Structural Basis for the Function of DCN-1 in Protein Neddylation

Abstract

Covalent modification by Nedd8 (neddylation) stimulates the ubiquitin-protein isopeptide ligase (E3) activities of Cullins. DCN-1, an evolutionarily conserved protein, promotes neddylation of Cullins in vivo, binds directly to Nedd8, and associates with Cdc53 in the budding yeast Saccharomyces cerevisiae. The 1.9Å resolution structure of yeast DCN-1 shows that the region encompassing residues 66–269 has a rectangular parallelepiped-like all α-helical structures, consisting of an EF-hand motif N-terminal domain and a closely juxtaposed C-terminal domain with six α-helices. The EF-hand motif structure is highly similar to that of the c-Cbl ubiquitin E3 ligase. We also demonstrate that DCN-1 directly binds to Rbx-1, a factor important for protein neddylation. The structural and biochemical results are consistent with the role of DCN-1 as a scaffold protein in a multisubunit neddylation E3 ligase complex. Covalent modification by Nedd8 (neddylation) stimulates the ubiquitin-protein isopeptide ligase (E3) activities of Cullins. DCN-1, an evolutionarily conserved protein, promotes neddylation of Cullins in vivo, binds directly to Nedd8, and associates with Cdc53 in the budding yeast Saccharomyces cerevisiae. The 1.9Å resolution structure of yeast DCN-1 shows that the region encompassing residues 66–269 has a rectangular parallelepiped-like all α-helical structures, consisting of an EF-hand motif N-terminal domain and a closely juxtaposed C-terminal domain with six α-helices. The EF-hand motif structure is highly similar to that of the c-Cbl ubiquitin E3 ligase. We also demonstrate that DCN-1 directly binds to Rbx-1, a factor important for protein neddylation. The structural and biochemical results are consistent with the role of DCN-1 as a scaffold protein in a multisubunit neddylation E3 ligase complex. Ubiquitin-like proteins, such as Nedd8/Rub1, can be covalently attached to substrate proteins in a manner similar to ubiquitination. Modification by Nedd8, a process known as neddylation, has been found in Cullins and several other cellular proteins (1Xirodimas D.P. Saville M.K. Bourdon J.C. Hay R.T. Lane D.P. Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 2Pan Z.Q. Kentsis A. Dias D.C. Yamoah K. Wu K. Oncogene. 2004; 23: 1985-1997Crossref PubMed Scopus (342) Google Scholar). Neddylation and ubiquitination use parallel mechanisms; each involves their own E1, 3The abbreviations used are: E1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseGSTglutathione S-transferasePBSphosphate-buffered salineSIRASsingle isomorphous replacement with anomalous signal 3The abbreviations used are: E1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseGSTglutathione S-transferasePBSphosphate-buffered salineSIRASsingle isomorphous replacement with anomalous signal E2, and E3 proteins, and both follow a sequential modification process. During neddylation, the Nedd8 E1 heterodimer activates Nedd8 in an ATP-dependent process, and the enzyme and Nedd8 form a reaction intermediate through a thioester bond. The activated Nedd8 is then transferred to the conjugating enzyme E2, which also covalently links Nedd8 through a thioester bond. Next, the E2 interacts with an E3 to transfer Nedd8 through an isopeptide bond to a lysine residue of the substrate. The Nedd8 acceptor lysine is located in the C-terminal region of the Cullin family of ubiquitin E3 proteins (2Pan Z.Q. Kentsis A. Dias D.C. Yamoah K. Wu K. Oncogene. 2004; 23: 1985-1997Crossref PubMed Scopus (342) Google Scholar). Cullins and p53 are the only neddylation substrates known to date (1Xirodimas D.P. Saville M.K. Bourdon J.C. Hay R.T. Lane D.P. Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 2Pan Z.Q. Kentsis A. Dias D.C. Yamoah K. Wu K. Oncogene. 2004; 23: 1985-1997Crossref PubMed Scopus (342) Google Scholar, 3Parry G. Estelle M. Semin. Cell Dev. Biol. 2004; 15: 221-229Crossref PubMed Scopus (71) Google Scholar).Protein ubiquitination usually leads to substrate degradation by the proteasome, whereas neddylation of Cullins increases their ubiquitin ligase activities (4Morimoto M. Nishida T. Honda R. Yasuda H. Biochem. Biophys. Res. Commun. 2000; 270: 1093-1096Crossref PubMed Scopus (123) Google Scholar, 5Osaka F. Saeki M. Katayama S. Aida N. Toh E.A. Kominami K. Toda T. Suzuki T. Chiba T. Tanaka K. Kato S. EMBO J. 2000; 19: 3475-3484Crossref PubMed Scopus (186) Google Scholar, 6Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Crossref PubMed Scopus (325) Google Scholar). At present, Rbx-1 is the only protein known to have a Nedd8 E3 function (7Morimoto M. Nishida T. Nagayama Y. Yasuda H. Biochem. Biophys. Res. Commun. 