A Single Mms2 “Key” Residue Insertion into a Ubc13 Pocket Determines the Interface Specificity of a Human Lys63 Ubiquitin Conjugation Complex

Human Ubc13 and Mms2 (or its homolog, Uev1) form a unique ubiquitin-conjugating enzyme (Ubc) complex that generates atypical Lys63-linked ubiquitin conjugates. Such conjugates are attached to specific targets that modulate the activity of various cellular processes including DNA repair, mitotic progression, and nuclear factor-κB signaling. Whereas Ubc13 is a typical Ubc, Mms2 is a non-catalytic Ubc variant. Substantial biochemical evidence has revealed a mechanism whereby Mms2 properly orients ubiquitin to allow for Lys63 conjugation by Ubc13; however, how this specific Ubc13-Mms2 complex is formed and why Mms2 does not form a complex with other Ubcs have not been reported. In order to address these questions, we used a structurebased approach to design mutations and characterize the human Ubc13-Mms2 interface. We used the yeast two-hybrid assay, glutathione S-transferase pull-downs, and surface plasmon resonance to test in vivo and in vitro binding. These experiments were paired with functional complementation and ubiquitin conjugation studies to provide in vivo and in vitro functional data. The results in this study allowed us to identify important residues of the Ubc13-Mms2 interface, determine a correlation between heterodimer formation and function, and conclude why Mms2 forms a specific complex with Ubc13 but not other Ubc proteins. Human Ubc13 and Mms2 (or its homolog, Uev1) form a unique ubiquitin-conjugating enzyme (Ubc) complex that generates atypical Lys63-linked ubiquitin conjugates. Such conjugates are attached to specific targets that modulate the activity of various cellular processes including DNA repair, mitotic progression, and nuclear factor-κB signaling. Whereas Ubc13 is a typical Ubc, Mms2 is a non-catalytic Ubc variant. Substantial biochemical evidence has revealed a mechanism whereby Mms2 properly orients ubiquitin to allow for Lys63 conjugation by Ubc13; however, how this specific Ubc13-Mms2 complex is formed and why Mms2 does not form a complex with other Ubcs have not been reported. In order to address these questions, we used a structurebased approach to design mutations and characterize the human Ubc13-Mms2 interface. We used the yeast two-hybrid assay, glutathione S-transferase pull-downs, and surface plasmon resonance to test in vivo and in vitro binding. These experiments were paired with functional complementation and ubiquitin conjugation studies to provide in vivo and in vitro functional data. The results in this study allowed us to identify important residues of the Ubc13-Mms2 interface, determine a correlation between heterodimer formation and function, and conclude why Mms2 forms a specific complex with Ubc13 but not other Ubc proteins. In recent years, the attachment of ubiquitin (Ub) 1The abbreviations used are: Ub, ubiquitin; Ubc, ubiquitin-conjugating enzyme; E2, ubiquitin carrier protein; Uev, ubiquitin-conjugating enzyme variant; E3, ubiquitin-protein isopeptide ligase; SPR, surface plasmon resonance; MMS, methylmethane sulfonate; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SD, synthetic dextrose; YPD, yeast extract-peptone-dextrose; RU, response unit(s); RUeq, response unit(s) at equilibrium. 1The abbreviations used are: Ub, ubiquitin; Ubc, ubiquitin-conjugating enzyme; E2, ubiquitin carrier protein; Uev, ubiquitin-conjugating enzyme variant; E3, ubiquitin-protein isopeptide ligase; SPR, surface plasmon resonance; MMS, methylmethane sulfonate; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SD, synthetic dextrose; YPD, yeast extract-peptone-dextrose; RU, response unit(s); RUeq, response unit(s) at equilibrium. to its target has become one of the cornerstones of covalent posttranslational modification in eukaryotes. The biochemical process, called ubiquitination, is a sequential three-step reaction whereby Ub is activated in an ATP-dependent manner by a ubiquitin-activating enzyme. The Ub C terminus then forms a thiolester intermediate with the catalytic Cys residue of a Ub-conjugating enzyme (Ubc or E2) active site. The final step involves a substrate-specific ubiquitin ligase (E3) and leads to the attachment of the Ub C terminus to an ∈-amino group of a Lys residue of the target (reviewed in Ref. 1Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2857) Google Scholar). Most often, the process is repeated, and subsequent Ub molecules are attached to one another so that poly-Ub chains are generated. Mono-Ub or poly-Ub chains are used as signals for numerous cellular processes such as cell cycle progression (2Wei W. Ayad N.G. Wan Y. Zhang G.J. Kirschner M.W. Kaelin Jr., W.G. Nature. 2004; 428: 194-198Crossref PubMed Scopus (389) Google Scholar), apoptosis (3Zhang H.G. Wang J. Yang X. Hsu H.C. Mountz J.D. Oncogene. 