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• W2085362167 abstract "The Bre5 protein is a cofactor for the deubiquitinating enzyme Ubp3, and it contains a nuclear transfer factor 2 (NTF2)-like protein recognition module that is essential for Ubp3 activity. In this study, we report the x-ray crystal structure of the Bre5 NTF2-like domain and show that it forms a homodimeric structure that is similar to other NTF2-like domains, except for the presence of an intermolecular disulfide bond in the crystals. Sedimentation equilibrium studies reveal that under non-reducing conditions, the Bre5 NTF2-like domain is exclusively dimeric, whereas a disulfide bond-deficient mutant undergoes a monomer-dimer equilibrium with a dissociation constant in the midnanomolar range, suggesting that dimer formation and possibly also disulfide bond formation may modulate Bre5 function in vivo. Using deletion analysis, we also identify a novel N-terminal domain of Ubp3 that is necessary and sufficient for interaction with Bre5 and use isothermal titration calorimetry to show that Bre5 and Ubp3 form a 2:1 complex, in contrast to other reported NTF2-like domain/protein interactions that form 1:1 complexes. Finally, we employ structure-based mutagenesis to map the Ubp3 binding surface of Bre5 to a region near the Bre5 dimer interface and show that this binding surface of Bre5 is important for Ubp3 function in vivo. Together, these studies provide novel insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding. The Bre5 protein is a cofactor for the deubiquitinating enzyme Ubp3, and it contains a nuclear transfer factor 2 (NTF2)-like protein recognition module that is essential for Ubp3 activity. In this study, we report the x-ray crystal structure of the Bre5 NTF2-like domain and show that it forms a homodimeric structure that is similar to other NTF2-like domains, except for the presence of an intermolecular disulfide bond in the crystals. Sedimentation equilibrium studies reveal that under non-reducing conditions, the Bre5 NTF2-like domain is exclusively dimeric, whereas a disulfide bond-deficient mutant undergoes a monomer-dimer equilibrium with a dissociation constant in the midnanomolar range, suggesting that dimer formation and possibly also disulfide bond formation may modulate Bre5 function in vivo. Using deletion analysis, we also identify a novel N-terminal domain of Ubp3 that is necessary and sufficient for interaction with Bre5 and use isothermal titration calorimetry to show that Bre5 and Ubp3 form a 2:1 complex, in contrast to other reported NTF2-like domain/protein interactions that form 1:1 complexes. Finally, we employ structure-based mutagenesis to map the Ubp3 binding surface of Bre5 to a region near the Bre5 dimer interface and show that this binding surface of Bre5 is important for Ubp3 function in vivo. Together, these studies provide novel insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding. The activity of enzymes that catalyze ubiquitin (Ub) 1The abbreviations used are: Ub, ubiquitin; USP and UBP, ubiquitin-specific processing protease in humans and yeasts, respectively; NTF2, nuclear transfer factor 2; RRM, RNA recognition motif; GST, glutathione S-transferase; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid. transfer on target proteins is correlated with the regulation of many cellular processes including protein degradation, cell cycle control, stress response, DNA repair, immune response, signal transduction, gene regulation, endocytosis, and vesicle trafficking (1Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2959) Google Scholar, 3Pickart C.M. Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar). Studies over the last decade have provided important mechanistic insights into catalysis and substrate specificity of Ub conjugation by the E1/E2/E3 multiprotein system (1Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). However, relatively little mechanistic information is available on the proteins that mediate deubiquitination, despite mounting evidence that these proteins play as important a regulatory role in cellular processes as their Ub-conjugating counterparts (4Fischer J.A. Int. Rev. Cytol. 