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• W1985891703 abstract "RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain. RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain. The intracellular destiny of many proteins in the cell is governed by their post-translational modification with the covalent attachment of ubiquitin. This process, known as ubiquitylation, plays a pivotal role in numerous cellular processes including protein turnover, cell cycle progression, transcriptional regulation, DNA repair, and signal transduction (1.Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6879) Google Scholar). Ubiquitylation involves the sequential transfer of a ubiquitin molecule through an enzyme cascade consisting of a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3) until it forms an isopeptide bond between the C terminus of ubiquitin and the ϵ-amino group of a lysine on a substrate protein. The E2-E3 combination governs the specificity of the target protein for modification and the site of attachment to the substrate protein as well as the chain length and type of linkage between the ubiquitin molecules attached (2.Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1875) Google Scholar). The really interesting new gene (RING) domain-containing E3 ligases function primarily as scaffolds that orient the E2∼ubiquitin thiol ester complex as well as the substrate protein for efficient ubiquitin transfer. To date, >650 different human RING E3 ligases have been identified (2.Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1875) Google Scholar). The cullin-RING-ligases (CRLs) 3The abbreviations used are: CRLCullin RING ligaseUbCysK48R/G76C substituted ubiquitinCDC34-UbCysdisulfide complex between CDC34 and UbCysRbx1/ROC1RING box protein 1SCFSkp1/Cullin 1/F-boxHSQCheteronuclear single quantum coherenceTEVtobacco etch virus. comprise the largest superfamily of these enzymes and consist of a cullin scaffold (CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, or CUL7) with a RING-containing catalytic protein, either RING-box protein 1 (Rbx1, also referred to as ROC1 and Hrt1), or RING-box protein 2 (Rbx2, aka ROC2/Hrt2) bound at its C-terminal catalytic core (3.Petroski M.D. Deshaies R.J. Function and regulation of cullin-RING ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1678) Google Scholar, 4.Zimmerman E.S. Schulman B.A. Zheng N. Structural assembly of cullin-RING ubiquitin ligase complexes.Curr. Opin. Struct. Biol. 2010; 20: 714-721Crossref PubMed Scopus (176) Google Scholar, 5.Duda D.M. Scott D.C. Calabrese M.F. Zimmerman E.S. Zheng N. Schulman B.A. Structural regulation of cullin-RING ubiquitin ligase complexes.Curr. Opin. Struct. Biol. 2011; 21: 257-264Crossref PubMed Scopus (152) Google Scholar). The Rbx1/ROC1 or Rbx2/ROC2 proteins are responsible for recognizing and recruiting the E2 conjugating enzyme during ubiquitin transfer to a target protein. Crystal structures of multiple CRLs demonstrate they form elongated structures with a long stalk-like N-terminal domain that binds to an adaptor (such as Skp1; CUL3 is the only exception to this rule) and various substrate recognition factors (such as F-box proteins) to recognize different substrate proteins (6.Angers S. Li T. Yi X. MacCoss M.J. Moon R.T. Zheng N. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.Nature. 2006; 443: 590-593Crossref PubMed Scopus (518) Google Scholar, 7.Zheng 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. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.Nature. 