2004; 23: 2009-2015Crossref PubMed Scopus (227) Google Scholar), mitochondrial inheritance (4Fisk H.A. Yaffe M.P. J. Cell Biol. 1999; 145: 1199-1208Crossref PubMed Scopus (158) Google Scholar), and transcriptional regulation (5Kao C.F. Hillyer C. Tsukuda T. Henry K. Berger S. Osley M.A. Genes Dev. 2004; 18: 184-195Crossref PubMed Scopus (177) Google Scholar). The versatility of ubiquitination and its ability to function in a wide variety of cellular roles are generally attributed to three main factors. First, whereas E1s are encoded by one or very few genes in the cell, E2s are a more plentiful family, with 13 such enzymes in budding yeast and many more in higher eukaryotes. Second, the most plentiful and diverse group of ubiquitination enzymes is the E3s, which function alone or as part of multi-subunit complexes. E3s are responsible for determining substrate specificity and act in either active or passive roles in ubiquitination by covalently binding Ub or acting as E2 substrate adapter proteins, respectively. A third important factor in providing versatility for ubiquitination involves the nature of the poly-Ub chains themselves. Conventional poly-Ub chains are built via Lys48 and provide a characteristic signal for substrate-specific degradation by the 26S proteasome (6Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1444) Google Scholar). Because protein turnover by the Ub proteasome system is a fundamental process, it is not surprising that Lys48 poly-Ub chains play such a pivotal role in the cell. However, because Ub has six other surface Lys residues, an even greater level of versatility is possible. Indeed, several studies have reported non-standard poly-Ub chains via the Lys6 (7Wu-Baer F. Lagrazon K. Yuan W. Baer R. J. Biol. Chem. 2003; 278: 34743-34746Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), Lys29 (8Arnason T. Ellison M.J. Mol. Cell. Biol. 1994; 14: 7876-7883Crossref PubMed Scopus (195) Google Scholar), Lys11 (9Baboshina O.V. Haas A.L. J. Biol. Chem. 1996; 271: 2823-2831Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), and Lys63 (10Spence J. Gali R.R. Dittmar G. Sherman F. Karin M. Finley D. Cell. 2000; 102: 67-76Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) residues of Ub. Furthermore, such atypical conjugates have been shown to function in roles other than proteasome degradation. Some of the best-documented examples of such poly-Ub chains involve those through Lys63, which have been demonstrated in a stress response (8Arnason T. Ellison M.J. Mol. Cell. Biol. 1994; 14: 7876-7883Crossref PubMed Scopus (195) Google Scholar), mitochondrial inheritance (4Fisk H.A. Yaffe M.P. J. Cell Biol. 1999; 145: 1199-1208Crossref PubMed Scopus (158) Google Scholar), plasma membrane protein endocytosis (11Galan J.M. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (320) Google Scholar), ribosome function (10Spence J. Gali R.R. Dittmar G. Sherman F. Karin M. Finley D. Cell. 2000; 102: 67-76Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), and DNA postreplication repair (12Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). Because all Ubcs, regardless of the poly-Ub chain synthesized, have a well-conserved core domain that houses the active site (13Cook W.J. Jeffrey L.C. Xu Y. Chau V. Biochemistry. PubMed Scopus Google Scholar), part of the in atypical Ub chains in how are The to as a in the it that conjugation via Lys63 through a mechanism a complex between Ubc13 and a Ubc enzyme Mms2 (12Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). are in and to but the catalytic active site that forms with Ub. it that the act as of ubiquitination Genet. 1997; 16: PubMed Scopus Google however, subsequent studies revealed and Lys63 poly-Ub chains were shown to for postreplication repair (12Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). by the of of Lys63 Ub the for the human S. W. Ellison M.J. Biol. 2001; PubMed Scopus Google Scholar) and yeast R.M. C. Pickart C.M. C. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) Ubc13-Mms2 were were and study a for chain The studies were by that revealed the unique Lys63 poly-Ub chain a heterodimer is formed that Mms2 to an Ub through such that its Lys63 residue is to the Ub at the Ubc13 active site. isopeptide then form between the and Lys63 is to through a the Ubc13 active site a binding site S. T. C. W. Ellison M.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google S. T. C. W. Ellison M.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google S. J. T. W. Ellison M.J. Biochemistry. 2003; PubMed Scopus Google W. Chem. 2004; PubMed Scopus Google Scholar). The of the of heterodimer formation between the E2 and however, studies to have not in the of the interface. we studies and a approach to provide a study of the human Ubc13-Mms2 interface. In the were as to identify residues in Ubc13 and Mms2 that are important for complex to a correlation between Ubc13-Mms2 and and to determine why Mms2 forms a specific complex with and not other and Cell used in this study a of A S. and used as the to the by a PubMed Scopus Google Scholar) an through as W. T. S. T. S. 1999; PubMed Scopus Google Scholar). A with the a J. Y. W. Genet. 