as a control that transcriptional in vivo on with factor binding D. Mol. Cell. Biol. PubMed Scopus Google Scholar). are to sumoylation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the sequence containing Lys-15 or at least with other as of this residue the effect D. Mol. Cell. Biol. PubMed Scopus Google Scholar). this we have on Lys-15 as the SUMO-1 lysine in with these in vivo studies, and UBC9 sumoylated in vitro of Lys-15 with an arginine or this In contrast, of or to arginine not sumoylation of The lysine within a sequence the is not of a consensus sequence to a sumoylation site in the PNT domain of human R. S. S. A. PubMed Scopus Google Lys-15 is the target of sumoylation within the N-terminal of and UBC9 by of with the SUMO-conjugating enzyme, by NMR spectroscopy. of protein upon with a protein. The chemical shift of an group is to its from the of a protein-protein chemical shift the structure of the protein a of the for its and of these to an a of their binding affinity PubMed Scopus Google Scholar). that the consensus sumoylation site containing Lys-15 in binds UBC9 its site Cys-93 and addition of in the of chemical shift of in the the structure of residues the shift the SUMO-1 site by The of chemical shift for the of as well as for with UBC9 that the binding of these proteins upon the of Lys-15 and these that the PNT domain not with UBC9. which this also UBC9 as by chemical shift Lys-15 similar to with and the for the Ets-1 a of for the complex and for the binding of UBC9 and in with chemical shift the consensus sumoylation site at Lys-15 in the N-terminal of Ets-1 is the for However, the small in the for may from a or of the PNT domain complex containing Lys-15 or UBC9. are chemical as at for and from the addition of a of UBC9 at is from for Ets-1 containing Lys-15 or as by chemical shift for this with or with residues not NMR also used to the Ets-1 binding on the of UBC9. in the site including the and and chemical shift with and but not with The two the to the PNT domain. similar of chemical shift in UBC9 upon with the of human W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google as well as with of the sumoylation from and D. Yu S. R.T. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). well with the in the complex of and human UBC9 Lima C.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the NMR that Ets-1 binds UBC9 such that Lys-15 is as for SUMO-1 NMR measurements also that sumoylation of Lys-15 of the of including the PNT domain. To that this in vitro in vivo we the in vivo sumoylation of and in the of with efficiency to a of SUMO-1 the for sumoylation of the N-terminal of Ets-1 within the unstructured N-terminal that Lys-15. Ets-1 with of Lys-15 with the sumoylation of the protein still UBC9. with chemical shift similar to with the wild type protein also of the a of the side chain group of an arginine residue for the amino group of a lysine in the binding of to UBC9. SUMO-1 and Ets-1 by a a understanding sumoylation the biological of we used NMR to the and dynamic of this post-translational and not as by the of or chemical shift in the of either protein upon with a of the other not covalent to SUMO-1gg, the of the complex with that of and the of the of a signal from the isopeptide the small of for the chemical of to which The modified to Using the the of the complex on that of shift for and Gly-97, as of the of the C-terminal to a isopeptide and no interactions of the two proteins, including the PNT domain. that upon sumoylation the of and not the site of their covalent similar for complex of and the C-terminal domain of W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The dynamic of Ets-1 sumoylation relaxation provide a of the of a protein, as well as the of its polypeptide on a In the heteronuclear is a of on a with at a of from to with of a group A. PubMed Scopus Google Scholar). the well PNT domain the N-terminal and C-terminal residues in C.D. S. A. PubMed Scopus Google Scholar). with the chemical of these this that the consensus sumoylation site at Lys-15 well as the within an unstructured and of the of a of this polypeptide Using the we and W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, P. A. S. R. Melchior F. R. J. J. Mol. Biol. 280: PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google have that the and C-terminal of SUMO-1gg, including and Gly-97, are also sumoylation, the relaxation of and not change for residues Lys-15. The in the for these residues is of the of the these the N-terminal of Ets-1 is This is with the of structure within this as by the of the isopeptide an of that the side chain of Lys-15 is also to of Ets-1 at Lys-15 within N-terminal the of SUMO addition to we a well characterized that a single site of In vitro and in vivo of wild type and of this that Lys-15 is the for SUMO-1 In NMR chemical shift for Lys-15 with UBC9. measurements with that the chemical of to Cys-93 upon addition of the binding of to UBC9 likely Lys-15 in to sumoylation by the conjugating Although not the efficiency of this be in vivo by a SUMO The N-terminal segment of Ets-1 containing Lys-15 is unstructured in as by NMR chemical C.D. S. A. PubMed Scopus Google and heteronuclear The dynamic properties of this sequence may facilitate its binding in an to the site of UBC9 Lima C.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, D. Yu S. R.T. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). conformational flexibility to be a general feature of many proteins that are modified with SUMO. to characterized sumoylation within the unstructured or C-terminal of proteins or in This may for of the target lysine to the as well as provide flexibility for SUMO-dependent interactions with or downstream effectors. However, (2.Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 7.Baba D. Maita N. Jee J.G. Uchimura Y. Saitoh H. Sugasawa K. Hanaoka F. Tochio H. Hiroaki H. Shirakawa M. Nature. 