2003; 229: 43-72Crossref PubMed Scopus (25) Google Scholar). Deubiquitination is catalyzed by deubiquitinating proteases, which are classified into five groups based on sequence homology: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific processing proteases (USPs in humans and UBPs in yeasts), OTU-domain ubiquitinaldehyde-binding proteins, Jab1/Pad1/MPN domain-containing metalloenzymes, and the ataxin-3-like proteases (5Kim J.H. Park K.C. Chung S.S. Bang O. Chung C.H. J. Biochem. (Tokyo). 2003; 134: 9-18Crossref PubMed Scopus (131) Google Scholar, 6Wilkinson K.D. FASEB J. 1997; 11: 1245-1256Crossref PubMed Scopus (512) Google Scholar, 7Maytal-Kivity V. Reis N. Hofmann K. Glickman M.H. BMC Biochem. 2002; 3: 28-40Crossref PubMed Scopus (184) Google Scholar, 8Verma R. Aravind L. Oania R. McDonald W.H. Yates III, J.R. Koonin E.V. Deshaies R.J. Science. 2002; 298: 611-615Crossref PubMed Scopus (841) Google Scholar, 9Yao T. Cohen R.E. Nature. 2002; 419: 403-407Crossref PubMed Scopus (602) Google Scholar, 10Balakirev M.Y. Tcherniuk S.O. Jaquinod M. Chroboczek J. EMBO Rep. 2003; 4: 517-522Crossref PubMed Scopus (212) Google Scholar, 11Amerik A.Y. Hochstrasser M. Biochim. Biophys. Acta. 2004; 1695: 189-207Crossref PubMed Scopus (761) Google Scholar). In addition, sequence analysis yields an additional putative deubiquitinating protease family (12Iyer L.M. Koonin E.V. Aravind L. Cell Cycle. 2004; 3: 1440-1450Crossref PubMed Scopus (89) Google Scholar). Among the deubiquitinating protease families, UBPs represent the most widespread deubiquitylating enzymes across evolution. In particular, the Saccharomyces cerevisiae genome encodes for 16 UBPs and only one ubiquitin C-terminal hydrolase (13Amerik A.Y. Li S.J. Hochstrasser M. Biol. Chem. 2000; 381: 981-992Crossref PubMed Scopus (159) Google Scholar). In the human genome, there appears to be at least 4 and 63 distinctive genes encoding ubiquitin C-terminal hydrolases and UBPs/USPs, respectively (14Wing S.S. Int. J. Biochem. Cell Biol. 2003; 35: 590-605Crossref PubMed Scopus (163) Google Scholar). The observation that UBPs are abundant and broadly conserved in many species including bacteria, yeast, and humans, suggests that they might play important specific roles in regulating diverse biological processes. The molecular basis for how different UBPs select their cognate substrates and mediate distinct cellular functions is still unclear. Unlike the highly conserved ubiquitin C-terminal hydrolases, the UBP family exhibits homology only in two regions that surround the catalytic Cys and His residues, thus called the Cys box (∼19-amino acid) and His box (60–90-amino acid) regions. Homology among the UBP enzymes is restricted to a roughly 350-residue catalytic core domain, whereas the UBPs contain variable N-terminal extensions, occasional C-terminal extensions, or insertions in the catalytic domains. Whereas the functions of these additional UBP domains are not well understood, recent studies have suggested that they function in substrate recognition, cofactor association (15Papa F.R. Amerik A.Y. Hochstrasser M. Mol. Biol. Cell. 1999; 10: 741-756Crossref PubMed Scopus (106) Google Scholar, 16Wilkinson K.D. Semin. Cell Dev. Biol. 2000; 11: 141-148Crossref PubMed Scopus (470) Google Scholar), and subcellular localization (17Lin H. Yin L. Reid J. Wilkinson K.D. Wing S.S. J. Biol. Chem. 2001; 276: 20357-20363Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 18Lin H. Keriel A. Morales C.R. Bedard N. Zhao Q. Hingamp P. Lefrancois S. Combaret L. Wing S.S. Mol. Cell. Biol. 2000; 20: 6568-6578Crossref PubMed Scopus (59) Google Scholar). Whereas the human herpesvirus-associated USP has been shown to be necessary and sufficient for deubiquitination of its specific substrate, the p53 tumor suppressor (19Li M. Chen D. Shiloh A. Luo J. Nikolaev A.Y. Qin J. Gu W. Nature. 2002; 416: 648-653Crossref PubMed Scopus (810) Google Scholar), other characterized UBPs have been shown to require additional protein cofactors for activity. For example, USP10, one of the human UBPs, required the Ras-GAP Src homology 3-binding protein, G3BP1, for catalytic activity (20Soncini C. Berdo I. Draetta G. Oncogene. 