2000; PubMed Scopus Google Scholar). were at either in a yeast or in a synthetic with as T. in Scholar). to either or to were a as Y. K. 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Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) that the Ubc13-Mms2 complex is in of and a of that a of these a that to a and the of the that is unique to Second, we a in the of the human Mms2 terminus heterodimer formation with Ubc13 S. W. Ellison M.J. Biol. 2001; PubMed Scopus Google Scholar), an for the Ubc13 residues in to the Mms2 of the residues that we is either or very protein various The of residues that were in these studies is in and C. of Ubc13 and in the all evidence to a specific complex between Mms2 and a Ubc we to studies with Ubc13 that this approach the for residues because Ubcs in very cellular roles have very core domain a to to test in we were to and in vitro by the approach as These residues been S. W. Ellison M.J. Biol. 2001; PubMed Scopus Google Scholar) to to of the However, a binding to Mms2 as by and T. 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Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), human Ubc13 has conjugation activity in and we that the mutations are not in this between and it that the residue have a role with to the interface, to with in and to form with the Mms2 via its We that the the we an binding by mutations that the and with the The yeast two-hybrid that whereas the of of the with its in that and mutations the The in a complementation in which yeast with and were more to than the in to the Ubc13-Mms2 we that the but the of to the the of the A and is of a important that is of the In order to address this however, we a that but a for with is in the of and mutations Ubc13-Mms2 and DNA repair function in yeast with the In not the in binding with In the have the of In the to as as Ubc13 in functional complementation experiments not these results the of in to a Ubc13 for The heterodimer is the E2 that atypical poly-Ub chains through Lys63 in The of the complex in Ub chain to numerous biochemical studies S. 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In this we the to human Ubc13 and Mms2 in order to address how the complex is We a of in vivo and in vitro including the yeast two-hybrid assay, pull-downs, and in order to binding In an in vitro ubiquitination and a complementation approach were to binding ability with biochemical and an correlation between these results The Ubc13-Mms2 is and and a surface of study with the of a of residues the and the terminus of all of which that Ubc13 in the human S. W. Ellison M.J. Biol. 2001; PubMed Scopus Google Scholar) A and and yeast R.M. C. Pickart C.M. C. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) We with the of a of to the of which to for complex formation and of and of Ubc13 in to the of the Mms2 to the core of the Ubc13-Mms2 interface. the and mutations have the Ubc13-Mms2 In as a between Mms2 because it to the of Mms2 and an with the its as another the binding and residues in the Mms2 of the core the and mutations that the with not have the as the the other binding and in vivo the Ubc13-Mms2 is to a formed by the Ubc13 and several chain of a in a the of this we three very mutations at this in the of binding in all of in vivo and in vitro the of these and more we and a more We that the of this group to the Ubc13-Mms2 interface, and this the of the for the the Ubc13-Mms2 heterodimer is with an that the characteristic but the Ubc13 surface to Whereas studies have revealed a of Ubc13 residues, to the residue with a role in binding to A and this study that is between and and is by which form a is in with the demonstrated of Ubc13-Mms2 as as is that such as those in the via the and residues, not to binding and play more roles such as Because of functional the human S. W. Ellison M.J. Biol. 2001; PubMed Scopus Google Scholar) and yeast R.M. C. Pickart C.M. C. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) of the Ubc13-Mms2 are very W. S. PubMed Scopus Google C. S. Ellison M.J. W. PubMed Scopus Google Scholar). In the residues that we in the human are and in A is the human which to a in however, has its group the The us to to determine the residues in other yeast Ubc that to human and The of these residues for of S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), (13Cook W.J. Jeffrey L.C. Xu Y. Chau V. Biochemistry. PubMed Scopus Google Scholar), and W.J. Chau V. Biochemistry. 1997; PubMed Scopus Google Scholar) are such that the for is not Ellison M.J. S. C. M. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) allow an the of its and residues at the and not allow chains of to in studies these Ubc13 residues are not but to with We for the of and for in and for of the with

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