2003; 301: 392-398Crossref PubMed Scopus (79) Google Scholar, 8Kamura T. Conrad M.N. Yan Q. Conaway R.C. Conaway J.W. Genes Dev. 1999; 13: 2928-2933Crossref PubMed Scopus (233) Google Scholar). Rbx-1 contains a single RING motif, and it is an inefficient ubiquitin E3 on its own. This is reminiscent of many E3 ubiquitin a single RING domain is of inefficient to ubiquitin T. N. F. K. M. PubMed Scopus Google that a protein, DCN-1 in Cullin is important for Cdc53 neddylation in the budding DCN-1 is a conserved protein found in to a DCN-1 protein in also known as is an Y. M. A. R. R. A. Y. Res. PubMed Scopus Google and in the by DCN-1 contains a N-terminal motif and a conserved C-terminal domain of yeast and that DCN-1 directly binds Nedd8 is and with The of Cdc53 neddylation is in yeast structural and biochemical that DCN-1 is to as a scaffold protein in a neddylation E3 ligase that and yeast DCN-1 in as a protein a on a by the of the with in with for and the with the protein with and of DCN-1 on and by in DCN-1 by and proteins and similar to that for the yeast Rbx-1 in and a by the in a and by the in the of and a with an and the Scopus Google Scholar). The to the with of a and and is are in of is the and is the of The is all in are that of the resolution the single anomalous as used for used for of the in is the and is the of The is all in are that of the resolution the single anomalous as of the in in a and structure of DCN-1 by the of single isomorphous replacement with anomalous signal the J. Cell 2003; PubMed Scopus Google Scholar). for The Google used to and G. PubMed Scopus Google Scholar). The to and with and and Biol. PubMed Scopus Google Scholar). with of the by A. R. Biol. 1999; PubMed Scopus Google J. M. T. Biol. PubMed Scopus Google Scholar, J. M. A. PubMed Scopus Google Scholar). of the the J. M. T. Biol. PubMed Scopus Google Scholar, J. M. A. PubMed Scopus Google Scholar). The contains residues and has an of and an of for The and have been in the with on by the of of DCN-1 DCN-1 of the with proteins, and the protein the with The then by an The in with yeast DCN-1 a and the of the by have a structure of yeast The protein has and structure an domain encompassing residues 66–269 The N-terminal region contains an The has an of and an of and also has with of the residues in the region of the J. Google Scholar). of the and can be found in all α-helical structure in and can be an N-terminal domain encompassing and a C-terminal domain The are in and with and The rectangular parallelepiped-like structure has of of a the structure of The structure is the to the with to a of the DCN-1 and the of conserved residues on the of the protein conserved residues are in and highly conserved residues are in the in the for The to the of which is in on the protein is in and which are the as in and are in are in and are in of the N-terminal domain and EF-hand The EF-hand is of and which are through a residues whereas and form the with a residues the The N-terminal of and their to the and it and in a the have in the is a EF-hand and it conserved and has a it also conserved C-terminal domain contains six of which are in an manner with on of the and on the The C-terminal and are to and on the to and and form of the structure of DCN-1 the of conserved and residues on the protein of the rectangular parallelepiped-like protein The which and is with of which are conserved and The is by and and of the protein is with many of such as and are highly conserved and The of conserved residues on the of the protein an evolutionarily conserved role of of the protein C-terminal domain is highly conserved with residues located the which and are residues the of in yeast DCN-1, it highly The of and the a role of the C-terminal domain of DCN-1, the of structural is DCN-1 and for similar protein the J. 2000; PubMed Scopus Google structural a of yeast DCN-1 residues as the the N-terminal domain of DCN-1 and the EF-hand domain of which is an ubiquitin E3 ligase that activated N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The can be with a of the of residues located in for the in DCN-1, the EF-hand of c-Cbl a The EF-hand of c-Cbl binds the that found in a and the of a in c-Cbl are also in of c-Cbl and a of DCN-1 and