2001; 20: 3869-3879Crossref PubMed Scopus (142) Google Scholar). Ubp3, the yeast homologue of human USP10, has been shown to form a complex with the Bre5 cofactor to specifically deubiquitinate the Sec23 (21Cohen M. Stutz F. Belgareh N. Haguenauer-Tsapis R. Dargemont C. Nat. Cell Biol. 2003; 5: 661-667Crossref PubMed Scopus (133) Google Scholar) and β′-COP (22Cohen M. Stutz F. Dargemont C. J. Biol. Chem. 2003; 278: 51989-51992Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) subunits of the COPII and COPI complexes that regulate anterograde and retrograde transport between the endoplasmic reticulum and the Golgi apparatus (21Cohen M. Stutz F. Belgareh N. Haguenauer-Tsapis R. Dargemont C. Nat. Cell Biol. 2003; 5: 661-667Crossref PubMed Scopus (133) Google Scholar), respectively. Further analysis of the Ubp3-Bre5 complex reveals that Bre5 is an essential cofactor for Ubp3-mediated catalysis (21Cohen M. Stutz F. Belgareh N. Haguenauer-Tsapis R. Dargemont C. Nat. Cell Biol. 2003; 5: 661-667Crossref PubMed Scopus (133) Google Scholar). Taken together, it appears that Bre5 is a key component of Ubp3-mediated function, although the molecular basis for this is currently not known. Yeast Bre5 has two recognizable domains, a nuclear transfer factor 2 (NTF2) domain at the N terminus (residues 8–140) and an RNA recognition motif (RRM) at the C terminus (residues 419–481), and Ubp3 contains no recognizable domains outside of its C-terminal catalytic domain. Although NTF2-like domains are found in many proteins with diverse functions, (23Bayliss R. Leung S.W. Baker R.P. Quimby B.B. Corbett A.H. Stewart M. EMBO J. 2002; 21: 2843-2853Crossref PubMed Scopus (139) Google Scholar, 24Bullock T.L. Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 260: 422-431Crossref PubMed Scopus (119) Google Scholar, 25Fribourg S. Conti E. EMBO Rep. 2003; 4: 699-703Crossref PubMed Scopus (42) Google Scholar, 26Senay C. Ferrari P. Rocher C. Rieger K.J. Winter J. Platel D. Bourne Y. J. Biol. Chem. 2003; 278: 48395-48403Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), a common function of these domains appears to be that they mediate protein-protein interactions. Indeed, deletion analysis of Bre5 reveals that its NTF2 domain, but not the RNA-binding domain, is necessary and sufficient to bind Ubp3 and is required for Ubp3 function in vivo (21Cohen M. Stutz F. Belgareh N. Haguenauer-Tsapis R. Dargemont C. Nat. Cell Biol. 2003; 5: 661-667Crossref PubMed Scopus (133) Google Scholar, 22Cohen M. Stutz F. Dargemont C. J. Biol. Chem. 2003; 278: 51989-51992Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), suggesting that the NTF2 domain of Bre5 may directly associate with Ubp3 to regulate its deubiquitination activity. The NTF2-like domains from NTF2 (23Bayliss R. Leung S.W. Baker R.P. Quimby B.B. Corbett A.H. Stewart M. EMBO J. 2002; 21: 2843-2853Crossref PubMed Scopus (139) Google Scholar, 24Bullock T.L. Clarkson W.D. Kent H.M. Stewart M. J. Mol. Biol. 1996; 260: 422-431Crossref PubMed Scopus (119) Google Scholar, 27Bayliss R. Ribbeck K. Akin D. Kent H.M. Feldherr C.M. Gorlich D. Stewart M. J. Mol. Biol. 1999; 293: 579-593Crossref PubMed Scopus (152) Google Scholar, 28Stewart M. Kent H.M. McCoy A.J. J. Mol. Biol. 1998; 284: 1517-1527Crossref PubMed Scopus (59) Google Scholar), TAP and P15 (29Fribourg S. Braun I.C. Izaurralde E. Conti E. Mol. Cell. 2001; 8: 645-656Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), and Mtr2 (25Fribourg S. Conti E. EMBO Rep. 2003; 4: 699-703Crossref PubMed Scopus (42) Google Scholar) and Mex67 (26Senay C. Ferrari P. Rocher C. Rieger K.J. Winter J. Platel D. Bourne Y. J. Biol. Chem. 2003; 278: 48395-48403Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) alone and, in some cases, in complex with their cognate protein targets have been characterized at both the biochemical and structural levels. These studies reveal that this domain forms homo- or heterodimers with low micromolar affinity (30Chaillan-Huntington C. Butler P.J. Huntington J.A. Akin D. Feldherr C. Stewart M. J. Mol. Biol. 2001; 314: 465-477Crossref PubMed Scopus (28) Google Scholar) and that the NTF2-like dimers bind protein targets, some of