Crystal Structure of the Human Ubiquitin-like Protein NEDD8 and Interactions with Ubiquitin Pathway Enzymes

The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-Å resolution to anR factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2–25K. E2–25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways. The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-Å resolution to anR factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2–25K. E2–25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways. Ubiquitin (Ub) 1The abbreviations used are: Ub, ubiquitin; DHFR, dihydrofolate reductase; DUBs, deubiquitinating enzymes; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; Ubn, Lys-48-linked polyubiquitin chain composed of n ubiquitins; polyUb, polyubiquitin; Ubl, ubiquitin-like protein; His10-NEDD8, recombinant human NEDD8 that contains an N-terminal extension of 10 histidine residues. is a small intracellular protein of 76 amino acid residues that is found both as a monomer and covalently conjugated to other protein molecules. Conjugation results from a covalent isopeptide linkage between the C terminus of Ub and a lysine side chain(s) of the target protein. Conjugation often involves the attachment of a polyubiquitin (polyUb) chain, in which a series of Ub molecules are linked one to another through isopeptide bonds between the C terminus of one Ub and a lysine residue of the adjacent Ub (Refs. 1Hershko A. Heller H. Biochem. Biophys. Res. Commun. 1985; 128: 1079-1086Crossref PubMed Scopus (194) Google Scholar and 2Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1122) Google Scholar and reviewed in Ref. 3Pickart C.M. Peters J.-M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell. Plenum Press, New York1998: 19-63Crossref Google Scholar). PolyUb chains linked through Lys-48 play a well defined role as a signal that targets substrate proteins to the 26 S proteasome for degradation (2Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1122) Google Scholar, 4Rechsteiner M. Peters J.-M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell. Plenum Press, New York1998: 147-189Crossref Google Scholar). In its capacity as a degradation signal Ub plays a key role both in housekeeping functions and in tightly regulated processes such as progression of the cell cycle. In the latter case, Ub and 26 S proteasome-mediated degradation accomplishes the synchronized removal of various activators and inhibitors of cyclin-dependent kinases (5Deshaies R.J. Curr. Opin. Genet. Dev. 1997; 7: 7-16Crossref PubMed Scopus (83) Google Scholar). Other functions of ubiquitination, distinct from that of targeting to the 26 S proteasome, have also been identified (6Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar, 7Hicke L. Riezman H. Cell. 1996; 84: 277-287Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). The very high conservation of the Ub amino acid sequence (only three residue substitutions between human and yeast) is presumably a function of the specific interactions that Ub makes with many other proteins. The C terminus of monomeric Ub is activated by the ubiquitin-activating (or E1) enzyme, which in turn passes the Ub to a ubiquitin conjugating (or E2) enzyme. Inspection of the Saccharomyces cerevisiaegenome sequence indicates the presence of 13 different E2 enzymes, each of which is thought to be associated with a distinct set of substrates (8Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1461) Google Scholar). It has recently been shown that two of these E2 enzymes mediate the conjugation of Ub-like proteins, rather than the conjugation of Ub (9Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 10Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 19: 2208-2216Crossref Scopus (307) Google Scholar). A further level of ubiquitination specificity is provided by the numerous ubiquitin ligases (E3 enzymes), some of which are known to make direct contact with Ub (11Reiss Y. Heller H. Hershko A. J. Biol. Chem. 1989; 264: 10378-10383Abstract Full Text PDF PubMed Google Scholar). In the context of a polyUb chain, Ub also binds to one or more components of the 26 S proteasome regulatory complex. Finally, Ub also makes critical interactions with deubiquitinating enzymes, 17 of which have been identified in the genome of S. cerevisiae (12Wilkinson K.D. Hochstrasser M. Peters J.