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• W1996656575 abstract "E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an α/β fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation. E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an α/β fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation. Looking at Ubiquitin Chain Assembly♦Journal of Biological ChemistryVol. 279Issue 45PreviewUbiquitin-conjugating enzymes help to activate ubiquitin for labeling damaged or misfolded proteins for degradation. The most basic (class I) ubiquitin-conjugating enzymes consist of a 150-residue catalytic domain responsible for forming a thiol ester ubiquitin intermediate. The more complex class II proteins have C-terminal extensions on their catalytic domains and are involved in creating polyubiquitin chains. Although three-dimensional structures have been solved for numerous ubiquitin-conjugating enzymes, these structures all consist solely of the catalytic core domain. Full-Text PDF Open Access Labeling of proteins with the molecule ubiquitin is an important cellular function that is required for protein degradation, cell cycle control, stress response, DNA repair, signal transduction, transcriptional regulation, and vesicular traffic (1Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1257) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar, 3Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (960) Google Scholar, 4Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (230) Google Scholar). In this process, the number and topology of the ubiquitin molecule chains underscores the fate of the substrate and determines the biochemical pathway followed. For example, labeling the substrate with a single ubiquitin (monoubiqutination) is important for cellular regulation (5Hicke L. Nat. Rev. Mol. Cell. Biol. 2001; 2: 195-201Crossref PubMed Scopus (994) Google Scholar, 6Di Fiore P.P. Polo S. Hofmann K. Nat. Rev. Mol. Cell. Biol. 2003; 4: 491-497Crossref PubMed Scopus (262) Google Scholar). On the other hand ubiquitin-dependent proteolysis, the process responsible for the turnover of damaged or misfolded proteins in the cell, involves labeling the substrate protein with a polyubiquitin chain that is subsequently recognized by the 26 S proteasome facilitating substrate degradation. The most common polyubiquitin chains are formed via isopeptide bond linkages between the C-terminal (Gly76) of one ubiquitin molecule and the side chain ϵ-NH2 from Lys48 of another. However, other configurations are possible including the Lys63 linkage, which are important in the postreplicative DNA repair pathway (7Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). The ubiquitination degradation pathway is described as a cascade of events in which ubiquitin is passed through three enzymes until it reaches a protein selected for degradation. The first step involves an ATP-dependent activation of ubiquitin by an ubiquitin-activating enzyme (E1) 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; NOE, nuclear Overhauser effect; r.m.s.d., root mean square deviation. forming a high energy E1-ubiquitin thiol ester complex. The activated ubiquitin is then passed from the E1 to an ubiquitin-conjugating enzyme (E2) forming a second thiol ester intermediate between the E2 and ubiquitin. Labeling the target protein with ubiquitin is catalyzed by an E3 ligase protein, either by direct transfer of the ubiquitin to the substrate from the E2 (RING E3) or by thiol ester formation between ubiquitin to an E3 (HECT E3) and subsequent transfer to the substrate (4Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (230) Google Scholar). Some details of this ubiquitin-mediated cascade have been garnered from the structures of several E2 enzymes showing a core 150-residue catalytic α/β fold that is maintained upon complexation with either a HECT (8Huang L. Kinnucan E. Wan G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Science. 