which contain FXFG or FG repeats. Interestingly, although the various NTF2-like domain structures have similar overall dimeric folds, the region of the NTF2-like domain that is used for recognition of these repeats is not conserved among the different proteins, possibly reflecting the relatively low primary sequence homology between the NTF2-like domains (25Fribourg S. Conti E. EMBO Rep. 2003; 4: 699-703Crossref PubMed Scopus (42) Google Scholar) and their diverse biological roles (26Senay C. Ferrari P. Rocher C. Rieger K.J. Winter J. Platel D. Bourne Y. J. Biol. Chem. 2003; 278: 48395-48403Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Indeed, the observation that Ubp3 does not contain FXFG or FG repeats within its primary sequence suggests that its mode of recognition by the NTF2-like domain of Bre5 may also be novel. In this study, we employ biochemical and structural analysis to show that the NTF2-like domain of Bre5 forms a tight homodimer that directly associates with a folded domain within the N terminus of Ubp3. We also use the crystal structure of the Bre5 NTF2-like domain to inform structure-based mutagenesis to further characterize the molecular basis for the Bre5-Ubp3 interaction in vitro and show the importance of this interaction in vivo. Together, these studies provide new insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding. Protein Preparation—The UBP3 gene was amplified by PCR from yeast genomic DNA and inserted into the pGEX5T expression vector for further subcloning. Ubp3 fragments encoding residues 1–912 (full-length), 50–912, 104–912, 189–912, and 260–912 were subcloned into the PET28a expression vector as C-terminal His6-tagged fusion proteins. DNA encoding residues 181–260 and 181–282 of Ubp3 was subcloned into the PGEX4T-1 expression vector for the preparation of N-terminal GST fusion proteins. DNA encoding residues 1–146 of Bre5 was PCR-amplified from pBTM116 and cloned into the pGEX4T-1 expression vector for the preparation of the N-terminal GST fusion protein. All Ubp3 and Bre5 expression plasmids were transformed into Escherichia coli strain BL21 (DE3) for protein expression. Transformed bacteria were initially grown at 37 °C to an absorbance of 0.7–0.9 at 600 nm, and protein overexpression induced by addition of 0.1 mm isopropyl 1-thio-β-d-galactopyranoside followed by overnight growth at 15 °C. Cells were disrupted by sonication in a solution containing PBS buffer supplemented with 10 mm β-mercaptoethanol and 1 mm phenylmethylsulfonyl fluoride. For the purification of His-tagged protein, the protein was partially purified using an Ni2+-NTA resin as described by the manufacturer and further purified using anion exchange (Q-Sepharose) and gel filtration (Superdex-200) in PBS buffer. For purification of GST-tagged proteins, the supernatant was partially purified using glutathione resin (Novagen) as described by the manufacturer. Unfused Bre5-(1–146) was prepared by treating a slurry of the resin-bound GST fusion proteins with thrombin (10 units/mg fused protein) overnight at 4 °C, and the untagged Bre5 protein was eluted with PBS buffer. Bre5-(1–146) was further purified with gel filtration (Superdex-75) in PBS buffer. Protein purity was judged to be greater than 90% by SDS-PAGE, and the protein was concentrated to 10–20 mg/ml in PBS for storage at -70 °C. Selenomethionine-derivatized Bre5-(1–146) was overexpressed from pGEX4T-Bre5-(1–146) transformed bacterial strain B834 (DE3) (Novagen) and grown in MOPS-based minimal medium. Selenomethionine-derivatived protein was purified and stored essentially as described for the underivatized protein. All site-directed Bre5-(1–146) mutations were prepared with the appropriate primers and the QuikChange mutagenesis kit essentially as described by the manufacturer (Stratagene), and the mutant proteins were purified essentially as described for the wild-type protein. The secondary structure of Bre5 protein fragments was analyzed using circular dichroism on a 62A DS spectropolarimeter (Aviv Associates, Lakewood, NJ) by using 300 μl of the respective sample at 10 °C. The protein concentration was about 30 μm in a buffer containing 20 mm Hepes, pH 