2005; 435: 979-982Crossref PubMed Scopus (187) Google Scholar, 8.Pichler A. Knipscheer P. Oberhofer E. van Dijk W.J. Korner R. Olsen J.V. Jentsch S. Melchior F. Sixma T.K. Nature Struct. Mol. Biol. 2005; 12: 264-269Crossref PubMed Scopus (166) Google as by the sumoylation of at a lysine within an in its PNT domain S. A. PubMed Scopus Google Scholar, R. S. S. A. PubMed Scopus Google Scholar). Ets-1 is not modified at the in its PNT domain. similar to with the that its M. S. J. PubMed Scopus Google is sumoylated in vitro at a consensus site within its unstructured N-terminal but not within its PNT domain. S. J. M. D. and P. with this is also to no longer sumoylation in vivo S. A. PubMed Scopus Google Scholar). the conjugation of SUMO to the PNT domain of may its and may be in vivo by an on NMR the for the of the complex with UBC9 is ∼400 This is than that of for the binding of the C-terminal domain of human with UBC9 S. Yu E. J. J. R.T. Y. PubMed Scopus Google and that of for of sumoylation from and D. Yu S. R.T. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in these may in the the SUMO lysine in protein. However, the binding of to which with its of sumoylation in arise from two the SUMO site in is located in an and of its C-terminal domain Lima C.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Although this is flexible to the in relaxation measurements that is dynamic than the unstructured N-terminal of Ets-1 W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). the sumoylation site of may be in an for binding the site of UBC9. This to a for with UBC9 than by the target in or the of and in addition to the consensus sumoylation sequence of and the site of UBC9 interactions are also with residues in and of and of UBC9 Lima C.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). This binding not be present with sumoylation in unstructured of target proteins such as Sumoylation with to of Lys-15 with arginine the sumoylation of However, demonstrate that containing binds with similar affinity to the site of UBC9 as does the wild type In the structure of the the amino group of the SUMO is to the side chain carboxyl of of UBC9 and to an also in to the of Cys-93 Lima C.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). is that an arginine these interactions. of this a sequence as a of a consensus sumoylation site for binding to UBC9. This may provide an for sumoylation in Ets-1 and SUMO-1 and are with no interactions the two proteins the site of their covalent attachment for this from and dynamic NMR of the The of protein in the complex that of its with chemical shift to the C-terminal and of and to Lys-15 in no in either protein the site of their via the isopeptide This the of structure within the N-terminal of and a of interactions the PNT domain and relaxation measurements reveal that the unstructured N-terminal of on a upon sumoylation, with Lys-15 of from the a of for its isopeptide the side chain of Lys-15 is also on this to of for the covalent complex of with a containing the unstructured N-terminal sequence of SUMO have been characterized to NMR that the C-terminal domain of and are by a flexible isopeptide and remain structurally and independent W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The of interactions and SUMO-1 is also in the complex of with UBC9 and a of the D. Lima C.D. Nature. 2005; 435: 687-692Crossref PubMed Scopus (383) Google Scholar). In contrast, the structure of SUMO-1 with the enzyme a small but well these two proteins at the site of their isopeptide linkage. The isopeptide is also with a in the of SUMO-1 at its C-terminal A. Knipscheer P. Oberhofer E. van Dijk W.J. Korner R. Olsen J.V. Jentsch S. Melchior F. Sixma T.K. Nature Struct. Mol. Biol. 2005; 12: 264-269Crossref PubMed Scopus (166) Google Scholar). However, this the of and SUMO-1 are not the is located within the N-terminal of that a with UBC9 may be for sumoylation at this the of DNA to SUMO-1 two D. Maita N. Jee J.G. Uchimura Y. Saitoh H. Sugasawa K. Hanaoka F. Tochio H. Hiroaki H. Shirakawa M. Nature. 2005; 435: 979-982Crossref PubMed Scopus (187) Google Scholar). DNA and SUMO-1 with a from the to a motif J. Y. S. A. 2004; PubMed Scopus Google to the of SUMO-1. sumoylation to of an in DNA preceding its lysine In to DNA does not a motif, and its sumoylation does not to on these studies, several be for SUMO-dependent interactions of a target protein with other macromolecules a (2.Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar). their covalent the of the target protein SUMO may or a recognition on of the The is by DNA the upon sumoylation is to of the from its product complex with DNA D. Maita N. Jee J.G. Uchimura Y. Saitoh H. Sugasawa K. Hanaoka F. Tochio H. Hiroaki H. Shirakawa M. Nature. 2005; 435: 979-982Crossref PubMed Scopus (187) Google Scholar). SUMO and the target protein may largely their independent within the of an with SUMO such as or of an lysine residue R.T. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Sumoylation also the of a target protein with This is by for which SUMO attachment to with its with the E1-activating enzyme, bond A. Knipscheer P. Oberhofer E. van Dijk W.J. Korner R. Olsen J.V. Jentsch S. Melchior F. Sixma T.K. Nature Struct. Mol. Biol. 2005; 12: 264-269Crossref PubMed Scopus (166) Google Scholar). SUMO as a or docking module, the protein to a protein. However, in the where a the sumoylated protein, but not SUMO or the protein is likely that SUMO and the modified protein to a binding for that or The isopeptide may also be present in this the of is likely that the biological of Ets-1 sumoylation arise through these rather than conformational the flexibility of the polypeptide segment linking Ets-1 and SUMO-1 may for the binding of or signaling or transcriptional regulatory on NMR of sumoylated and W.J. M. M. C.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google we hypothesize that such flexibility may be a general feature contributing to the recognition of and other ubiquitin-like proteins by their downstream effectors. In we have characterized the sumoylation of Ets-1 at the within its unstructured N-terminal This consensus site binds to UBC9 its the of SUMO from this E2-conjugating The covalent attachment of to or does not perturb the structure or dynamic properties of either protein, that are tethered by a flexible isopeptide linkage. provide a framework for understanding the molecular mechanisms by which sumoylation affects Ets-1 in We and for with