1999; 286: 1321-1326Crossref PubMed Scopus (440) Google Scholar) or RING (9Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar, 10Zheng 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 (1169) Google Scholar) E3 ligase or in the E2-ubiquitin thiol ester intermediate (11Hamilton K.S. Ellison M.J. Barber K.R. Williams R.S. Huzil J.T. McKenna S. Ptak C. Glover M. Shaw G.S. Structure (Lond.). 2001; 9: 897-904Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). While these structures show the interactions between the E2, E3, and ubiquitin proteins, details how the transfer of ubiquitin to the substrate occurs and how the polyubiquitin chain is constructed are more uncertain. More recently, three-dimensional structures of the heterodimeric complex between the canonical E2 protein Ubc13 in complex with an E2 variant protein Mms2 have shown how these proteins function together to assemble Lys63-linked polyubiquitin chains (12VanDemark A.P. Hofmann R.M. Tsui C.M.P.C. Wolberger C. Cell. 2001; 105: 711-720Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 13Moraes T.F. Edwards R.A. McKenna S. Pastushok L. Xiao W. Glover J.N. Ellison M.J. Nat. Struct. Biol. 2001; 8: 669-673Crossref PubMed Scopus (138) Google Scholar, 14McKenna S. Moraes T. Pastushok L. Ptak C. Xiao W. Spyracopoulos L. Ellison M.J. J. Biol. Chem. 2003; 278: 13151-13158Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Similar structural details for construction of Lys48-linked polyubiquitin chains are not available, although it has been suggested that dimeric forms of other E2 proteins might also be required (15Ptak C. Prendergast J.A. Hodgins R. Kay C.M. Chau V. Ellison M.J. J. Biol. Chem. 1994; 269: 26539-26545Abstract Full Text PDF PubMed Google Scholar, 16Gwozd C.S. Arnason T.G. Cook W.J. Chau V. Ellison M.J. Biochemistry. 1995; 34: 6296-6302Crossref PubMed Scopus (24) Google Scholar). The E2 conjugating proteins are considered key enzymes in the ubiquitination pathway. All E2 proteins have a 150-residue catalytic domain that is structurally conserved through many species. For example, 11 E2 proteins have been identified in Saccharomyces cerevisiae, and at least 25 are known in mammals. E2 proteins are divided into three classes in which class I enzymes are the simplest and are comprised exclusively of the core catalytic domain that contains the active site cysteine residue required for thiol ester formation with ubiquitin. Several E2 proteins are more complex than the class I members and have either N- or C-terminal extensions. Class II E2 proteins have a C-terminal extension or a “tail,” whereas class III E2 proteins have an additional N-terminal sequence (17Jentsch S. Seufert W. Sommer T. Reins H.A. Trends Biochem. Sci. 1990; 15: 195-198Abstract Full Text PDF PubMed Scopus (124) Google Scholar). One of the key functions of the class II E2 conjugating enzymes is the creation of the polyubiquitin chain required for protein labeling and subsequent degradation. For example the mammalian E2 protein E2-25K is able to synthesize free Lys48-linked polyubiquitin chains in the absence of an E3 enzyme (18Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar). The E2 proteins Ubc1 and Ubc3 (Cdc34) from S. cerevisiae are able to assemble polyubiquitin chains in conjunction with an auto-ubiquitination activity (16Gwozd C.S. Arnason T.G. Cook W.J. Chau V. Ellison M.J. Biochemistry. 