7.0, 100 mm NaCl, and 10 mm β-mercaptoethanol. Sedimentation Equilibrium Analysis of Bre5—The oligomerization properties of Bre5-(1–146) and the C117A mutant were analyzed using sedimentation equilibrium on a Beckman XL-I analytical ultracentrifuge in a buffer containing 20 mm HEPES, pH 7.0, and 100 mm NaCl. Three starting protein concentrations (0.5, 0.75, and 1.0 mg/ml) and three centrifugation speeds (22,000, 30,000, and 40,000 rpm) were used with absorption optics for analysis. After equilibrium at each speed, as assessed by comparison of absorbance optics using the MATCH program, data editing was performed using the REEDIT program (both programs were provided by the National Analytical Ultracentrifugation Facility, Storrs, CT). The NONLIN program (31Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (779) Google Scholar) was used to globally fit several scans for each set of experiments. NONLIN fits used an effective reduced molecular weight of σ. For models of associating systems, σ was held at the correct value based on the known monomer molecular weight of the proteins and equilibrium constants were fitted as lnK. These values were converted to dissociation constants with the appropriate molar units. The fit quality of the models for all of the experiments was determined by examination of residuals and by minimization of the fit variance. In Vitro Bre5-Ubp3 Binding Studies—For pull-down assays, GST-Bre5-(1–146) protein (50 μm in PBS buffer) was incubated with purified His-Ubp3 deletion constructs (50 μm in PBS buffer) for 1 h at 4 °C, followed by immobilization on 30 μl of glutathione-Sepharose beads for 1 h at 4 °C. Beads were washed three times with 1 ml of PBS buffer, and 10-μl aliquots were mixed with an equal volume of 2× SDS-loading buffer and analyzed on SDS-PAGE. All isothermal titration calorimetry measurements were carried out using a MicroCal VP-ITC isothermal titration calorimeter (MicroCal, Inc.), and each experiment was carried out in duplicate. Untagged Ubp3-(181–282) protein was diluted to 50 μm in PBS buffer and added to the 1.4-ml sample cell, and a 0.3–0.5 mm solution of wild-type or mutant Bre5-(1–146) protein titrant was loaded into the injection syringe. For each titration experiment, a 60-s delay at the start of the experiment was followed by 35 injections of 10 μl of the titrant solution. The sample cell was stirred at 300 rpm throughout and maintained at 15 °C. Titration data were analyzed using the Origin 5.0 software supplied by MicroCal Inc., and data sets were corrected for base-line heats of dilutions from control runs as appropriate. The corrected data were then fit to a theoretical titration curve describing one binding site per titrant. The area under each peak of the resultant heat profile was integrated and plotted against the molar ratio of Ubp3 to Bre5. A nonlinear best fit binding isotherm for the data was used to calculate Ubp3/Bre5 stoichiometry, dissociation constant, and standard enthalpy change. In Vivo Sec23 Deubiquitination and Growth Assays—Yeast cultures were grown either in rich medium (YPD; Q-Biogen) or in minimal medium (SD) containing 0.67% yeast nitrogen base with ammonium sulfate, 2% dextrose, and supplemented with appropriate nutrients (32Orci L. Ravazzola M. Meda P. Holcomb C. Moore H.P. Hicke L. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8611-8615Crossref PubMed Scopus (149) Google Scholar). Yeast transformations were performed with standard procedures and preparation of the Bre5Δ strain as previously described (22Cohen M. Stutz F. Dargemont C. J. Biol. Chem. 2003; 278: 51989-51992Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The preparation of fusion proteins between Bre5 or its truncated versions with the LexA DNA binding domain expressed from the pBTM116 plasmid has been previously described (22Cohen M. Stutz F. Dargemont C. J. Biol. Chem. 2003; 278: 51989-51992Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The point mutations in Bre5-HisY42D, Bre5-HisR139D, and Bre5-HisY42R139D as well as the C-terminal His tag were introduced by PCR, and the resulting products were cloned into BamH1/XhoI sites of the P426-ADH plasmid (33Clemens K.E. Brent R. Gyuris J. Munger K. Virology. 