Ubiquitin Manipulation by an E2 Conjugating Enzyme Using a Novel Covalent Intermediate
2005; Elsevier BV; Volume: 280; Issue: 36 Linguagem: Inglês
10.1074/jbc.m505205200
ISSN1083-351X
AutoresNadine Merkley, Kathryn R. Barber, Gary S. Shaw,
Tópico(s)Click Chemistry and Applications
ResumoDegradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes. Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes. The ubiquitin-dependent proteolysis pathway controls the removal of damaged and misfolded proteins in the cell. One of the key steps in this pathway is the assembly of a polyubiquitin chain that ultimately targets a substrate for degradation (1Pickart C.M. Eddins M.J. Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1020) Google Scholar, 2Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). This process involves tagging of a substrate protein with an arrangement of four to eight ubiquitin molecules linked in series through the C-terminal Gly76 of one ubiquitin molecule and the side-chain ϵ-NH2 from Lys48 of another (3Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1309) Google Scholar). Once tagged, the polyubiquitinated protein is recognized by the 26 S proteasome, where it is degraded. The building of a polyubiquitin chain and subsequent labeling of a substrate protein is a complex process potentially involving the passage of ubiquitin through three different enzymes (1Pickart C.M. Eddins M.J. Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1020) Google Scholar, 4Fang S. Weissman A.M. Cell Mol. Life Sci. 2004; 61: 1546-1561Crossref PubMed Google Scholar, 5Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (230) Google Scholar). Initially, ubiquitin is activated in an ATP-dependent step forming a high energy E1 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; Ubc11-150, S. cerevisiae Ubc1 (residues 1-150); Ub, S. cerevisiae K48R-ubiquitin; UbCys, K48R-ubiquitin with a G76C mutation; UBA, ubiquitin-associated; UbCys-Ubc1, disulfide-linked complex between UbCys and Ubc1 (or Ubc11-150).1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; Ubc11-150, S. cerevisiae Ubc1 (residues 1-150); Ub, S. cerevisiae K48R-ubiquitin; UbCys, K48R-ubiquitin with a G76C mutation; UBA, ubiquitin-associated; UbCys-Ubc1, disulfide-linked complex between UbCys and Ubc1 (or Ubc11-150).-ubiquitin thioester complex. Ubiquitin is then transferred to an E2 or ubiquitin-conjugating enzyme forming a thioester intermediate. E2 proteins have been demonstrated recently to bind to the ubiquitin-like domain of the E1 providing insight into the mechanism in which the thioester-bound ubiquitin is passed to the E2 (6Huang D.T. Paydar A. Zhuang M. Waddell M.B. Holton J.M. Schulman B.A. Mol. Cell. 2005; 17: 341-350Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7Lois L.M. Lima C.D. EMBO J. 2005; 24: 439-451Crossref PubMed Scopus (237) Google Scholar, 8Walden H. Podgorski M.S. Schulman B.A. Nature. 2003; 422: 330-334Crossref PubMed Scopus (178) Google Scholar). Two different E3 ligase proteins can catalyze the final passage of ubiquitin to the substrate. For RING E3 ligases, ubiquitin or a polyubiquitin chain is transferred directly from the E2 to the substrate, whereas HECT E3 ligases form an ubiquitin-E3 thioester prior to ubiquitin transfer to the substrate (5Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (230) Google Scholar). The E2 conjugating proteins are the key enzymes in this pathway because they are required to transfer ubiquitin either to the E3 ligase (HECT E3) or to the substrate (RING E3). All E2 proteins have a 150-residue catalytic domain that is structurally conserved throughout many species and contains the cysteine residue necessary for thioester formation with Gly76 of ubiquitin. Structures of E2 proteins show that the catalytic domain has an α/β-fold that is maintained upon complexation with either HECT (9Huang L. Kinnucan E. Wan G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Science. 1999; 286: 1321-1326Crossref PubMed Scopus (439) Google Scholar) or RING (10Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar, 11Zheng N. Schulman B.A. Song L. Miller J.J. Jeffrey P.D. Wang P. Chu C. Koepp D.M. Elledge S.J. Pagano M. Conaway R.C. Conaway J.W. Harper J.W. Pavletich N.P. Nature. 2002; 416: 703-709Crossref PubMed Scopus (1164) Google Scholar) E3 ligases. Details on the involvement of the E2 thioester in the polyubiquitin chain-building process or the mechanism for transfer of ubiquitin from an E2 protein to an E3 or substrate are less certain. The E2 proteins Ubc1 and Ubc3 (Cdc34) from Saccharomyces cerevisiae assemble polyubiquitin chains and exhibit autoubiquitination activities (12Hodgins R. Gwozd C. Arnason R. Cummings M. Ellison M.J. J. Biol. Chem. 1996; 271: 28766-28771Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Gwozd C.S. Arnason T.G. Cook W.J. Chau V. Ellison M.J. Biochemistry. 