1995; 34: 6296-6302Crossref PubMed Scopus (24) Google Scholar, 19Hodgins 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). A variety of other mechanistically distinct functions have been identified for the C-terminal extensions in class II E2 enzymes including dimerization (20Leggett D.S. Candido E.P.M. Biochem. J. 1997; 327: 357-361Crossref PubMed Scopus (14) Google Scholar), substrate recognition (21Gosink M.M. Vierstra R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9117-9121Crossref PubMed Scopus (37) Google Scholar), and anchoring of specific E2 enzymes to the cystolic side of the endoplasmic reticulum (22Sommer T. Jentsch S. Nature. 1993; 365: 176-179Crossref PubMed Scopus (282) Google Scholar). Despite the diverse range of functions, and the integral nature of class II E2 conjugating proteins in the ubiquitination pathway, a three-dimensional structure of one of these proteins has not been reported. In this work we present the solution structure of the 215-residue protein Ubc1, a class II E2 conjugating enzyme from S. cerevisiae shown to be important for degradation of short-lived proteins especially during the G0-G1 transition accompanying spore germination (23Seufert W. McGrath J.P. Jentsch S. EMBO J. 1990; 9: 4535-4541Crossref PubMed Scopus (122) Google Scholar). We show that Ubc1 is a unique two-domain E2 enzyme containing a canonical catalytic domain and a C-terminal UBA domain separated by a flexible tether. Furthermore, we have used NMR chemical shift analysis to determine that ubiquitin binds to the Ubc1 UBA domain with little or no perturbation of the catalytic domain. Our structural results and sequence analysis indicate several other class II E2 proteins, including mammalian E2-25K, likely have this two domain architecture. We have used this information to provide insights into Lys48-linked polyubiquitin chain formation by these class II E2 enzymes. Protein Expression and Purification—Uniformly 15N- and 15N,13C-labeled Ubc1(R48K) was overexpressed in Escherichia coli BL21(DE3)pLysS strain and purified as described previously (24Merkley N. Shaw G.S. J. Biomol. NMR. 2003; 26: 147-155Crossref PubMed Scopus (8) Google Scholar). Uniformly 2H,15N,13C-labeled and Val, Leu, Ile (δ1) methyl-protonated 15N,13C,2H-labeled Ubc1 were expressed in 1 liter of M9 minimal media containing 1.0g/L 99% of 15NH4Cl, 2.0 g/liter of 99% [13C6; 1,2,3,4,5,6,6-2H7]glucose in 100% D2O (25Gardner K.H. Kay L.E. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 357-406Crossref PubMed Scopus (511) Google Scholar). For the Val, Leu, Ile (δ1) methyl-protonated 15N,13C,2H-labeled protein, [13C5;3-2H]α-ketoisovaleric acid, sodium salt (85 mg/liter) and [13C4;3,3-2H2]α-ketobutyric acid, sodium salt (50 mg/liter) were added 1 h before induction with isopropyl β-d-thiogalactopyranoside and induced for a maximum of 4 h (26Goto N.K. Gardner K.H. Mueller G.A. Willis R.C. Kay L.E. J. Biomol. NMR. 1999; 13: 369-374Crossref PubMed Scopus (440) Google Scholar). Fractionally 13C-labeled Ubc1 was prepared by growing the cells in minimal M9 media with a 1:10 mixture of 99% [13C6]glucose and unlabeled glucose (27Neri D. Szyperski T. Otting G. Senn H. Wuthrich K. Biochemistry. 1989; 28: 7510-7516Crossref PubMed Scopus (567) Google Scholar). Pure protein fractions from gel filtration chromatography were pooled and concentrated. NMR samples were prepared at pH 7.5 in 90% H2O/10% D2O (v/v) with a final concentration of 0.48 mm. Samples in 100% D2O were prepared by exchanging buffer with 25 mm Tris, 1 mm EDTA, 1 mm dithiothreitol, and 150 mm NaCl in 100% D2O using an ultrafiltration cartridge (Mr cutoff 10,000). NMR Spectroscopy and Structure Determination—NMR experiments were acquired at 35 °C on Varian 500-, 600-, or 800-MHz spectrometers with pulse field gradient triple resonance probes. Sequential assignments for the backbone residues were made from HNCA, HNCACB, CBCA(CO)NH, HNCO, 15N-edited TOCSY, and 1H-15N HSQC experiments (28Grzesiek S. Bax A. J. Magn. Reson. 