-M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell. Plenum Press, New York1998: 99-125Crossref Google Scholar). Thus, the functions of Ub result from a series of highly specific macromolecular interactions. Taken together, these interactions are likely to involve most of the protein surface. Several ubiquitin-like proteins (Ubls) have been identified that share sequence similarity with Ub. One such protein, known as NEDD8 in mammals (13Kumar S. Tomooka Y. Noda M. Biochem. Biophys. Res. Commun. 1992; 185: 1155-1161Crossref PubMed Scopus (450) Google Scholar, 14Kamitani T. Kito K. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) and Rub1 in yeast (8Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1461) Google Scholar), also identified in plants (15Callis J. Carpenter T. Sun C. Vierstra R.D. Genetics. 1995; 139: 921-939Crossref PubMed Google Scholar), shares ∼60% sequence identity with Ub (16Kumar S. Yoshida Y. Noda M. Biochem. Biophys. Res. Commun. 1993; 195: 393-399Crossref PubMed Scopus (117) Google Scholar). Invariant residues in the Ub and NEDD8 sequences include most of those near the C terminus and most of the lysine residues, including Lys-48 which is critical for Ub's degradative signaling function (above). NEDD8 undergoes a metabolism which parallels that of Ub, including activation by a distinct E1-like enzyme (10Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 19: 2208-2216Crossref Scopus (307) Google Scholar, 17Schwarz S.E. Matuschewski K.D.L. Scheffner M. Jentsch S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 560-564Crossref PubMed Scopus (189) Google Scholar) and conjugation mediated by a dedicated E2 enzyme (10Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 19: 2208-2216Crossref Scopus (307) Google Scholar). NEDD8 and its orthologs also form conjugates with intracellular proteins, a process that, as for Ub, requires the C-terminal Gly-76 residue of the mature processed protein (10Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 19: 2208-2216Crossref Scopus (307) Google Scholar, 14Kamitani T. Kito K. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 18Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 916Crossref Scopus (279) Google Scholar). One of the best characterized Ubls is the mammalian protein SUMO-1 (19Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar,20Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar) and its yeast ortholog Smt3 (21Meluh P.B. Koshland D. Mol. Biol. Cell. 1995; 6: 793-807Crossref PubMed Scopus (354) Google Scholar). Like NEDD8, SUMO-1/Smt3 undergoes a metabolism that parallels that of Ub, with distinct E1-like (22Johnson E.S. Schienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (445) Google Scholar) and E2 enzymes (9Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). SUMO-1 also performs an intracellular targeting function, but this function involves neither the 26 S proteasome nor degradation. At least in some cases, SUMO-1 conjugation is a signal for targeting to the nuclear pore complex (19Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (961) Google Scholar, 20Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar) or for specific locations within the nucleus (23Müller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (581) Google Scholar). Despite the presence of only 18% sequence identity, the recently reported three-dimensional structure of SUMO-1 closely resembles that of Ub (24Bayer P. Arndt A. Metzger S. Mahajan R. Melchoir F. Jaenicke R. Becker J. J. Mol. Biol. 1998; 280: 275-286Crossref PubMed Scopus (328) Google Scholar). Another Ubl, ISG15(UCRP), also serves a targeting role by localizing conjugated proteins to intermediate filaments (25Loeb K.R. Haas A.L. Mol. Cell. Biol. 1994; 14: 8408-8419Crossref PubMed Scopus (92) Google Scholar). 