1995; 34: 6296-6302Crossref PubMed Scopus (24) Google Scholar), whereas the mammalian E2 protein E2-25K can assemble polyubiquitin chains in the absence of an E3 enzyme (14Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar). Furthermore, Ubc1 has been shown to be important for the creation of polyubiquitin chains required for protein labeling and subsequent degradation (12Hodgins R. Gwozd C. Arnason R. Cummings M. Ellison M.J. J. Biol. Chem. 1996; 271: 28766-28771Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The three-dimensional structure of Ubc1 from S. cerevisiae, determined by NMR spectroscopy, provides some insights into these biological activities (15Merkley N. Shaw G.S. J. Biol. Chem. 2004; 279: 47139-47147Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The structure shows that Ubc1 and its related class II conjugating enzymes form a unique two-domain protein having a typical α/β-fold catalytic domain connected via a flexible tether to a C-terminal UBA (ubiquitin-associated) domain. UBA domains, such as the one in Ubc1, are capable of interaction with mono- or polyubiquitin chains in a non-covalent fashion and may result in either inhibition of degradation or transfer enhancement via ubiquitin interaction (16Hofmann K. Bucher P. Trends Biochem. Sci. 1996; 21: 172-173Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 17Raasi S. Orlov I. Fleming K.G. Pickart C.M. J. Mol. Biol. 2004; 341: 1367-1379Crossref PubMed Scopus (131) Google Scholar, 18Raasi S. Pickart C.M. J. Biol. Chem. 2003; 278: 8951-8959Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 19Chen L. Shinde U. Ortolan T.G. Madura K. EMBO Rep. 2001; 2: 933-938Crossref PubMed Scopus (173) Google Scholar). The E2 conjugating protein Ubc1, is a well positioned candidate to examine the transfer of ubiquitin from the ubiquitin-E2 thioester because of its ability to moderate and build polyubiquitin chains. Mechanistic experiments that examine the role of the ubiquitin-E2 thioester in polyubiquitin chain assembly have been difficult because of the inherent instability of the thioester complex. To date the best details have been garnered from models derived from NMR chemical shift perturbation data for ubiquitin-E2 thioester intermediates (20Hamilton K.S. Ellison M.J. Shaw G.S. J. Biomol. NMR. 2000; 18: 319-327Crossref PubMed Scopus (29) Google Scholar). Attempts to stabilize this complex for more detailed structural and mechanistic experiments have met with limited success. To circumvent this we have created a novel disulfide-linked ubiquitin-Ubc1 complex that mimics the ubiquitin-E2 thioester intermediate. We show that this ubiquitin-Ubc1 complex can be purified in high amounts, is stable for long periods of time, and has similar structural characteristics to the ubiquitin-E2 thioester intermediate. We have used this complex and NMR spectroscopy to show that the UBA domain can bind ubiquitin in a non-covalent fashion even in the presence of an ubiquitin molecule covalently bound at the catalytic domain. These results provide a glimpse at the first step of polyubiquitin chain formation by Ubc1 and its related class II E2 enzymes. Protein Expression and Purification—Recombinant S. cerevisiae Ubc11-150, Ubc1, and ubiquitin (Ub) were overexpressed in Escherichia coli BL21(DE3)pLysS and purified as described previously (12Hodgins R. Gwozd C. Arnason R. Cummings M. Ellison M.J. J. Biol. Chem. 1996; 271: 28766-28771Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Both Ubc1 proteins carried K93R mutations, and ubiquitin contained a K48R change to inhibit autoubiquitination. Uniform 15N labeling of Ubc1 (21Merkley N. Shaw G.S. J. Biomol. NMR. 2003; 26: 147-155Crossref PubMed Scopus (8) Google Scholar) and [Val,Leu,Ile-(δ1)-methyl-protonated-13C,2H,15N]Ubc1 (15Merkley N. Shaw G.S. J. Biol. Chem. 2004; 279: 47139-47147Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 22Goto N.K. Gardner K.H. Mueller G.A. Willis R.C. Kay L.E. J. Biomol. NMR. 1999; 13: 369-374Crossref PubMed Scopus (439) Google Scholar) were obtained from their overexpression in minimal M9 media and purified as described previously. The expression plasmid for ubiquitin carrying a G76C mutation (UbCys) was constructed by PCR from the K48R-ubiquitin template and purified as reported for ubiquitin (12Hodgins R. Gwozd C. Arnason R. Cummings M. Ellison M.J. J. Biol. Chem. 