1992; 99: 201-207Google Scholar). Side chain assignments were made from, C(CO)NH, HC(CO)NH (29Grzesiek S. Anglister J. Bax A. J. Magn. Reson. Ser. B. 1993; 101: 114-119Crossref Scopus (585) Google Scholar), HCCH-TOCSY (30Kay L.E. Xu G. Singer A.U. Muhandiram D.R. Forman-Kay J.D. J. Magn. Reson. 1993; 101: 333-337Crossref Scopus (558) Google Scholar) (100% D2O), and 1H-13C HSQC experiments. Resonance assignments for the 1H, 13C, and 15N atoms have been deposited in the BioMagResBank under the accession number BMRB-6202 (www.bmrb.wisc.edu). Stereospecific assignment of the prochiral methyl groups from valine and leucine was achieved from analysis of a constant time 1H-13C HSQC experiment using a fractionally 13C-labeled Ubc1 sample. Interproton distances were measured from 15N-NOESY (mixing time 150 ms) (31Zhang O. Kay L.E. Olivier J.P. Forman-Kay J.D. J. Biomol. NMR. 1994; 4: 845-858Crossref PubMed Scopus (612) Google Scholar) and 13C-NOESY (mixing time 100 ms, in 100% D2O) (32Muhandriam D.R. Farrow N.A. Xu G. Smallcombe S.H. Kay L.E. J. Magn. Reson. Ser. B. 1993; 102: 317-321Crossref Scopus (181) Google Scholar) experiments collected on a Varian INOVA 800-MHz spectrometer. Amide-amide interproton distances were measured from an 15N-NOESY with a mixing time of 300 ms, collected on a Varian INOVA 600-MHz spectrometer using an uniformly 2H,13C,15N-labeled Ubc1 sample. Assignment of the leucine, valine methyl and isoleucine (Cδ1, Hδ1) protons, and 13C resonances were facilitated using a sample of Val, Leu, Ile (δ1) methyl-protonated 15N,13C,2H-labeled Ubc1 in which the C(CO)NH and HC(CO)NH afforded correlations exclusively with these methyls. Methyl-methyl interproton distances were measured from a methyl-methyl NOE experiment (mixing time: 100 ms) (33Zwahlen C. Gardner K.H. Sarma S.P. horita D.A. Byrd R.A. Kay L.E. J. Am. Chem. Soc. 1998; 120: 7617-7625Crossref Scopus (82) Google Scholar) and 13C-NOESY (13C carrier set to 19.6 ppm; 13C spectral window: 3200 Hz; mixing time: 100 ms) using the selective methyl-protonated sample of Ubc1. Heteronuclear 15N{1H} NOEs were measured using the methods of Farrow et al. (34Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2013) Google Scholar) using a 3-s irradiation period and a 2-s or a 5-s relaxation delay. Experiments were done in triplicate and averaged. 1H-15N residual dipolar couplings were measured using IPAP-HSQC experiments (35Ottiger M. Delaglio F. Bax A. J. Magn. Reson. 1998; 131: 373-378Crossref PubMed Scopus (841) Google Scholar) using a 0.4 mm sample of 15N-labeled Ubc1 in 12 mg/ml pf1 phage. NMR spectra were processed using NMRPipe (36Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11566) Google Scholar) and analyzed using Pipp and Stapp (37Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar) or NMRView (38Johnson B.A. Belvins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2678) Google Scholar) software using a Sun Ultra 10 workstation. Structures of Ubc1 were calculated using the simulated annealing protocol in the program CNS (39Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). The structures of Ubc1 were generated from 1864 NOEs, including 858 intraresidue, 363 sequential, 357 short range and 286 long range and 63 hydrogen bond distance restraints, 194 dihedral angle restraints, and included 29 1H-15N residual dipolar couplings for residues 170–215. Interproton distances were calibrated for all proton pairs using maximum and minimum nOe intensities for known possible dNHα distances. Dihedral restraints were determined from the program TALOS (torsion angle likelihood obtained from shifts and sequence similarity) (40Cornilescu G. Delagio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar) based on the Cα, Cβ, C′, Hα, and N chemical shift information for Ubc1 where 9 out of 10 predictions fell in the same region of the Ramachandran plot. The resulting φ and Ψ angles were restricted to two times the error from the TALOS output for the structure calculations. Hydrogen bonds were identified from the slowly exchanging NH residues after 2-h incubation after solvent exchange. For each hydrogen bond, two distance restraints were used, NH–O (1.8–2.3 Å) and N–O (2.3–3.3 Å). 