2A. L. Haas, personal communication. A possible function for NEDD8 in the regulation of cell cycle progression is suggested by the observation that the S. cerevisiae ortholog, Rub1, is found covalently attached to the cullin protein Cdc53 (10Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 19: 2208-2216Crossref Scopus (307) Google Scholar, 18Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 916Crossref Scopus (279) Google Scholar), which is a component of the SCFCdc4 ubiquitin-ligase complex. The activity of this E3 is critical for progression from G1 to S phase (26Feldman R.M. Correll C.C. Kaplan K.B. Deshaies R.J. Cell. 1997; 91: 221-230Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, 27Skowyra D. Craig K.L. Tyres M. Elledge S.J. Harper J.W. Cell. 1997; 91: 209-219Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). SCFCdc4, which in addition to Cdc53 contains the Skp1 and Cdc4 proteins and the E2 enzyme Cdc34, functions by conjugating Ub to the cyclin-dependent kinase inhibitor Sic1, thereby targeting Sic1 for degradation. Yeast cells are apparently healthy following deletion of the genes for Rub1, the Rub1-specific E1-like enzyme, or the Rub1-specific E2 enzyme. Nevertheless, a role for Rub1 in SCFCdc4 function is indicated by the synthetic enhancement of mutations in the CDC34, CDC4,CDC53, and SKP1 genes by deletion of theRUB1 or ENR2 genes (ENR2 encodes a component of the heterodimeric Rub1-specific activating enzyme). Overproduction of Cdc34 or Cdc53 also sensitizes cells to loss of the Rub1 modification. Furthermore, a C-terminal truncation of Cdc53 renders this protein resistant to Rub1 modification and at the same time makes the cells sensitive to mutation of CDC34. Cullins are also conjugated to NEDD8 in higher organisms. 3E. T. H. Yeh, personal communication. Further evidence for a NEDD8 function in cell cycle progression is provided by a hamster cell line carrying a temperature-sensitive allele of a gene nearly identical to that encoding one of the subunits of the human NEDD8-activating enzyme. At nonpermissive temperatures these cells traverse multiple S phases without intervening mitoses (cited in Ref. 18Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 916Crossref Scopus (279) Google Scholar). In an effort to understand the biochemical and biological functions of NEDD8, we have determined the crystal structure of recombinant human NEDD8. Biochemical analysis demonstrates that Ala-72 of NEDD8 (Arg-72 in Ub), performs a key role in preventing the interaction of NEDD8 with the ubiquitin E1 enzyme. In the context of a chimeric polyUb molecule, NEDD8 is competent to interact with the 26 S proteasome. The distribution of conserved and divergent residues on the surface of the NEDD8 structure provides a framework for considering the interactions of NEDD8 with other proteins. A human NEDD8-encoding gene comprising 76 amino acids was generated by PCR amplification using pcDNA/hNEDD8 (14Kamitani T. Kito K. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) as template. The 5′ primer (5′-TGGAAGACATATGCTAATTAAAGTGAAG-3′) and the 3′ primer (5′-CTGGATCCTCATCCTCCTCTCAGAGCCA-3′) harbored sites for NdeI and BamHI, respectively. The PCR product was digested with these two enzymes and ligated into pET3a (to produce pET3a-NEDD8). The sequence of the insert was verified by automated sequencing (Hopkins Core Facility). The insert was excised and ligated into pET16b to allow for the expression of a His10-tagged version of NEDD8. A cDNA encoding NEDD8-A72R was created by PCR amplification with the 5′ primer described above and a 3′ primer specifying the mutation (5′-CTGGATCCTCATCCTCCTCTCAGACGCAACACCAGGTG-3′). The PCR product was cloned into pET3a as described above. The presence of the A72R mutation was verified by automated DNA sequence analysis. The plasmid pET3a-NEDD8 was expressed inEscherichia coli strain BL21(DE3)pLysS at 37 °C as described previously (28Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). The cell pellet from a 2-liter culture was suspended in 20 ml of lysis buffer (28Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar); cell lysis and digestion of DNA were carried out as before (28Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). The crude lysate was centrifuged at 15,000 × g for 20 min. The pellet was resuspended in 10 ml of buffer containing 50 mm Tris-HCl (24% base, pH 7.6), 1 mm EDTA, 20% v/v sucrose, and 1% Triton X-100. NEDD8 inclusion bodies were pelleted by centrifugation at 15,000 × g for 20 min; this wash step was repeated twice. The purified inclusion bodies