c-Cbl EF-hand a to the c-Cbl EF-hand of c-Cbl EF-hand and are with DCN-1 to the of the is to that c-Cbl also has a as of the domain N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). it a the in the domain are in manner the of a of parallel that are also of of the in the C-terminal domain of the in c-Cbl are the C-terminal domain of DCN-1, are located N-terminal to the EF-hand The of the with to their EF-hand is also both of c-Cbl and DCN-1 their EF-hand through with the The structural DCN-1 and which is an ubiquitin E3 a parallel the proteins, DCN-1 function as a of the neddylation E3 DCN-1 and the that DCN-1 function in a Nedd8 E3 ligase for of yeast DCN-1 with the RING protein on and DCN-1 to the in with the proteins to the with a of the a shows that DCN-1 with whereas DCN-1 on in a DCN-1 interacts with Rbx-1 directly in We the DCN-1 and Rbx-1 the The results that DCN-1 binds to with an of This is consistent with and a that DCN-1, and Rbx-1 can in a T. N. F. K. M. PubMed Scopus Google of DCN-1 and DCN-1, and DCN-1 proteins with used for DCN-1 C-terminal domain with on Lane DCN-1 with the C-terminal domain of DCN-1 by of DCN-1 of DCN-1 is important for with Lane DCN-1 protein as a DCN-1, a DCN-1 and an of DCN-1 for by DCN-1 by as a DCN-1 used in the in and which region of DCN-1 interacts with Rbx-1, several of DCN-1 and the DCN-1 in and in a manner similar to that for the with that a C-terminal of DCN-1 encompassing residues binds to Rbx-1 with an with that of the DCN-1 the C-terminal domain of DCN-1 is for with the DCN-1 residues important for with Rbx-1, a of of highly conserved residues the C-terminal domain of single and and and and DCN-1 proteins used in and all of the DCN-1 only a of to Rbx-1 is important for Rbx-1, whereas the of other conserved residues to be The structure shows that its and a with and residues are also highly conserved the region by the residues have an evolutionarily conserved role of with structural an the of DCN-1 and an ubiquitin E3 have that DCN-1 also interacts with Nedd8, and ubiquitin T. N. F. K. M. PubMed Scopus Google Scholar). We have also that DCN-1 interacts directly with the C-terminal domain in results that DCN-1 as a of the Nedd8 E3 ligase is that DCN-1 function as a Nedd8 E3 ligase on its own. a RING domain to domain with all ubiquitin E3 known to and the protein ubiquitination and neddylation the of of in the Nedd8 E3 have that Rbx-1 as the RING domain protein in the Nedd8 E3 complex. with the RING domain ubiquitin E3 ligase it is that the RING domain of DCN-1 is in by This is a a structural of c-Cbl contains a RING domain in with the EF-hand and and the RING domain is to the similar of the RING domain of Rbx-1 with to the domain DCN-1 is as have that the biochemical and structural the of DCN-1, Rbx-1, and Ubiquitin-like proteins, such as Nedd8/Rub1, can be covalently attached to substrate proteins in a manner similar to ubiquitination. Modification by Nedd8, a process known as neddylation, has been found in Cullins and several other cellular proteins (1Xirodimas D.P. Saville M.K. Bourdon J.C. Hay R.T. Lane D.P. Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 2Pan Z.Q. Kentsis A. Dias D.C. Yamoah K. Wu K. Oncogene. 2004; 23: 1985-1997Crossref PubMed Scopus (342) Google Scholar). Neddylation and ubiquitination use parallel mechanisms; each involves their own E1, 3The abbreviations used are: E1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseGSTglutathione S-transferasePBSphosphate-buffered salineSIRASsingle isomorphous replacement with anomalous signal 3The abbreviations used are: E1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseGSTglutathione S-transferasePBSphosphate-buffered salineSIRASsingle isomorphous replacement with anomalous signal E2, and E3 proteins, and both follow a sequential modification process. During neddylation, the Nedd8 E1 heterodimer activates Nedd8 in an ATP-dependent process, and the enzyme and Nedd8 form a reaction intermediate through a thioester bond. The activated Nedd8 is then transferred to the conjugating enzyme E2, which also covalently links Nedd8 through a thioester bond. Next, the E2 interacts with an E3 to transfer Nedd8 through an isopeptide bond to a lysine residue of the substrate. The Nedd8 acceptor lysine is located in the C-terminal region of the Cullin family of ubiquitin E3 proteins (2Pan Z.Q. Kentsis A. Dias D.C. Yamoah K. Wu K. Oncogene. 