1H-15N residual dipolar couplings were included in the structure calculations with a precision equal to the digital resolution of the HSQC spectra (±0.75 Hz). Values for Da and R were estimated from the program DYNAMO using a preliminary fold from CNS trials and measured 1H-15N residual dipolar couplings. Final and initial force constants were 0.01 and 1.0 kcal/mol, respectively. Structural similarity searches were performed using the DALI web server (41Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3565) Google Scholar), and sequences were aligned using T-Coffee (42Notredame C. Higgins D.G. Heringa J. J. Mol. Biol. 2000; 302: 205-217Crossref PubMed Scopus (5476) Google Scholar). Ubc1-Ubiquitin Titrations—The equilibrium dissociation constant for the Ubc1-ubiquitin complex was measured by NMR spectroscopy using 1H-15N HSQC experiments. 15N,13C-labeled Ubc1 was titrated with increasing amounts of unlabeled ubiquitin up to 4 equivalents. Protein samples were made up in 25 mm Tris, 1 mm EDTA, 1 mm dithiothreitol, and 150 mm NaCl at pH 7.5 in 10% D2O/90% H2O (v/v), and protein concentration was determined by amino acid analysis, ubiquitin (14.9 mm) and Ubc1 (0.48 mm). 1H-15N HSQC spectra were recorded after each ubiquitin addition. Chemical shift deviations were calculated for each isolated residue that underwent a significant change. Non-linear regression analysis assumed a single binding site (43Marsden B.J. Hodges R.S. Sykes B.D. Biochemistry. 1988; 27: 4198-4206Crossref PubMed Scopus (62) Google Scholar) and were fitted using the software Prism 4. Five NMR samples of 100 μm15N-labeled ubiquitin in 25 mm Tris, 1 mm EDTA, 1 mm dithiothreitol, and 150 mm NaCl at pH 7.5 (10% D2O/90% H2O) with increasing concentrations of unlabeled Ubc1 (0, 0.25, 0.5, 1.0, 2.0, and 4.0 equivalents) were used to measure the chemical shift perturbation of ubiquitin upon binding the C-terminal tail. 1H-15N HSQC spectra were recorded for each sample and chemical shifts changes were quantified using the equation ΣΔδ = 0.5(|Δδ(1H)| + 0.125*|Δδ(15N)|). Coordinate Deposition—The atomic coordinates for the structures of Ubc1 have been deposited in the Protein Data Bank under the accession code 1TTE. The Class II E2 Enzyme Ubc1 Is a Flexible Two-domain Protein—The ubiquitin-conjugating enzyme Ubc1 from S. cerevisiae is a typical class II E2 enzyme possessing a 150-residue catalytic domain and a 65-residue C-terminal tail region. The length of the C terminus is intermediate of those found for Ubc2 (23 residues) (44Morrison A. Miller E.J. Prakash L. Mol. Cell. Biol. 1988; 8: 1179-1185Crossref PubMed Scopus (78) Google Scholar) and Ubc3 (125 residues) (15Ptak C. Prendergast J.A. Hodgins R. Kay C.M. Chau V. Ellison M.J. J. Biol. Chem. 1994; 269: 26539-26545Abstract Full Text PDF PubMed Google Scholar). The three-dimensional structure of Ubc1 was determined by NMR spectroscopy using standard triple resonance and 15N-edited, 13C-edited, and methyl-specific NOE experiments. A family of 21 structures was chosen (Fig. 1) based on their low energy and the absence of distance violations (Table I). The structure of Ubc1 contains two individually well defined domains tethered by a 22-residue linker. The first 150 residues of Ubc1 (Fig. 1, B and C) comprise the catalytic domain of the enzyme defined by 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). This domain is well defined having a backbone r.m.s.d. of 0.78 ± 0.12 Å. The α/β fold of Ubc1 is similar to that observed in crystal structures of class I E2 proteins including Ubc2 (45Worthylake D.K. Prakash S. Prakash L. Hill C.P. J. Biol. Chem. 1998; 273: 6271-6276Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), Ubc4 (46Cook W.J. Jeffrey L.C. Xu Y. Chau V. Biochemistry. 