were suspended in buffer containing 50 mm Tris-HCl (24% base), 2 mm EDTA, and 8m urea; the suspension was held at room temperature for 30 min, during which time it became clear. The solution was then dialyzed extensively at 5 °C against buffer containing 50 mmTris-HCl (24% base), 2 mm EDTA, and 1 mmdithiothreitol; any precipitate that formed was removed by centrifugation. The protein solution was passed successively through 10-ml columns of 1) Q-Sepharose and 2) SP-Sepharose (both from Amersham Pharmacia Biotech) which had been pre-equilibrated with dialysis buffer. The final flow-through fraction was concentrated (Millipore Ultra-free 4) to yield a NEDD8 concentration of ∼20 mg/ml. Aliquots (1 ml) of the concentrated protein were run on a 1 × 50-cm Sephacryl-200 column (Amersham Pharmacia Biotech) pre-equilibrated with dialysis buffer. Fractions (1 ml) were collected, and aliquots were analyzed by SDS-PAGE to identify the peak fractions. Usually about 30 mg of purified NEDD8 was recovered from 1 liter of cell suspension; the purity of the protein was >98% as evaluated by SDS-PAGE and Coomassie staining. Sometimes a fraction of the NEDD8 protein precipitated during repeated cycles of freezing and thawing. NEDD8-A72R was expressed and purified as described above, with the following differences. 1) The refolded mutant protein bound weakly to SP-Sepharose. Therefore the loaded column was washed with dialysis buffer containing 0.1 m NaCl prior to elution with buffer containing 0.15 m NaCl. 2) The gel filtration step was omitted. The purified mutant protein was >90% pure by SDS-PAGE and Coomassie staining. About 5 mg of purified NEDD8-A72R was obtained from 1 liter of cell suspension. His10-NEDD8 was expressed as described above. The cell pellet from a 400-ml culture was resuspended in 8 ml of buffer containing 50 mm Tris-HCl (24% base) and 8 murea. The suspension was held for 1 h at 60 °C, during which time the cells lysed. The lysate was passed through a 2.5-ml Ni2+-nitrilotriacetic acid column (Novagen). The loaded column was washed extensively with the same buffer, and His10-NEDD8 was eluted with 3 volumes of the same buffer containing 0.2 m imidazole. The protein was dialyzed and concentrated (above). Ubiquitin-activating enzyme (E1) was purified from rabbit reticulocytes (29Pickart C.M. Vella A.T. J. Biol. Chem. 1988; 263: 12028-12035Abstract Full Text PDF PubMed Google Scholar). The following E2s were purified from bovine erythrocytes or rabbit reticulocytes: E2–14K (29Pickart C.M. Vella A.T. J. Biol. Chem. 1988; 263: 12028-12035Abstract Full Text PDF PubMed Google Scholar), E2–17K (30Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar), E2–20K (29Pickart C.M. Vella A.T. J. Biol. Chem. 1988; 263: 12028-12035Abstract Full Text PDF PubMed Google Scholar), and E2–35K (29Pickart C.M. Vella A.T. J. Biol. Chem. 1988; 263: 12028-12035Abstract Full Text PDF PubMed Google Scholar). Purified recombinant human Ubc13 homolog was a gift of R. Hofmann (Johns Hopkins); purified recombinant human UbcH5B (31Scheffner M. Huibregtse J.M. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8797-8801Crossref PubMed Scopus (236) Google Scholar) was a gift of J. You (Johns Hopkins). C170S-E2–25K was expressed and purified as described (28Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar, 32Mastrandrea L.D. Kasperek E.M. Niles E.G. Pickart C.M. Biochemistry. 1998; 37: 9784-9792Crossref PubMed Scopus (13) Google Scholar). Bovine Ub (Sigma) and recombinant NEDD8 (above) were radioiodinated to ∼9000 and ∼6000 cpm/pmol, respectively (33Beal R.E. Toscano-Cantaffa D. Young P. Rechsteiner M. Pickart C.M. Biochemistry. 