1996; 271: 28766-28771Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) except that dithiothreitol was omitted from buffers. Isotopically labeled UbCys was achieved by overexpression in E. coli BL21(DE3)pLysS in 1 liter of M9 minimal media with 1.0 g/liter 15NH4Cl and 2.0 g/liter [13C6]glucose for uniformly labeled [13C,15N]UbCys. UbCys-Ubc1 Disulfide Complex Formation—Solutions (0.1 mm) of UbCys, Ubc11-150, and Ubc1 were fully reduced with 2 mm tris(2-carboxyethyl)phosphine. An excess of either Ubc11-150 or Ubc1 was combined with UbCys and dialyzed at 4 °C against several changes of 100 mm Na2HPO4/NaH2PO4, 100 mm NaCl, 10 μm CuCl2 at pH 7.5. The progress of disulfide complex formation was monitored by non-reducing SDS-PAGE and was considered complete when the reduced UbCys was exhausted. The protein solution was concentrated and purified by size exclusion chromatography on Sephadex G-75 with 25 mm Tris-HCl, 250 mm NaCl, 1 mm EDTA at pH 7.5. Fractions were analyzed on SDS-PAGE under non-reducing conditions, and those containing pure UbCys-Ubc11-150 or UbCys-Ubc1 were pooled and concentrated for NMR experiments. NMR Spectroscopy—NMR samples of the UbCys-Ubc1 complexes with the following isotopic labeling schemes were used: [13C,15N]UbCys-Ubc11-150, UbCys-[Val,Leu,Ile-(δ1)-methyl-protonated-13C,2H,15N]Ubc1, and UbCys-[15N]Ubc1. All samples were prepared at pH 7.5 in 90% H2O/10% D2O (v/v) with final concentrations of 0.5 mm. NMR experiments were acquired at 35 °C on a Varian INOVA 600 MHz spectrometer with a pulse field gradient triple resonance probe. Sequential assignments for the backbone residues for the [13C,2H,15N]Ubc1 component of UbCys-Ubc1 or the [13C,15N]UbCys portion of the UbCys-Ubc11-150 complex were made from HNCA and HNCACB experiments (23Grzesiek S. Bax A. J. Magn. Reson. 1992; 99: 201-207Google Scholar). NMR spectra were processed with NMRPipe (24Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11527) Google Scholar) and analyzed using NMRView (25Johnson B.A. Belvins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2677) Google Scholar) software on a Sun Ultra 10 work station. Titration of UbCys-[15N]Ubc1 with Ub—The equilibrium dissociation constant for Ub with the UbCys-Ubc1 complex was measured by NMR spectroscopy from 1H-15N HSQC experiments. A solution of the UbCys-[15N]Ubc1 complex (0.5 mm) was prepared in 25 mm Tris, 1 mm EDTA, and 250 mm NaCl at pH 7.5 in 10% D2O/90% H2O (v/v). Increasing amounts of unlabeled Ub (34.8 mm) to a maximum of 4.0 eq Ub:UbCys-Ubc1 were titrated into the sample. Chemical shift deviations were calculated for each isolated residue and analyzed by non-linear regression using the software Prism 4. All protein concentrations were determined in triplicate by amino acid analysis. The class II E2 enzyme Ubc1 from S. cerevisiae is a flexible two-domain protein comprising a canonical N-terminal catalytic domain and a C-terminal UBA domain (Fig. 1). The catalytic domain of Ubc1 contains four α-helices (α1, Lys5-Ala13; α2, Leu102-Leu113; α3, Ala124-Leu131; α4 Arg134-Leu147) and four β-strands (β1, Ile22-Phe26; β2, His34-Leu40; β3, Lys51-Val58; β4, Lys68-Gln70), and the fold is typical for E2 proteins (26Cook W.J. Jeffrey L.C. Xu Y. Chau V. Biochemistry. 1993; 32: 13809-13817Crossref PubMed Scopus (83) Google Scholar). The catalytic cysteine (Cys88) used in the formation of the ubiquitin-E2 thioester intermediate is found in a relatively unstructured region below helix α2 and is the only cysteine in the protein. In Ubc1 and other class II E2 proteins, a C-terminal UBA domain consisting of three α-helices (α5, His170-Glu177; α6, Lys183-Arg191; α7, Asn204-Leu213) is present. Unlike the catalytic domain, the UBA domain is able to interact with ubiquitin in a non-covalent manner (15Merkley N. Shaw G.S. J. Biol. Chem. 2004; 279: 47139-47147Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The ubiquitin-E2 thioester intermediate formed in the ubiquitin-mediated proteolysis pathway occurs between the C-terminal Gly76 carboxylate of ubiquitin and the side-chain thiol of Cys88. Based on a model of the Ubc1-ubiquitin thioester, the length of this linkage from Gly76 Cα to Cys88 Cα is ∼4.8 Å. In attempting to stabilize the linkage between Ubc1 and ubiquitin we wanted to preserve this distance while increasing the stability of this junction to a significant extent. To accomplish this, we replaced the ubiquitin Gly76 with a cysteine residue (UbCys) to form a disulfide linkage between the side chains of Cys76 in ubiquitin and Cys88 in Ubc1. In this process the geometry of Cys88 in Ubc1 should be unaffected. Ubc1 is an