1993; 32: 13809-13817Crossref PubMed Scopus (83) Google Scholar), Ubc7 (47Cook W.J. Martin P.D. Edwards B.F. Yamazaki R.K. Chau V. Biochemistry. 1997; 36: 1621-1627Crossref PubMed Scopus (60) Google Scholar), and Ubc2b (48Miura T. Klaus W. Ross A. Guntert P. Senn H. J. Biolmol. NMR. 2002; 22: 89-92Crossref PubMed Scopus (13) Google Scholar). For example, the r.m.s.d. for the backbone atoms (N, Cα, C′) in Ubc1 to those for the x-ray crystal structure of a truncated mutant of Ubc1 (residues 1–150) (11Hamilton K.S. Ellison M.J. Barber K.R. Williams R.S. Huzil J.T. McKenna S. Ptak C. Glover M. Shaw G.S. Structure (Lond.). 2001; 9: 897-904Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) is ∼1.40 Å. The catalytic cysteine in Ubc1, Cys88, is located below helix α2, in a position common to all E2 conjugating enzyme structures to date.Table IStructural statistics for the 21 lowest energy structures of Ubc1Restraints for structure calculationsaAll statistics were calculated from a family of 21 structuresIntraresidue NOEs858Sequential NOEs363Short range NOEs357Long range NOEs286Hydrogen bonds63Dihedral angles194NH residual dipolar couplings29b15N-1H residual dipolar couplings were included for α5, α6, and α7 of the tail domain of Ubc1Energies (kcal mol-1)ENOE17.9 ± 1.8Ecdih0.76 ± 0.23Erepel58.26 ± 9.35EL-JcLennard-Jones van der Waals energy was calculated using CHARMM empirical energy and was not included in the structure calculation-560.1 ± 29.3Esani20.6 ± 2.7r.m.s.d. from experimental restraintsDihedral angles (°)0.25 ± 0.04Distances (Å)0.011 ± 0.006r.m.s.d. from idealized geometryBond (Å)0.0014 ± 0.00008Bond angles (°)0.309 ± 0.007Improper torsions (°)0.168 ± 0.010Residual dipolar couplingsr.m.s.d. (Hz)0.87 ± 0.05Q-value0.20 ± 0.01Ramachandran plot statisticsResidues in favorable regionsdThis reflects residues in both most favored and additionally favored regions93.0%r.m.s.d. to mean (Å)Catalytic domain (1-150)eCalculation of r.m.s.d. included Cα, N, C′ for helices 1-4 and strands 1-4Backbone atoms0.78 ± 0.12Heavy atoms1.37 ± 0.10Tail domain (170-215)fCalculation of r.m.s.d. included Cα, N, C′ for helices 5-7Backbone atoms0.35 ± 0.06Heavy atoms0.99 ± 0.09a All statistics were calculated from a family of 21 structuresb 15N-1H residual dipolar couplings were included for α5, α6, and α7 of the tail domain of Ubc1c Lennard-Jones van der Waals energy was calculated using CHARMM empirical energy and was not included in the structure calculationd This reflects residues in both most favored and additionally favored regionse Calculation of r.m.s.d. included Cα, N, C′ for helices 1-4 and strands 1-4f Calculation of r.m.s.d. included Cα, N, C′ for helices 5-7 Open table in a new tab The C-terminal tail region of Ubc1 (Fig. 1, D and E) is comprised of a three α-helix bundle (α5, His170–Glu177; α6, Lys183–Arg191; α7, Asn204–Leu213) having a tightly packed, well defined structure (r.m.s.d. of 0.35 ± 0.06 Å). Helix α5, which starts immediately after the tether region, runs roughly in the same direction as α7 (Ω5,7 = 54 ± 5°). These two helices are separated by helix α6, which lies across both helices α5 and α7 (Ω5,6 = 128 ± 3°, Ω6,7 = 130 ± 3°). The core of the three-helix bundle in Ubc1 is composed of Ile173 and Phe176 (α5) that pack against residues Ile186 and Leu190 (α6) and Ile209, Ile210, and Leu213 (α7). A 12-residue segment located between helices α6 and α7 forms an extended loop. This region is largely solvent exposed consistent with its high proportion of polar residues. The exceptions are Leu193, Val195, and Leu198 that cluster with Thr205 (α7) and Leu172 (α5) and Lys196 that interacts with Ile168 prior to helix