1998; 37: 2925-2934Crossref PubMed Scopus (103) Google Scholar). Thiol ester assays were carried out at pH 7.3 and 37 °C as described previously (28Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar), using 4 μm labeled protein and ∼3 μm E2. The concentration of E1 was 0.1 μm (Ub assays) or 0.5 μm (NEDD8 assays). Assays (8 μl) were quenched after 3.5 min. (Ub) or 25 min (NEDD8) with an equal volume of sample buffer lacking β-mercaptoethanol. The E2 thiol esters were detected by electrophoresis and autoradiography and quantitated by band excision and γ-counting. The formation of a labile E2∼NEDD8 adduct was observed with each E2, but the appearance of the NEDD8 thiol ester (at 0.5 μm E1) was much slower than the appearance of the corresponding Ub thiol ester (at 0.1 μm E1). Ubc13 gave a higher yield of NEDD8 thiol ester than did the other E2s assayed. Pulse incubations (pH 7.3 and 37 °C) contained ∼4 μm C170S-E2–25K and 4 μm125I-Ub or 125I-NEDD8. The conditions of the 10-min pulse, and the concentration of E1, were the same as in thiol ester assays (28Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). At the end of the pulse, a 2.5-μl aliquot was removed to monitor thiol ester formation. The chase was then initiated by adding a mixture providing 1 mg/ml of unlabeled Ub or NEDD8 and 10 mm EDTA. Aliquots were quenched at increasing times in sample buffer without mercaptoethanol and analyzed by SDS-PAGE and autoradiography. Pseudo-first order rate constants for the disappearance of the E2 thiol ester were obtained from semi-log plots of thiol ester radioactivity versus time. Incubations were carried out under the same conditions as for E2 thiol ester formation, except that E2 was omitted and the concentration of E1 was 0.15 μm.125I-Ub was 1.3 μm; the concentration of unlabeled NEDD8 or Ub was varied (see “Results”). The reaction was initiated by adding E1 and quenched after 3 min with sample buffer lacking mercaptoethanol. The E1∼125I-Ub thiol ester was detected following electrophoresis and autoradiography and quantitated by band excision and counting. E2–25K was used to conjugate His10-NEDD8 to Lys-48 at the distal terminus of Lys-48-linked Ub3. The Ub3 was purified by cation exchange from a mixture of chains assembled using E2–25K (34Pickart C.M. Haldeman M.T. Kasperek E.M. Chen Z. J. Biol. Chem. 1992; 267: 14418-14423Abstract Full Text PDF PubMed Google Scholar); it was largely des-GlyGly at its proximal terminus, which prevented self-conjugation. The incubation (200 μl, pH 7.3, and 37 °C) contained 5 μm C170S-E2–25K, 0.25 mmdithiothreitol, 0.2 μm E1, 58 μmUb3, and 150 μm His10-NEDD8. Other conditions were as described previously (32Mastrandrea L.D. Kasperek E.M. Niles E.G. Pickart C.M. Biochemistry. 1998; 37: 9784-9792Crossref PubMed Scopus (13) Google Scholar). Incubation was for 2 h (37 °C), and 0.2 μm additional E1 was added at 40 and 80 min. The poor acceptor activity of NEDD8 (“Results”) prevented the ligation of more than one His10-NEDD8 to the chain. The chimeric His10-NEDD8-Ub3 tetramer (and free His10-NEDD8) were resolved from unutilized Ub3 and enzymes by chromatography on nickel resin. Bovine serum albumin was added to the column eluate as a carrier prior to concentration and buffer exchange. Due to the small amount of chimeric tetramer, we did not attempt to purify this species further. Based on the failure of mono-Ub to inhibit the 26S proteasome (35Piotrowski J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Crossref PubMed Scopus (191) Google Scholar), we considered it unlikely that the residual mono-Nedd8 would interfere in our assays, and this proved to be the case (“Results”). The competition assay was similar to that described previously (35Piotrowski J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Crossref PubMed Scopus (191) Google Scholar), except that it employed a substrate in which Lys-48-linked Ub4 was conjugated to Lys-48 of the Ub moiety in a linear Ub-DHFR fusion protein (the DHFR fusion protein was metabolically labeled with35S-Met, in E. coli). Degradation was monitored by the appearance of acid-soluble radioactivity and was linear in time and proteasome concentration. The properties of this substrate, and the assay, will be described in detail elsewhere. 