ideal candidate to model this disulfide reaction because its sequence contains exclusively the catalytic cysteine preventing any multiple ubiquitin adducts or disulfide exchange between different sites from occurring. Furthermore, in UbCys the cysteine side chain, being at the C terminus of the protein, should be flexible enough to adopt a preferred conformation that may be dictated by interaction with Ubc1. In modeling studies, the span between the Cα atoms of Cys76 in UbCys and Cys88 in Ubc1 was ∼4.9-5.5 Å, a distance similar to that observed in the native ubiquitin-E2 thioester. Formation of a Stable, Covalent Ubiquitin-E2 "Intermediate"—A disulfide-linked complex between UbCys and Ubc1 (UbCys-Ubc1) was formed by mixing fully reduced UbCys with an excess of fully reduced E2 protein in phosphate buffer containing a catalytic amount of Cu2+ as an oxidizing agent. In both cases the K93R mutant of Ubc1 and the K48R mutant of ubiquitin were used. These mutations are utilized to prevent autoubiquitination and polyubiquitin chain formation reactions from occurring, although these reactions were not a factor in our experiments. A disulfide complex between UbCys and a truncated version of Ubc1 lacking the C-terminal domain (Ubc11-150) was also synthesized in addition to the complex utilizing the full-length Ubc1 protein containing the C-terminal UBA domain. Formation of the UbCys-E2 complex occurred for both Ubc1 and Ubc11-150 over a period of 10 h at room temperature at which point the UbCys had been exhausted (Fig. 2A). In both experiments two products were observed, the UbCys-Ubc1 disulfide (or UbCys-Ubc11-150) as the major species and a minor amount of UbCys-UbCys disulfide. Over the same time period and under identical conditions there was little difference between the rates or extent of formation for UbCys-Ubc1 compared with that of the truncated UbCys-Ubc11-150 indicating that the C-terminal UBA domain in Ubc1 does not appear to enhance or inhibit formation of the disulfide. Both UbCys-Ubc1 and UbCys-Ubc11-150 could be readily purified by size exclusion chromatography, and their identities were confirmed by mass spectrometry (UbCys-Ubc1 MWobs 33,270.7, MWcalc 33,274.2; UbCys-Ubc11-150 MWobs 25,421.4, MWcalc 25,420.1). Because Ubc1 and Ubc11-150 contain a single cysteine residue, which is at the catalytic site, the UbCys-Ubc1 (and UbCys-Ubc11-150) disulfide must be formed using the catalytic site cysteine. The complexes were stable in solution for several weeks at room temperature. Formation of a disulfide-linked complex between UbCys and other E2 proteins was tested to assess the general applicability of this method. Both of the human E2 proteins, Ubc13 and UbcH7, formed stable disulfide complexes using the identical conditions for synthesis of UbCys-Ubc1. The sequence of Ubc13 has exclusively the catalytic cysteine and like Ubc1, the reaction afforded a single product, UbCys-Ubc13 (Fig. 2B) identified by mass spectrometry (UbCys-Ubc13 MWobs 26,180.8, MWcalc 26,180.2). A disulfide reaction between UbCys and UbcH7 yielded a single UbCys-UbcH7 disulfide, and multiubiquitin adducts were not observed by non-reducing gel electrophoresis (Fig. 2B), although UbcH7 has a catalytic cysteine residue and two additional cysteine residues. UbCys-Ubc1 Mimics an Ubiquitin-E2 Thioester Intermediate—The instability of the ubiquitin-E2 thioester precluded three-dimensional structure determination of this species and limited characterization of the interface between the two proteins. NMR spectroscopy was used to identify residues in ubiquitin or Ubc11-150 that decreased in intensity during formation of the thioester. However, the stability of the thioester (∼1 h) did not allow sequence-specific assignment of 1H-15N resonances in the Ub-E2 thioester complex thereby limiting this approach (27Hamilton K.S. Ellison M.J. Barber K.R. Williams R.S. Huzil J.T. McKenna S. Ptak C. Glover M. Shaw G.S. Structure (Camb.). 