2002; 416: 703-709Crossref PubMed Scopus (1164) Google Scholar, 8.Goldenberg S.J. Cascio T.C. Shumway S.D. Garbutt K.C. Liu J. Xiong Y. Zheng N. Structure of the Cand1-Cul1-Roc1 complex reveals regulatory mechanisms for the assembly of the multisubunit cullin-dependent ubiquitin ligases.Cell. 2004; 119: 517-528Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 9.Duda D.M. Borg L.A. Scott D.C. Hunt H.W. Hammel M. Schulman B.A. Structural insights into NEDD8 activation of cullin-RING ligases. Conformational control of conjugation.Cell. 2008; 134: 995-1006Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar), whereas the C terminus forms a globular α/β domain that binds to the Rbx/ROC protein through an intermolecular β-sheet (7.Zheng 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. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.Nature. 2002; 416: 703-709Crossref PubMed Scopus (1164) Google Scholar). Recent structural and biochemical studies show that the covalent attachment of the ubiquitin-like modifier protein NEDD8 to a conserved lysine residue near the C terminus of the cullin scaffold protein causes a conformational change in the C-terminal domain of CUL1 liberating the RING-domain of Rbx1/ROC1 (9.Duda D.M. Borg L.A. Scott D.C. Hunt H.W. Hammel M. Schulman B.A. Structural insights into NEDD8 activation of cullin-RING ligases. Conformational control of conjugation.Cell. 2008; 134: 995-1006Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). The increased mobility of Rbx1/ROC1 is believed to enhance its rate of ubiquitin transfer from the E2 to the substrate protein (10.Deshaies R.J. Emberley E.D. Saha A. Control of cullin-ring ubiquitin ligase activity by nedd8.Subcell Biochem. 2010; 54: 41-56Crossref PubMed Scopus (76) Google Scholar, 11.Boh B.K. Smith P.G. Hagen T. Neddylation-induced conformational control regulates Cullin RING ligase activity in vivo.J. Mol. Biol. 2011; 409: 136-145Crossref PubMed Scopus (47) Google Scholar). This modulation of Rbx1/ROC1-based E3 ligases by cullin and NEDD8 is unique among the RING E3 ligases and represents one of the most intriguing and important regulation steps in the ubiquitination process. Cullin RING ligase K48R/G76C substituted ubiquitin disulfide complex between CDC34 and UbCys RING box protein 1 Skp1/Cullin 1/F-box heteronuclear single quantum coherence tobacco etch virus. The best characterized CRL E3 complex to date both biochemically and structurally is the Skp1/Cullin 1/F-box (SCF) ligase complex, which consists of Rbx1/ROC1 bound to the CUL1 catalytic C-terminal domain. UbcH5 and CDC34 (cell division cycle protein 34) are the two E2 enzymes known to directly interact and function with the SCF in humans (12.Saha A. Deshaies R.J. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation.Mol. Cell. 2008; 32: 21-31Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) with each being responsible for different facets in ubiquitylation on a target protein; UbcH5 attaches the initial ubiquitin to the substrate, whereas CDC34 is responsible for the successive addition of ubiquitin molecules to the substrate during polyubiquitin chain formation (13.Wu K. Kovacev J. Pan Z.Q. Priming and extending. A UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate.Mol. Cell. 2010; 37: 784-796Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). A recent structural and functional study demonstrated that CDC34 can be allosterically inhibited by a small molecule that impedes the release of its ubiquitin cargo at the E2-E3 step (14.Ceccarelli D.F. Tang X. Pelletier B. Orlicky S. Xie W. Plantevin V. Neculai D. Chou Y.C. Ogunjimi A. Al-Hakim A. Varelas X. Koszela J. Wasney G.A. Vedadi M. Dhe-Paganon S. Cox S. Xu S. Lopez-Girona A. Mercurio F. Wrana J. Durocher D. Meloche S. Webb D.R. Tyers M. Sicheri F. An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.Cell. 2011; 145: 1075-1087Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), demonstrating that inhibition of CDC34-dependent polyubiquitylation might represent a possible step for therapeutic intervention. The acidic C terminus of CDC34 has recently been shown to interact with ubiquitin (15.Spratt D.E. Shaw G.S. Association of the disordered C terminus of CDC34 with a catalytically bound ubiquitin.J. Mol. Biol. 2011; 407: 425-438Crossref PubMed Scopus (15) Google Scholar) as well as the C-terminal domain of CUL1 (16.Kleiger G. Saha A. Lewis S. Kuhlman B. Deshaies R.J. Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates.Cell. 2009; 139: 957-968Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 17.Kleiger G. Hao B. Mohl D.A. Deshaies R.J. The acidic tail of the Cdc34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCdc4.J. Biol. Chem. 2009; 284: 36012-36023Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Although these observations suggest that the C terminus of CDC34 is important for bringing CDC34 and the SCF into closer proximity, the structural basis for the functional interaction between the catalytic domain of CDC34 and Rbx1/ROC1 remains unclear. Determination of the structural rationale for the enhanced activity of CDC34 bound to the RING E3 adaptor protein Rbx1/ROC1 would help to further clarify the mechanism of processive ubiquitination for this E2-E3 complex. In this study the solution structure of Rbx1/ROC1 in the absence of its cullin scaffold was determined. The structure shows that the N terminus of Rbx1/ROC1 undergoes a large structural rearrangement from a disordered conformation to a structured β-strand in the CRL complex. Furthermore, we used NMR spectroscopy to show that Rbx1/ROC1 displays poor affinity for CDC34 alone that is increased by more than 50-fold for CDC34 in its ubiquitin-loaded form. Using biochemical reconstitution assays, we demonstrate that the cullin-free Rbx1/ROC1 is capable of activating the catalytic core of CDC34. We also identify key residues in helix α2 of Rbx1/ROC1 that are important for binding and activating CDC34∼ubiquitin. This study provides the first structural details showing that the CDC34∼ubiquitin complex is recruited by Rbx1/ROC1 and functions within the sequential model for polyubiquitylation (18.Petroski M.D. Kleiger G. Deshaies R.J. Evaluation of a diffusion-driven mechanism for substrate ubiquitination by the SCF-Cdc34 ubiquitin ligase complex.Mol. Cell. 2006; 24: 523-534Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 19.Pierce N.W. Kleiger G. Shan S.O. Deshaies R.J. Detection of sequential polyubiquitylation on a millisecond timescale.Nature. 2009; 462: 615-619Crossref PubMed Scopus (161) Google Scholar). The open reading frame of Rbx1/ROC1 was PCR-amplified from pcDNA3-Myc3-ROC1 (Addgene, Cambridge MA) with flanking NdeI and HindIII restriction sites. Primers used for cloning full-length Rbx1/ROC1 were Rbx1_fr (5′-AATCTAGACATATGGCGGCAGCGATGGATGTGGATACCC-3′) and Rbx1_rv (5′-AACTCGAGAAGCTTTTATTACTAGTGCCCATACTTTTGG-3′). The resulting PCR product was then cloned into pET28a that contained an N-terminal 6-histidine tag followed by a TEV cleavage site (MGSSHHHHHHSSGGRENLYFQ) to facilitate the removal of the tag during purification. This initial construct was then shortened from residues 1–108 to 12–108 and 36–108 using site-directed mutagenesis based upon the observed residues in the crystal structure of CUL1-Rbx1/ROC1, PDB code 1LDJ (7.Zheng 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. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.Nature. 