2004; 23: 1985-1997Crossref PubMed Scopus (342) Google Scholar). Cullins and p53 are the only neddylation substrates known to date (1Xirodimas D.P. Saville M.K. Bourdon J.C. Hay R.T. Lane D.P. Cell. 2004; 118: 83-97Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 2Pan Z.Q. Kentsis A. Dias D.C. Yamoah K. Wu K. Oncogene. 2004; 23: 1985-1997Crossref PubMed Scopus (342) Google Scholar, 3Parry G. Estelle M. Semin. Cell Dev. Biol. 2004; 15: 221-229Crossref PubMed Scopus (71) Google Scholar). enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase single isomorphous replacement with anomalous signal enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase single isomorphous replacement with anomalous signal ubiquitination usually leads to substrate degradation by the proteasome, whereas neddylation of Cullins increases their ubiquitin ligase activities (4Morimoto M. Nishida T. Honda R. Yasuda H. Biochem. Biophys. Res. Commun. 2000; 270: 1093-1096Crossref PubMed Scopus (123) Google Scholar, 5Osaka F. Saeki M. Katayama S. Aida N. Toh E.A. Kominami K. Toda T. Suzuki T. Chiba T. Tanaka K. Kato S. EMBO J. 2000; 19: 3475-3484Crossref PubMed Scopus (186) Google Scholar, 6Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Crossref PubMed Scopus (325) Google Scholar). At present, Rbx-1 is the only protein known to have a Nedd8 E3 function (7Morimoto M. Nishida T. Nagayama Y. Yasuda H. Biochem. Biophys. Res. Commun. 2003; 301: 392-398Crossref PubMed Scopus (79) Google Scholar, 8Kamura T. Conrad M.N. Yan Q. Conaway R.C. Conaway J.W. Genes Dev. 1999; 13: 2928-2933Crossref PubMed Scopus (233) Google Scholar). Rbx-1 contains a single RING motif, and it is an inefficient ubiquitin E3 on its own. This is reminiscent of many E3 ubiquitin a single RING domain is of inefficient to ubiquitin T. N. F. K. M. PubMed Scopus Google that a protein, DCN-1 in Cullin is important for Cdc53 neddylation in the budding DCN-1 is a conserved protein found in to a DCN-1 protein in also known as is an Y. M. A. R. R. A. Y. Res. PubMed Scopus Google and in the by DCN-1 contains a N-terminal motif and a conserved C-terminal domain of yeast and that DCN-1 directly binds Nedd8 is and with The of Cdc53 neddylation is in yeast structural and biochemical that DCN-1 is to as a scaffold protein in a neddylation E3 ligase that and yeast DCN-1 in as a protein a on a by the of the with in with for and the with the protein with and of DCN-1 on and by in DCN-1 by and proteins and similar to that for the yeast Rbx-1 in and a by the in a and by the in the of and a with an and the Scopus Google Scholar). The to the with of a and and is are in of is the and is the of The is all in are that of the resolution the single anomalous as used for used for of the in is the and is the of The is all in are that of the resolution the single anomalous as of the in in a and structure of DCN-1 by the of single isomorphous replacement with anomalous signal the J. Cell 2003; PubMed Scopus Google Scholar). for The Google used to and G. PubMed Scopus Google Scholar). The to and with and and Biol. PubMed Scopus Google Scholar). with of the by A. R. Biol. 1999; PubMed Scopus Google J. M. T. Biol. PubMed Scopus Google Scholar, J. M. A. PubMed Scopus Google Scholar). of the the J. M. T. Biol. PubMed Scopus Google Scholar, J. M. A. PubMed Scopus Google Scholar). The contains residues and has an of and an of for The and have been in the with on by the of of DCN-1 DCN-1 of the with proteins, and the protein the with The then by an The in with yeast DCN-1 a and the of the by and yeast DCN-1 in as a protein a on a by the of the with in with for and the with the protein with and of DCN-1 on and by in DCN-1 by and proteins and similar to that for the yeast Rbx-1 in and a by the in a and by the in the of and a with an and the Scopus Google Scholar). The to the with of a and and is are in and structure of DCN-1 by the of single isomorphous replacement with anomalous signal the J. Cell 2003; PubMed Scopus Google Scholar). for The Google used to and G. PubMed Scopus Google Scholar). The to and with and and Biol. PubMed Scopus Google Scholar). with of the by A. R. Biol. 1999; PubMed Scopus Google J. M. T. Biol. PubMed Scopus Google Scholar, J. M. A. PubMed Scopus Google Scholar). of the the J. M. T. Biol. PubMed Scopus Google Scholar, J. M. A. PubMed Scopus Google Scholar). The contains residues and has an of and an of for The and have been in the with on by the of of DCN-1 DCN-1 of the with proteins, and the protein the with The then by an The in with yeast DCN-1 