2001; 9: 897-904Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Because UbCys-Ubc1 is orders of magnitude more stable than its thioester counterpart this has now allowed the 1H, 13C, and 15N resonance (backbone) assignment of the 33-kDa UbCys-Ubc1 complex using conventional triple resonance techniques. Fig. 3 shows the 1H-15N HSQC spectrum of the UbCys-Ubc1 complex using 15N-labeled Ubc1 and unlabeled UbCys. In general the spectrum is similar to Ubc1 alone indicating that disulfide formation between UbCys and Ubc1 did not result in global conformational changes in Ubc1. In addition calculation of the Chemical Shift Index (CSI) using CA, CB, and C′ shifts (28Wishart D.S. Sykes B.D. J. Biomol. NMR. 1994; 4: 171-180Crossref PubMed Scopus (1908) Google Scholar) indicated that there was little change in the secondary structure of Ubc1 upon formation of the disulfide complex. Although the inter-residue distance between the active site Cys88 in Ubc1 and the C-terminal Cys76 in UbCys is similar to that in the thioester, it is possible that intermolecular contacts between the two proteins could be modified. A subtle difference also exists between the UbCys-Ubc1 disulfide and the Ub-Ubc1 thioester. Because of the difference in covalent linkage, the UbCys-Ubc1 disulfide possesses a free C-terminal carboxylate that would normally be used to form the thioester bond. Previously, the ubiquitin-Ubc1 interface for the Ub-Ubc11-150 thioester has been partially characterized by following changes in line widths from 1H-15N HSQC spectra upon thioester formation (20Hamilton K.S. Ellison M.J. Shaw G.S. J. Biomol. NMR. 2000; 18: 319-327Crossref PubMed Scopus (29) Google Scholar). The protein-protein interface for the Ub-Ubc11-150 thioester complex was compared with that of UbCys-Ubc11-150 using 15N-labeled UbCys to probe the similarity of the two interfaces for the two complexes. In the absence of Ubc11-150, the 1H-15N HSQC for UbCys is well resolved with most resonances in similar positions to those found in the wild-type protein (Fig. 4A). Upon formation of UbCys-Ubc11-150 several resonances shift to new positions including Arg48, Val70, Leu71, Arg72, Leu73, and Cys76 in UbCys (Fig. 4B). These residues are essentially the same as those identified in the Ub-Ubc11-150 thioester (Fig. 4C) (20Hamilton K.S. Ellison M.J. Shaw G.S. J. Biomol. NMR. 2000; 18: 319-327Crossref PubMed Scopus (29) Google Scholar) and are found in a tight cluster in the 1H-15N HSQC spectra. Histograms of the chemical shift perturbations for both UbCys (Fig. 4D) and Ub (Fig. 4E) upon complex formation have similar patterns indicating that the same residues are affected in ubiquitin at the ubiquitin-E2 interface in both complexes. Minor variations between the histograms may be a result of the changes in experimental conditions (pH and ionic strength) upon sequential addition of the reaction mixture (E1 and ATP) to the Ub, E2 sample, which is required for the synthesis of the Ub-Ubc11-150 thioester in situ (20Hamilton K.S. Ellison M.J. Shaw G.S. J. Biomol. NMR. 2000; 18: 319-327Crossref PubMed Scopus (29) Google Scholar). The UbCys-Ubc11-150 on the other hand could be purified, and the pH and ionic strength was controlled. In addition, residual Ub correlations are present in the 1H-15N HSQC spectrum of the thioester (Fig. 4C) as a result of incomplete thioester formation hindering the analysis of the chemical shift perturbations. Several of the Ubc1 amide resonances in the UbCys-Ubc1 disulfide complex underwent chemical shift changes (residues Lys74, Ser81, Cys88, Trp96, Ile100, Ala105, Ser115, and Asn119) or extensive line broadening (residues Ile87, Leu89, Ile91, and Leu92) as a result of complex formation. These are common to those reported to undergo a decrease in peak intensity in the Ub-Ubc1 thioester intermediate (27Hamilton K.S. Ellison M.J. Barber K.R. Williams R.S. Huzil J.T. McKenna S. Ptak C. Glover M. Shaw G.S. Structure (Camb.). 2001; 9: 897-904Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), indicating that the interacting surface in Ubc1 is similar to that of the thioester intermediate. The interacting residues in both Ubc1 and ubiquitin in the disulfide complex are similar to those observed in the thioester, indicating that the disulfide complex mimics the thioester intermediate, and a minimal influence on the E2 surface is created by the presence of a charged carboxylate group near the active site. Two major advantages exist for the UbCys-Ubc1 disulfide complex. First, the complex contains no side products such as uncomplexed ubiquitin or E2 that can hamper data analysis. For example, in the UbCys-Ubc1 complex, resonances resulting from residues in the "tail" region of ubiquitin (Val70, Leu71, Arg72, and Leu73) are completely absent (Fig. 4B) from their original positions in UbCys (Fig. 4A). However in the Ub-Ubc11-150 thioester (Fig. 4C), remnants of these peaks exist because formation of the thioester only occurs to ∼90% completion. Second, the stability of UbCys-Ubc11-150 has allowed backbone NMR assignments to be obtained for the UbCys component enabling identification of the new resonance positions in the UbCys-Ubc11-150 complex. This was not possible in the thioester due to hydrolysis of the Ub-Ubc11-150 thioester bond under aqueous conditions. Overall, the