4J. Piotrowski, L. Hoffman, M. Rechsteiner, and C. M. Pickart, manuscript in preparation. Purified 26 S proteasome was generously provided by L. Hoffman and M. Rechsteiner (University of Utah). Ub4 was synthesized as described (35Piotrowski J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Crossref PubMed Scopus (191) Google Scholar). Recombinant human NEDD8 was crystallized by vapor diffusion in sitting drops at 21 °C. The initial protein solution was 10 or 15 mg/ml NEDD8, 40 mm Tris, pH 7.6, 50 mm NaCl, 0.4 mmEDTA, and 0.2 mm dithiothreitol. The reservoir solution was 500 μl of 100 mm citric acid, pH 4.8, and 2.2m ammonium sulfate. Drops were made by mixing 2 μl each of protein and reservoir solutions. Clusters of thin crystalline plates formed after 24 to 36 h, and small prism-shaped crystals up to 0.01 mm in the longest dimension appeared a few days later. The small prism-shaped crystals were transferred to fresh crystallization drops that had equilibrated for less than 24 h. Crystals grew from these seeds to typical dimensions of 0.1 × 0.1 × 0.1 mm. Two closely related crystal forms with indistinguishable morphology grew in the same drop. The two crystal forms belong to space groups P1 and P21, and they both contain 4 molecules in the asymmetric unit. The P1 crystals diffracted to approximately 2.10-Å resolution and had unit cell dimensions a = 34.1 Å,b = 45.3 Å, c = 48.4 Å, α = 83.8°, β = 73.2°, γ = 79.5°. The P21 crystals diffracted to 1.60-Å resolution and had unit cell dimensionsa = 45.8 Å, b = 65.0 Å,c = 48.6 Å, β = 96.6°. The P1 and P21crystals are closely related to each other and have very similar Matthews' V m coefficients (36Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7926) Google Scholar) of 2.06 and 2.10 Å3/Da, which correspond to solvent contents of 39.7 and 41.0%, respectively. All x-ray diffraction data were collected at −170 °C using a copper rotating anode source and an RAXIS-IV image plate area detector. Prior to data collection the crystals were immersed for 2 (P21) or 30 (P1) min in a solution containing 100 mm citric acid, pH 4.8, 2.6 m ammonium sulfate, and 15% glycerol. The crystals were suspended in a small rayon loop attached to a metal pin and cryo-cooled by plunging into liquid nitrogen. The crystals were transferred to the data collection instrument, and complete data sets were collected from each of the crystals. Data were collected as 0.6 to 1.0o rotation images with typical exposure times of 10–60 min. Data were indexed, integrated, and scaled with DENZO and SCALEPACK (37Otwinowski Z. Sawyer L. Isaacs N. Bailey S. Data Collection and Processing. SERC Daresbury Laboratory, Warrington, Great Britain1993: 56-62Google Scholar). Data collected from crystal form P1 extend to 2.10-Å resolution with an R sym of 10.6%. The data from crystal form P21 extend to 1.60-Å resolution with anR sym of 6.7%. See Table I for data processing statistics.Table IData collection statisticsCrystal formP1P21No. of reflections observed76,227232,354No. of reflections unique15,22336,579d min (Å)2.101.60High resolution shell2.10–2.141.60–1.63Completeness (%)96.0 (93.8)97.8 (93.7)R sym(%)aR sym = 100 ∗ ∑‖I − 〈I〉‖/∑I.10.6 (36.8)6.7 (45.2)AverageI/ς(I)9 (2.5)>20 (1.9)Mosaicity (°)0.7520.449Data were collected on a rotating anode x-ray source using an RAXIS-IV image plate detector. Values in parentheses refer to the highest resolution shell.a R sym = 100 ∗ ∑‖I − 〈I〉‖/∑I. Open table in a new tab Data were collected on a rotating anode x-ray source using an RAXIS-IV image plate detector. Values in parentheses refer to the highest resolution shell. Most crystallographic computations used programs from the Collaborative Computing Project 4 suite (38Collaborative Computing Project 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The scaled diffraction intensities were converted to structure-factor amplitudes using the program TRUNCATE (39French S. Wilson K. Acta Crystallogr. Sect. A. 1978; 34: 517-525Crossref Scopus (895) Google Scholar). Structure determination was by Molecular Replacement using the program AMoRe (40Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar). A single molecule of Ub (41Vijay-Kumar S. Bugg C.E. Cook W.J. J. Mol. Biol. 1987; 194: 531-544Crossref PubMed Scopus (1386) Google Scholar) (PDB entry 1ubq) (modified by deleting the C-terminal 6 residues) was used as the search probe. This gave a clear solution against the P1 data; fitting of all four molecules in the asymmetric unit gave a correlation coefficient of 0.327 and an R factor of 48.5% against data in the resolution range 8.0–3.5 Å. Refinement was performed using the program XPLOR (42Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR, Version 3. Yale University Press, New Haven, CT1992Google Scholar). Preliminary refinement against the P1 data of a NEDD8 model consisting o