a and the of the by have a structure of yeast The protein has and structure an domain encompassing residues 66–269 The N-terminal region contains an The has an of and an of and also has with of the residues in the region of the J. Google Scholar). of the and can be found in all α-helical structure in and can be an N-terminal domain encompassing and a C-terminal domain The are in and with and The rectangular parallelepiped-like structure has of of the N-terminal domain and EF-hand The EF-hand is of and which are through a residues whereas and form the with a residues the The N-terminal of and their to the and it and in a the have in the is a EF-hand and it conserved and has a it also conserved C-terminal domain contains six of which are in an manner with on of the and on the The C-terminal and are to and on the to and and form of the structure of DCN-1 the of conserved and residues on the protein of the rectangular parallelepiped-like protein The which and is with of which are conserved and The is by and and of the protein is with many of such as and are highly conserved and The of conserved residues on the of the protein an evolutionarily conserved role of of the protein C-terminal domain is highly conserved with residues located the which and are residues the of in yeast DCN-1, it highly The of and the a role of the C-terminal domain of DCN-1, the of structural is DCN-1 and for similar protein the J. 2000; PubMed Scopus Google structural a of yeast DCN-1 residues as the the N-terminal domain of DCN-1 and the EF-hand domain of which is an ubiquitin E3 ligase that activated N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The can be with a of the of residues located in for the in DCN-1, the EF-hand of c-Cbl a The EF-hand of c-Cbl binds the that found in a and the of a in c-Cbl are also in of c-Cbl and a of DCN-1 and c-Cbl EF-hand a to the c-Cbl EF-hand of c-Cbl EF-hand and are with DCN-1 to the of the is to that c-Cbl also has a as of the domain N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). it a the in the domain are in manner the of a of parallel that are also of of the in the C-terminal domain of the in c-Cbl are the C-terminal domain of DCN-1, are located N-terminal to the EF-hand The of the with to their EF-hand is also both of c-Cbl and DCN-1 their EF-hand through with the The structural DCN-1 and which is an ubiquitin E3 a parallel the proteins, DCN-1 function as a of the neddylation E3 DCN-1 and the that DCN-1 function in a Nedd8 E3 ligase for of yeast DCN-1 with the RING protein on and DCN-1 to the in with the proteins to the with a of the a shows that DCN-1 with whereas DCN-1 on in a DCN-1 interacts with Rbx-1 directly in We the DCN-1 and Rbx-1 the The results that DCN-1 binds to with an of This is consistent with and a that DCN-1, and Rbx-1 can in a T. N. F. K. M. PubMed Scopus Google of DCN-1 and DCN-1, and DCN-1 proteins with used for DCN-1 C-terminal domain with on Lane DCN-1 with the C-terminal domain of DCN-1 by of DCN-1 of DCN-1 is important for with Lane DCN-1 protein as a DCN-1, a DCN-1 and an of DCN-1 for by DCN-1 by as a DCN-1 used in the in and which region of DCN-1 interacts with Rbx-1, several of DCN-1 and the DCN-1 in and in a manner similar to that for the with that a C-terminal of DCN-1 encompassing residues binds to Rbx-1 with an with that of the DCN-1 the C-terminal domain of DCN-1 is for with the DCN-1 residues important for with Rbx-1, a of of highly conserved residues the C-terminal domain of single and and and and DCN-1 proteins used in and all of the DCN-1 only a of to Rbx-1 is important for Rbx-1, whereas the of other conserved residues to be The structure shows that its and a with and residues are also highly conserved the region by the residues have an evolutionarily conserved role of with We have a structure of yeast The protein has and structure an domain encompassing residues 66–269 The N-terminal region contains an The has an of and an of and also has with of the residues in the region of the J. Google Scholar). of the and can be found in all α-helical structure in and can be an N-terminal domain encompassing and a C-terminal domain The are in and with and The rectangular parallelepiped-like structure has of of the N-terminal domain and EF-hand The EF-hand is of and which are through a residues whereas and form the with a residues the The N-terminal of and their to the and it and in a the have in the is a EF-hand and it conserved and has a it also conserved The