formation of the UbCys-Ubc11-150 complex not only mimics the Ub-Ubc11-150 thioester structure but also facilitates its analysis. Ubc1 Coordinates Two Ubiquitin Molecules at Different Sites—In addition to the catalytic domain, Ubc1 has a C-terminal UBA domain that non-covalently binds ubiquitin (15Merkley N. Shaw G.S. J. Biol. Chem. 2004; 279: 47139-47147Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). UBA domains bind mono- or polyubiquitin chains with a dissociation constant between 300 and 500 μm (29Mueller T.D. Kamionka M. Feigon J. J. Biol. Chem. 2004; 279: 11926-11936Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 30Ryu K.S. Lee K.J. Bae S.H. Kim B.K. Kim K.A. Choi B.S. J. Biol. Chem. 2003; 278: 36621-36627Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Ubc1 has the potential to coordinate two ubiquitin molecules simultaneously, suggesting that during the Ub-Ubc1 thioester formation process, it is possible that the UBA domain might guide or perturb the interaction of ubiquitin with Ubc1. Alternatively, the UBA domain could also interact with the thioester-bound ubiquitin once the bond is formed at Cys88 of Ubc1. Identification of either of these steps would provide insight into the first step of polyubiquitin chain formation. The UbCys-Ubc1 disulfide allowed us to examine the ubiquitin binding properties of the UBA domain of Ubc1 in the UbCys-Ubc1 disulfide complex. A similar investigation of the ubiquitin binding properties of the UBA in the Ub-Ubc1 thioester complex has not been possible because of the transient nature of the thioester conjugate. We probed the UbCys-Ubc1 complex to determine whether this enzyme could coordinate two ubiquitin molecules concurrently. The UbCys-[15N]Ubc1 complex was titrated with unlabeled ubiquitin, and each addition was followed by 1H-15N HSQC spectroscopy. Fig. 5 shows a portion of the 1H-15N HSQC spectrum of UbCys-[15N]Ubc1 in which resonances Gly180, Asn201, and Asn207 are perturbed upon ubiquitin binding (Fig. 5A). In addition, residues Glu177, Ser178, and Glu211 in the UBA domain of the UbCys-Ubc1 complex are strongly affected by ubiquitin binding (Fig. 5B). Several other residues (His170, Asp199, Asn203, Thr205, Ala206, Arg208, Ile209, and Leu214) in the UBA domain of the UbCys-Ubc1 complex underwent smaller but significant chemical shift changes. Overall the pattern of residues most affected in UbCys-Ubc1, upon interaction with ubiquitin, was specific to the UBA domain of Ubc1. This is remarkably similar to that observed in Ubc1 alone (Fig. 5, B and C). To quantify the interaction of ubiquitin with the UbCys-Ubc1 complex, the 15N chemical shift changes were analyzed by non-linear least-squares fitting (Fig. 6A) for a number of affected residues. This resulted in a dissociation constant of 259 ± 105 μm. The dissociation constant calculated from 15N chemical shift changes for Ubc1 titrated with Ub was 280 ± 69 μm using comparable residues. When the data for the ubiquitin interaction with UbCys-Ubc1 were normalized and compared with the interaction of ubiquitin with Ubc1 alone, the curves were nearly superimposable, reinforcing the similarity of the strength and stoichiometries of the interactions (Fig. 6B).Fig. 6Ubiquitin interacts in a similar manner with both Ubc1 and UbCys-Ubc1. A, changes in 15N chemical shifts for residues Gly180, Asn201, Asn207, and Glu211 as a function of added Ub. B, normalized changes in 15N and 1H chemical shifts for Gly180 for both UbCys-[15N]Ubc1 (□) and [15N]Ubc1 (▪) as a function of added Ub.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Polyubiquitin chains linked through the C-terminal glycine of one ubiquitin to Lys48 of another are a required recognition motif for substrate degradation by the 26 S proteasome. The mechanism in which these polyubiquitin chains are assembled is unclear. Two possibilities exist in which either the polyubiquitin chain is constructed on the E2 protein and transferred to an E3 or substrate, or the chain is built on the target substrate. In both cases the ubiquitin-E2 thioester complex is a key intermediate in this process. The transient nature of this thioester species has not allowed its three-dimensional structure to be determined or permitted direct experimentation showing how the thioester participates in the assembly of polyubiquitin chains. To date, the best structural analysis of the ubiquitin-E2 thioester complex has been found from peak intensity changes in NMR experiments upon thioester formation. This has indicated that the surface on ubiquitin at the protein-protein interface encompasses residues Val70-Gly76 and Arg48 (27Hamilton K.S. Ellison M.J. Barber K.R. Williams R.S. Huzil J.T. McKenna S. Ptak C. Glover M. Shaw G.S. Structure (Camb.). 