2002; 416: 703-709Crossref PubMed Scopus (1164) Google Scholar). The designed Rbx1/ROC112–108 and Rbx1/ROC136–108 constructs also had three extra glycine residues added between the TEV cleavage site and G12 to improve flexibility for TEV cleavage. The primers used to make Rbx1 12–108 were Rbx1d12fr (5′-AACTTGTATTTCCAGGGCGGCGGCGGCACCAACAGCGGCG-3′) and Rbx1d12rv (5′-CGCCGCTGTTGGTGCCGCCGCCGCCCTGGAAATACAAGTT-3′. The primers used to make Rbx1/ROC136–108 were Rbx1d36fr (5′-AACTTGTATTTCCAGGGCGGCGGCGGCGATATTGTGGTTGATAACTG-3′) and Rbx1d36rv (5′-CAGTTATCAACCACAATATCGCCGCCGCCGCCCTGGAAATACAAGTT3′). To improve the solubility of Rbx1/ROC112–108, four mutations (W27S, V30S, L32Q, and W33S) were incorporated into the N-terminal region of Rbx1/ROC1 that was predicted to have aggregation propensity by the online algorithm TANGO (20.Fernandez-Escamilla A.M. Rousseau F. Schymkowitz J. Serrano L. Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins.Nat. Biotechnol. 2004; 22: 1302-1306Crossref PubMed Scopus (1192) Google Scholar, 21.Linding R. Schymkowitz J. Rousseau F. Diella F. Serrano L. A comparative study of the relationship between protein structure and β-aggregation in globular and intrinsically disordered proteins.J. Mol. Biol. 2004; 342: 345-353Crossref PubMed Scopus (307) Google Scholar, 22.Rousseau F. Schymkowitz J. Serrano L. Protein aggregation and amyloidosis. Confusion of the kinds?.Curr. Opin. Struct. Biol. 2006; 16: 118-126Crossref PubMed Scopus (283) Google Scholar). These mutations were made using the primers Rbx1SSQSfr (5′-CGCTTTGAAGTGAAAAAGAGTAATGCAAGTGCTCAGAGTGCCTGGGATATTGTGG-3′) and Rbx1SSQSrv (5′-CCACAATATCCCAGGCACTCTGAGCACTTGCATTACTCTTTTTCACTTCAAAGCG-3′).The resulting vectors, pET28a-HisTEV-sRbx112–108 (where “sRbx1” stands for solubilized Rbx1) and pET28a-HisTEV-Rbx136–108, were verified by DNA sequencing (Robarts Research Institute, University of Western Ontario). Site-directed mutagenesis was used to substitute Trp-87, Lys-89, and Lys-91 to alanines using the following primers: for W87A, Rbx1W87Afr (5′-CCACTGCATCTCTCGGGCACTCAAAACACGACAGG-3′) and Rbx1W87Arv (5′-CCTGTCGTGTTTTGAGTGCCCGAGAGATGCAGTGG-3′); for K89A, Rbx1K89Afr (5′-GCATCTCTCGCTGGCTCGCAACACGACAGGTGTGTCC-3′) and Rbx1K89Arv (5′-GGACACACCTGTCGTGTTGCGAGCCAGCGAGAGATGC-3′); for K89A/R91A, Rbx1K89R91fr (5′-CATCTCTCGCTGGCTCGCAACCGCACAGGTGTGTCCATTGG-3′) and Rbx1K89R91rv (5′-CCAATGGACACACCTGTGCGGTTGCGAGCCAGCGAGAGATG-3′); for R91A, Rbx1R91Afr (5′-CTCGCTGGCTCAAAACAGCACAGGTGTGTCCATTGG-3′)and Rbx1R91Arv (5′-CCAATGGACACACCTGTGCTGTTTTGAGCCAGCGAG-3′). The resulting vectors coding for sRbx112–108 W87A, K89A, K89A/R91A, and R91A were verified by DNA sequencing. To express unlabeled sRbx112–108 protein (where “s” stands for soluble Rbx112–108 with solubilizing mutations), a 10-ml overnight culture of BL21(DE3)-RIL transformed with pET28a-HisTEV-sRbx112–108 was used to inoculate 1 liter of prewarmed LB media supplemented with 30 μg/ml kanamycin and 34 μg/ml chloramphenicol. To prepare 13C,15N-labeled sRbx112–108, cells were grown in 1 liter of M9 minimal media supplemented with 15NH4Cl (1 g/liter) alone or with [13C6]glucose (2 g/liter) in the presence of 30 μg/ml kanamycin and 34 μg/ml chloramphenicol. The cultures were grown at 37 °C with shaking at 200 rpm. At an A600 of 0.5, the unlabeled and 15N- or 13C,15N-labeled growth was supplemented with 300 μm ZnCl2. Once the cells reached an A600 of 0.7, the cultures were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at 16 °C for 20 h. Cells were then harvested by centrifugation at 6000 × g for 15 min at 4 °C. The cell pellets for unlabeled and 13C,15N-labeled sRbx112–108 were resuspended in 10 ml of wash buffer (50 mm Na2HPO4, 300 mm NaCl, 10 mm imidazole (pH 8.0)) with 1 mm PMSF and an EDTA-free protease inhibitor tablet (Roche Applied Science). The resuspended cells were then sonicated and centrifuged at 38,000 rpm for 1 h at 4 °C. The supernatant was then