C-terminal domain contains six of which are in an manner with on of the and on the The C-terminal and are to and on the to and and form of the structure of DCN-1 the of conserved and residues on the protein of the rectangular parallelepiped-like protein The which and is with of which are conserved and The is by and and of the protein is with many of such as and are highly conserved and The of conserved residues on the of the protein an evolutionarily conserved role of of the protein The C-terminal domain is highly conserved with residues located the which and are residues the of in yeast DCN-1, it highly The of and the a role of the C-terminal domain of DCN-1, the of structural is DCN-1 and for similar protein the J. 2000; PubMed Scopus Google structural a of yeast DCN-1 residues as the the N-terminal domain of DCN-1 and the EF-hand domain of which is an ubiquitin E3 ligase that activated N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The can be with a of the of residues located in for the in DCN-1, the EF-hand of c-Cbl a The EF-hand of c-Cbl binds the that found in a and the of a in c-Cbl are also in is to that c-Cbl also has a as of the domain N. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). it a the in the domain are in manner the of a of parallel that are also of of the in the C-terminal domain of the in c-Cbl are the C-terminal domain of DCN-1, are located N-terminal to the EF-hand The of the with to their EF-hand is also both of c-Cbl and DCN-1 their EF-hand through with the The structural DCN-1 and which is an ubiquitin E3 a parallel the proteins, DCN-1 function as a of the neddylation E3 ligase. DCN-1 and the that DCN-1 function in a Nedd8 E3 ligase for of yeast DCN-1 with the RING protein on and DCN-1 to the in with the proteins to the with a of the a shows that DCN-1 with whereas DCN-1 on in a DCN-1 interacts with Rbx-1 directly in We the DCN-1 and Rbx-1 the The results that DCN-1 binds to with an of This is consistent with and a that DCN-1, and Rbx-1 can in a T. N. F. K. M. PubMed Scopus Google Scholar). which region of DCN-1 interacts with Rbx-1, several of DCN-1 and the DCN-1 in and in a manner similar to that for the with that a C-terminal of DCN-1 encompassing residues binds to Rbx-1 with an with that of the DCN-1 the C-terminal domain of DCN-1 is for with the DCN-1 residues important for with Rbx-1, a of of highly conserved residues the C-terminal domain of single and and and and DCN-1 proteins used in and all of the DCN-1 only a of to Rbx-1 is important for Rbx-1, whereas the of other conserved residues to be The structure shows that its and a with and residues are also highly conserved the region by the residues have an evolutionarily conserved role of with structural an the of DCN-1 and an ubiquitin E3 have that DCN-1 also interacts with Nedd8, and ubiquitin T. N. F. K. M. PubMed Scopus Google Scholar). We have also that DCN-1 interacts directly with the C-terminal domain in results that DCN-1 as a of the Nedd8 E3 ligase is that DCN-1 function as a Nedd8 E3 ligase on its own. a RING domain to domain with all ubiquitin E3 known to and the protein ubiquitination and neddylation the of of in the Nedd8 E3 have that Rbx-1 as the RING domain protein in the Nedd8 E3 complex. with the RING domain ubiquitin E3 ligase it is that the RING domain of DCN-1 is in by This is a a structural of c-Cbl contains a RING domain in with the EF-hand and and the RING domain is to the similar of the RING domain of Rbx-1 with to the domain DCN-1 is as have that the biochemical and structural the of DCN-1, Rbx-1, and structural an the of DCN-1 and an ubiquitin E3 have that DCN-1 also interacts with Nedd8, and ubiquitin T. N. F. K. M. PubMed Scopus Google Scholar). We have also that DCN-1 interacts directly with the C-terminal domain in results that DCN-1 as a of the Nedd8 E3 ligase complex. is that DCN-1 function as a Nedd8 E3 ligase on its own. a RING domain to domain with all ubiquitin E3 known to and the protein ubiquitination and neddylation the of of in the Nedd8 E3 have that Rbx-1 as the RING domain protein in the Nedd8 E3 complex. with the RING domain ubiquitin E3 ligase it is that the RING domain of DCN-1 is in by This is a a structural of c-Cbl contains a RING domain in with the EF-hand and and the RING domain is to the similar of the RING domain of Rbx-1 with to the domain DCN-1 is as have that the biochemical and structural the of DCN-1, Rbx-1, and We are to for with and and for and for with with