2001; 9: 897-904Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In the current work, the ubiquitin-E2 disulfide is the first thioester intermediate synthesized that mimics this surface. In the disulfide the ubiquitin surface includes residues Arg48, Val70-Leu73, and Cys76, which are common not only to those in the thioester but also in the UBA domain interface. This indicates the ubiquitin-E2 disulfide is a suitable mimic of the transient ubiquitin-E2 thioester. The ubiquitin-Ubc1 disulfide complex is straightforward to synthesize in high yields and may be purified from any remaining starting materials by size exclusion chromatography. The product is stable for several weeks at room temperature. This is a remarkable improvement from other attempts to stabilize the ubiquitin-E2 linkage. For example, mutation of the active site cysteine in the E2, HsUbc2b, to a serine residue has allowed formation of an ester linkage between ubiquitin and the E2 (31Miura T. Klaus W. Gsell B. Miyamoto C. Senn H. J. Mol. Biol. 1999; 290: 213-228Crossref PubMed Scopus (85) Google Scholar, 32Sullivan M.L. Vierstra R.D. J. Biol. Chem. 1993; 268: 8777-8780Abstract Full Text PDF PubMed Google Scholar, 33Sung P. Prakash S. Prakash L. J. Mol. Biol. 1991; 221: 745-749Crossref PubMed Scopus (52) Google Scholar). Although the resulting complex could be purified, it was short-lived at pH 6.7 and unstable at alkaline pH impeding any attempts to model the association of the two proteins or to use the complex for biochemical characterization (31Miura T. Klaus W. Gsell B. Miyamoto C. Senn H. J. Mol. Biol. 1999; 290: 213-228Crossref PubMed Scopus (85) Google Scholar). However, chemical shift analysis revealed that residues Val70-Gly76 and Lys48 on ubiquitin were most significantly perturbed upon HsUbc2b binding, a similar observation to that obtained for the ubiquitin-Ubc1 thioester and the disulfide complex described here. An alternative procedure traps the thioester intermediate by reducing it to a hemithioacetal with sodium borohydride (NaBH4). This approach was successful in stabilizing the complex between ubiquitin and an ubiquitin hydrolase for several days allowing for structural characterization by NMR spectroscopy (34Rajesh S. Sakamoto T. Iwamoto-Sugai M. Shibata T. Kohno T. Ito Y. Biochemistry. 1999; 38: 9242-9253Crossref PubMed Scopus (21) Google Scholar). Our method for mimicking the ubiquitin-E2 thioester by forming a stable disulfide complex with S. cerevisiae Ubc1, human Ubc13, and UbcH7 E2 proteins can be used for mechanistic or structural studies with ubiquitin and other ubiquitin-conjugating enzymes. Approximately 20% of E2 conjugating enzymes contain exclusively the catalytic cysteine residue (35Winn P.J. Battey J.N. Schleinkofer K. Banerjee A. Wade R.C. Proteins. 2005; 58: 367-375Crossref PubMed Scopus (7) Google Scholar), and this procedure can be used as described in this work. For other conjugating enzymes such as UbcH7, which contains multiple cysteine residues, site-directed mutagenesis may be required to mutate the non-catalytic cysteine residues. In addition, the generality of this procedure illustrates that the disulfide complex designed could be used to investigate the intermediates between ubiquitin and other ubiquitin pathway enzymes, for example, E1 enzymes or HECT E3 ligases. A unique feature of Ubc1 is that it is a flexible two-domain protein that consists of catalytic and UBA domains, representative of several other class II E2 proteins. Upon titration of UbCys-Ubc1 with Ub, the UBA domain is able to non-covalently bind a second ubiquitin molecule. Therefore, Ubc1 has the ability to bind an ubiquitin molecule as a thioester at the active site of the catalytic domain and a second ubiquitin at the C-terminal UBA domain. The ability of Ubc1 to interact with two ubiquitin proteins may explain how Ubc1 and its related class II E2 proteins can build or manipulate polyubiquitin chains. The presence of a UBA domain affects the chain-building properties of two homologous class II E2 proteins in vitro, Ubc1 and E2-25K. Ubc1 undergoes autoubiquitination by assembling polyubiquitin chains at Lys93 near the Cys88 active site (12Hodgins R. Gwozd C. Arnason R. Cummings M. Ellison M.J. J. Biol. Chem. 1996; 271: 28766-28771Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Polyubiquitin chains comprising up to 10-12 ubiquitin molecules are assembled in the absence of the UBA domain. Shorter chains averaging only four ubiquitin molecules