α5. Together these contacts force the loop between helices α6 and α7 to lean toward the N terminus of helix α5. A 22-residue flexible tether links the catalytic domain of Ubc1 to its C-terminal domain. During the structure calculations it became apparent this connecting region contained little regular structure due to the absence of long range NOE contacts to either the catalytic or C-terminal domains and the observation of very few sequential and short range NOEs. Furthermore, no long range NOEs between the catalytic domain and C-terminal domain were observed, indicating that they must be isolated from each other. Previous chemical shift perturbation studies indicated that the C-terminal domain might interact with the catalytic domain, proximal to the start of the tether region (Ser150). The structure of Ubc1 (Fig. 1A) shows that the C-terminal domain adopts many orientations with respect to the catalytic domain, most which envelope the β1, β2, and α4 regions, the sites of the largest chemical shift changes observed previously. Since NOEs were not observed between the two domains it would appear that the chemical shift changes are largely a result of transient interactions or the proximity of the C-terminal domain (in sequence) to the catalytic domain junction. To establish the relationship between the domains, 15N{1H} heteronuclear NOE experiments were performed to determine whether the two domains had similar relaxation properties that would indicate a more globular structure or different properties that would indicate a more elongated structure with two independent domains. Analysis of the 15N{1H} heteronuclear NOE measurements indicated there was a clear difference between the average NOE from the catalytic and the C-terminal domains (Fig. 2). The catalytic domain had an average NOE of about 0.72, consistent with a τm of about 4.4 ns. This average NOE was very similar to that obtained for UBC9 (0.74), a 15-kDa ubiquitin-like conjugating enzyme that lacks a C-terminal domain (49Liu Q. Yuan Y.-C. Shen B. Chen D.J. Chen Y. Biochemistry. 1999; 38: 1415-1425Crossref PubMed Scopus (28) Google Scholar). UBC9 displays a similar α/β tertiary fold when compared with the catalytic domain of Ubc1 and also exhibits a more oblate shape, typical of the structures of other E2 catalytic domains. In contrast the C-terminal domain of Ubc1 had an average NOE of about 0.56, indicating that it has a smaller volume than the catalytic domain and is not associated with it. The average NOE for the C-terminal domain translates to a τm of about 2.6 ns. From this value, an approximate molecular mass of 5 kDa is estimated, in excellent agreement with that expected for residues His170–Lys215 (4.9 kDa). Furthermore, the heteronuclear NOE experiments showed that residues Asn154, Gly155, Gln156, and Lys157 had negative NOE values indicative of a region with high mobility on the nanosecond time scale. These observations are similar to those found for other bilobate proteins such as calmodulin (50Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (890) Google Scholar) and the cytokenesis protein Cdc4p (51Slupsky C.M. Desautels M. Huebert T. Zhao R. Hemmingsen S.M. McIntosh L.P. J. Biol. Chem. 2001; 276: 5943-5951Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Both of these prot" @default.
• W1996656575 created "2016-06-24" @default.
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• W1996656575 date "2004-11-01" @default.
• W1996656575 modified "2023-10-04" @default.
• W1996656575 title "Solution Structure of the Flexible Class II Ubiquitin-conjugating Enzyme Ubc1 Provides Insights for Polyubiquitin Chain Assembly" @default.
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• W1996656575 doi "https://doi.org/10.1074/jbc.m409576200" @default.
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