syringe-filtered through a 0.45-mm filter (Millipore, Mississauga, ON, Canada) and loaded onto a 5-ml HisTrap column (GE Healthcare) pre-equilibrated with wash buffer at a flow rate of 0.5 ml/min using an AKTA FPLC (GE Healthcare). After the column was washed with wash buffer containing 30 mm imidazole for 15 column volumes at 3 ml/min, the protein was eluted with elution buffer (50 mm Na2HPO4, 300 mm NaCl, 250 mm imidazole, pH 8.0) at a flow rate of 2 ml/min. Fractions containing eluted sRbx112–108 protein were pooled, TEV protease was added to cleave the His tag, and the protein was dialyzed against wash buffer overnight at 4 °C. The TEV-cleaved sRbx112–108 protein was then reloaded onto the HisTrap column at a flow rate of 1 ml/min. The flow-through containing purified sRbx112–108 was pooled and dialyzed against Rbx1 dialysis buffer (20 mm Na2HPO4, 100 mm NaCl, 1 mm DTT, pH 7.5, or 6.0) in preparation for NMR experiments. The resulting sRbx112–108 contained an additional GGG at its N termini as a result of its cloning and TEV cleavage. After purification, the concentration of sRbx112–108 was determined by the better Bradford method (Bio-Rad). Purified sRbx112–108 was also submitted for ICP-AES Zn2+ analysis (Laboratory for Geochemical Analysis, University of Western Ontario) to confirm that three zinc ions were bound per sRbx112–108 molecule, similar to a previous study (23.Hristova V.A. Beasley S.A. Rylett R.J. Shaw G.S. Identification of a novel Zn2+ binding domain in the autosomal recessive juvenile Parkinson-related E3 ligase parkin.J. Biol. Chem. 2009; 284: 14978-14986Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Unlabeled full-length CDC34 with C191S/C223S substitutions and ubiquitin K48R/G76C (UbCys) were expressed and purified as previously described (15.Spratt D.E. Shaw G.S. Association of the disordered C terminus of CDC34 with a catalytically bound ubiquitin.J. Mol. Biol. 2011; 407: 425-438Crossref PubMed Scopus (15) Google Scholar). The CDC34-UbCys thiol ester mimetic complex was formed using full-length CDC34 disulfide linked to UbCys, as previously described (15.Spratt D.E. Shaw G.S. Association of the disordered C terminus of CDC34 with a catalytically bound ubiquitin.J. Mol. Biol. 2011; 407: 425-438Crossref PubMed Scopus (15) Google Scholar, 24.Serniwka S.A. Shaw G.S. The structure of the UbcH8-ubiquitin complex shows a unique ubiquitin interaction site.Biochemistry. 2009; 48: 12169-12179Crossref PubMed Scopus (41) Google Scholar, 25.Merkley N. Barber K.R. Shaw G.S. Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate.J. Biol. Chem. 2005; 280: 31732-31738Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). After purification, CDC34 and CDC34-UbCys were dialyzed against 20 mm Na2HPO4, 100 mm NaCl, pH 7.5, in preparation for interaction studies with Rbx1/ROC1. NMR samples for assignment and structure calculation of sRbx112–108 were prepared in 20 mm Na2HPO4, 100 mm NaCl, 1 mm DTT, 10% D2O, 90% H2O at pH 7.5. 13C,15N-labeled sRbx112–108 was concentrated to 700 μm by ultrafiltration (Millipore) in a volume of 300 μl and transferred into a Shigemi tube. All NMR data were collected at 25 °C using a Varian Inova 600-MHz NMR spectrometer equipped with a triple resonance probe and z-field gradients. Backbone and side chain assignments for sRbx112–108 were determined from the following experiments collected using the standard pulse sequences from the Varian Biopack: 1H,15N HSQC (26.Kay L.E. Keifer P. Saarinen T. Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity.J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2429) Google Scholar), HNCO, HNCA, HNHA, HNCACB (27.Grzesiek S. Bax A. Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR.J. Am. Chem. Soc. 1992; 114: 6291-6293Crossref Scopus (926) Google Scholar), CBCA(CO)NH (28.Grzesiek S. Anglister J. Bax A. Correlation of backbone amide and aliphatic side-chain resonances in 13C,15N-enriched proteins by isotropic mixing of 13C magnetization.J. Magn. Reson. 1993; 101 (Series B): 114-119Crossref Scopus (585) Google Scholar), HCC(CO)NH, C(CO)NH, and HCCH-TOCSY spectroscopy (29.Kay L.E. Xu G. Singer A.U. Muhandirum D.R. Forman-Kay J.D. A gradient-enhanced HCCH-TOCSY experiment for recording side-chain 1H and 13C correlations in H2O samples of proteins.J. Magn. Reson. 1993; 101: 333-337Crossref Scopus (558) Google Scholar). The 15N-NOESY-HSQC experiment was collected with a mixing time of 150 ms. After exchanging the sample into 100% D2O, 13C-NOESY-HSQC aliphatic and aromatic experiments were collected using a mixing time of 100 ms. All of the three-dimensional experiments were collected with 16–32 transients. Heteronuclear 15N{1H} NOEs for sRbx112–108 were measured in 20 mm Na2HPO4, 100 mm NaCl, 1 mm DTT, 10% D2O, 90% H2O at pH 6.0 as previously described (15.Spratt D.E. Shaw G.S. Association of the disordered C terminus of CDC34 with a catalytically bound ubiquitin.J. Mol. Biol. 2011; 407: 425-438Crossref PubMed Scopus (15) Google Scholar, 30.Merkley N. Shaw G.S. Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly.J. Biol. Chem. 2004; 279: 47139-47147Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 31.Farrow 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. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2012) Google Scholar) using a 3-s irradiation period and 2-s relaxation delay (NOE) or a 5-s relaxation delay (no NOE). Data processing was performed using NMRPipe and NMRDraw (32.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. NMRPipe. A multidimensional spectral processing system based on UNIX pipes.J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11533) Google Scholar). NMRViewJ (33.Johnson B.A. Belvins R.A. NMRView. A computer program for the visualization and analysis of NMR data.J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2677) Google Scholar) was used for manual spectral analysis and resonance assignment. 2,2-Dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) was used as the internal standard with 1H chemical shifts referenced at 0 ppm, whereas the 13C and 15N chemical shifts were indirectly referenced to DSS, as previously described (34.Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. 1H, 13C, and 15N chemical shift referencing in biomolecular NMR.J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2068) Google Scholar). The NMR assignments for sRbx112–108 have been deposited in the BioMagResBank and assigned the identifier BMRB 17824. Structures were determined using a combination of manually assigned NOEs and automatic NOE assignment with the program CYANA (35.Güntert P. Mumenthaler C. Wüthrich K. Torsion angle dynamics for NMR structure calculation with the new program DYANA.J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2553) Google Scholar). Seven iterations of refinement of 100 structures per cycle were completed by using distance calibrations and parameters as previously described (36.Herrmann T. Güntert P. Wüthrich K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA.J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1327) Google Scholar). After each cycle of refinement, the 20 structures with the lowest calculated target function were used to seed the subsequent rounds of structure calculations and were used in automated NOE assignment by CYANA. Dihedral angular restraints (ϕ,ψ) were determined by inputting the Hα, Cα, Cβ, and C′ chemical shift assignments of sRbx112–108 into TALOS+ (37.Shen Y. Delaglio F. Cornilescu G. Bax A. TALOS+. A hybrid method for predicting backbone torsion angles from NMR chemical shifts.J. Biomol. NMR. 2009; 44: 213-223Crossref PubMed Scopus (2008) Google Scholar). Cysteine residues in sRbx112–108 involved in Zn2+ coordination were identified using their Cα and Cβ chemical shifts (38.Kornhaber G.J. Snyder D. Moseley H.N. Montelione G.T. Identification of zinc-ligated cysteine residues based on 13Cα and 13Cβ chemical shift data.J. Biomol. NMR. 2006; 34: 259-269Crossref PubMed Scopus (77) Google Scholar). After the initial fold of the protein was determined, a modified CYANA amino acid library that included Zn2+-ligated cysteine residues with S-Zn2+ distance constraints was used to incorporate the three Zn2+ ions into the subsequent sRbx112–108 structure calculations, as previously described (39.Beasley S.A. Hristova V.A. Shaw G.S. Structure of the Parkin in-between ring domain provides insights for E3 ligase dysfunction in autosomal recessive Parkinson disease.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 3095-3100Crossref PubMed Scopus (78) Google Scholar). The final 20 structures were subjected to water refinement using the RECOORD protocols for Xplor-NIH (40.Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. The Xplor-NIH NMR molecular structure determination package.J. Magn Reson. 2003; 160: 65-73Crossref PubMed Scopus (1861) Google Scholar, 41.Schwieters C.D. Kuszewski J.J. Clore G.M. Using Xplor-NIH for NMR molecular structure determination.Prog. Nucleic Acid Res. Mol. Biol. 2006; 48: 47-62Google Scholar) and were chosen as representative of the calculation. The atomic coordinates for sRbx112–108 were deposited in the Protein Data Bank and assigned the identifier PDB code 2LGV. The elongation assay required the assembly of two reaction mixtures that contained CDC34∼ubiquitin and Nedd8-SCFβTrCP-IκBα-ubiquitin, respectively. The CDC34∼ubiquitin charge mix (5 μl) contained 50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 0.5 mm DTT, 2 mm ATP, 0.1 mg/ml of BSA, 2 mm NaF, 10 nm okadaic acid, 60 μm ubiquitin, 0.1 μm E1 enzyme, and CDC34 in concentrations as indicated. The mixture was incubated for 5 min at 37 °C before mixing with the Nedd8-SCFβTrCP-IκBα-ubiquitin complex. The Nedd8-SCFβTrCP-IκBα-ubiquitin complex was assembled in sequential steps. First, Rbx1/ROC1-CUL1 was neddylated in a reaction mixture (3 μl) containing 50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 0.5 mm DTT, 2 mm ATP, 0.1 mg/ml of BSA, 2 mm NaF, 10 nm okadaic acid, 1.5 pmol of Rbx1/ROC1-CUL1, 20 μm Nedd8, 83 nm Nedd8 E1 (APPBP1/Uba3), and 15 μm Ubc12. This mixture was incubated at room temperature for 10 min. βTrCP/Skp1 (3 pmol) was then added to the mix, and the mixture was incubated at room temperature for 10 min to allow for the assembly of Nedd8-SCFβTrCP. 32P-Labeled IκBα-Ub (1 pmol), prepared as described previously (13.Wu K. Kovacev J. Pan Z.Q. Priming and extending. A UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate.Mol. Cell. 2010; 37: 784-796Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), was added and incubated for an additional 10 min at room temperature to form the Nedd8-SCFβTrCP-IκBα-ubiquitin complex. The final volume was adjusted to 5 μl. The ubiquitylation reaction was initiated by mixing CDC34∼ubiquitin and Nedd8-SCFβTrCP-IκBα-ubiquitin and incubating the mixture for 10 min at 37 °C. To assess the effects of CDC34-UbCys, CDC34-UbCys, in concentrations as indicated, was added t" @default.
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• W1985891703 title "Selective Recruitment of an E2∼Ubiquitin Complex by an E3 Ubiquitin Ligase" @default.
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