are built when full-length Ubc1 is utilized. A flexible tether (22Goto N.K. Gardner K.H. Mueller G.A. Willis R.C. Kay L.E. J. Biomol. NMR. 1999; 13: 369-374Crossref PubMed Scopus (439) Google Scholar amino acids) links the catalytic and UBA domains in Ubc1. This tether is long enough to allow the UBA domain to reach the thioester-bound ubiquitin thereby either aiding or interfering with thioester formation or polyubiquitin chain formation. However, our data show that the UBA domain does not interfere with disulfide formation. Furthermore, the affinity of the UBA domain for ubiquitin is not affected by the presence of a covalently attached ubiquitin at the catalytic site. This indicates that any interaction between the thioester-bound ubiquitin and the UBA domain is either very weak or does not occur. It is possible that the UBA domain may perturb the chain elongation step by potentially interacting with the growing thioester-bound polyubiquitin chain upon assembly of longer polyubiquitin chains at the thioester or on Lys93. A related class II E2 protein, E2-25K, has a flexible two-domain structure similar to Ubc1 and does not autoubiquitinate itself. Instead unanchored polyubiquitin chains are detected in vitro (14Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar). Deletion of the UBA domain in E2-25K results in termination of polyubiquitin chain formation, although ubiquitin-E2 thioester formation proceeds (36Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (120) Google Scholar). Both of these observations are consistent with our observations that the UBA domain in Ubc1 does not affect initial thioester formation and must be involved in construction of polyubiquitin chains. Further structural characterization of the ubiquitin-Ubc1 disulfide should provide some insight into how the UBA domain influences polyubiquitin chain assembly by Ubc1 and other related class II E2 proteins. From a mechanistic perspective, the ability of Ubc1 to bind two ubiquitin molecules may allow the thioester-bound acceptor ubiquitin (Gly76) to be in close proximity to the UBA-bound (donor) ubiquitin, which contains the nucleophilic side-chain ϵ-NH2 of Lys48. Recently, mechanisms for the construction of polyubiquitin chains have been proposed in which the acceptor ubiquitin is near the donor ubiquitin in other E2 complexes. Assembly of Lys63-linked polyubiquitin chains by the canonical E2 (Ubc13) proceeds through a heterodimer of Ubc13 with an inert E2 variant (Mms2) that non-covalently binds ubiquitin (37McKenna S. Hu J. Moraes T. Xiao W. Ellison M.J. Spyracopoulos L. Biochemistry. 2003; 42: 7922-7930Crossref PubMed Scopus (42) Google Scholar). A thioester formed between Ubc13 and ubiquitin accepts a second ubiquitin from Mms2, building up a Lys63-linked chain. The Mms2-Ubc13 complex binds the two ubiquitin molecules required for chain building, keeping them in close proximity. A mechanism for multiubiquitin chain synthesis has been proposed for S. cerevisiae Cdc34 (Ubc3), which has been observed to self-associate in vitro. Dimerization of the Ubc3 is driven by formation of the ubiquitin thioester, and this Ub-Ubc3 thioester dimer with or without E3 participation directs polyubiquitin assembly (38Varelas X. Ptak C. Ellison M.J. Mol. Cell. Biol. 2003; 23: 5388-5400Crossref PubMed Scopus (44) Google Scholar). Both of these mechanisms involve association of two ubiquitin molecules by dimerization of an ubiquitin-E2 thioester or an E2 thioester and an inert E2 variant. Similar to the mechanisms discussed above, self-association of a glutathione S-transferase-tagged E2-25K protein lacking its UBA domain initiates assembly of polyubiquitin chains, thus reversing the effect of deleting the UBA domain (36Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (120) Google Scholar). This may indicate that in E2-25K and Ubc1 the UBA domain and ubiquitin binding could facilitate dimerization of these E2 proteins in a manner similar to Ubc13-Mms2 or Ubc3. Although dimerization of Ubc1 has not been observed (21Merkley N. Shaw G.S. J. Biomol. NMR. 2003; 26: 147-155Crossref PubMed Scopus (8) Google Scholar) our design of the ubiquitin-E2 disulfide thioester mimic is well positioned to probe the potential association of the Ub-Ubc1 intermediate and provide further information on polyubiquitin chain building. We thank L. Spyracopoulos (University of Alberta) for a sample of purified human Ubc13 and P. A. Robinson (University of Leeds) for the plasmid encoding UbcH7. We thank Frank Delaglio (NMRPipe) and Bruce Johnson (NMRView) for software.
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