1995; 214: 289-293Crossref PubMed Scopus (52) Google Scholar). Yeast cells grown in YPD or minimal medium were collected during the exponential growth phase (A600 = 2 or 0.8, respectively). Total protein extracts were prepared by the NaOH-TCA lysis method (21Cohen M. Stutz F. Belgareh N. Haguenauer-Tsapis R. Dargemont C. Nat. Cell Biol. 2003; 5: 661-667Crossref PubMed Scopus (133) Google Scholar). Protein samples were separated by 7% SDS-PAGE, transferred to nitrocellulose membranes, probed with appropriate antibodies, and detected with chemiluminescence protein immunoblotting reagents (Pierce). Rabbit polyclonal antibodies to Sec23p (1:400 dilution) were kindly provided by B. Lesch and R. Schekman, and mouse monoclonal antibody anti-His and rabbit polyclonal antibody anti-Lex were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Invitrogen, respectively. Growth assays of vector-transformed cells at different pH values were carried out as described above except that parallel growths were carried out at pH 6.5 and 8.0, and growth was monitored by cell plating. Crystallization and Structure Determination of Bre5-(1–146)—Crystals of Bre5-(1–146) were grown at room temperature using the hanging drop vapor diffusion method. 2 μl of protein solution at 14 mg/ml (0.87 mm) in buffer (20 mm HEPES, pH 7.0, 5 mm DTT, and 100 mm NaCl) was mixed with an equal volume of reservoir solution containing 25% polyethylene glycol 3350, 100 mm sodium citrate, pH 5.5, 0.25 m ammonium sulfate and equilibrating over 0.5 ml of reservoir solution. Crystals grew to a typical size of 200 × 100 × 100 μm over 3 days and were flash frozen in a reservoir solution supplemented with 20% glycerol for storage in solid propane prior to data collection. Single wavelength native and three-wavelength (peak, inflection, and remote) multiple anomalous diffraction data for selenium-derivatized crystals were collected on beamline X25 at the Brookhaven National Laboratories using an ADSC Quantum-4 CCD detector at 100 K. All data were processed with the HKL 2000 suite (HKL Research Inc.), and the relevant statistics are summarized in Table I. Three selenium sites were identified using CNS and SOLVE (34Brunger 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 (16991) Google Scholar, 35Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Phases were combined using CNS, and the resulting experimental electron density map was improved by solvent flipping density modification. The program O (36Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A Found. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13016) Google Scholar) was used to build the model of the protein using the selenomethionine positions as guides. Model refinement employed simulated annealing and torsion angle dynamic refinement protocols in CNS with iterative manual adjustments of the model using the program O, with reference to 2Fo - Fc and Fo - Fc electron density maps. Toward the later stages of refinement, individual atomic B-factors were adjusted, and solvent molecules were modeled into the electron density map. The final model was checked for errors with a composite-simulated annealing omit map. A final round of refinement resulted in a model with good refinement statistics and geometry (Table I).Table IData collection, phasing, and structure refinement statisticsData statisticsSe-MADNativeSpace groupP6122P6122a (Å)90.90190.909b (Å)90.90190.909c (Å)195.220194.866Wavelength (Å)0.97950.97940.95000.9790Resolution (Å)50-2.150-2.150-2.150-2.1Unique reflections28,58028,70028,80328,566Completeness (%)aValues in parentheses are from the highest resolution shell99.5 (100)99.5 (100)99.5 (100)99.2(99.6)Multiplicity21.221.221.810.4I/Io38.1 (13.5)46.5 (10.5)43.8 (7.4)28.3 (8.7)Rmerge (%)bRmerge = Σ|I - 〈I〉|/Σ〈I〉7.3 (28.9)6.7 (37.0)8.0 (51.7)7.6 (37.9)Phasing (SOLVE)FOM0.53Z-score23.3Refinement statisticsResolution (Å)50-2.1Rfree (%)cRfree = ΣT||Fo| - |Fc||/ΣTb|Fo| (where T is a test data set of 5% for Bre5 of the total reflections randomly chosen and set aside prior to refinement)21.8Rworking (%)dRworking = Σ||Fo| - |Fc||/Σ|Fo|19.4No. of protein atoms/B-factors (Å2)2103/33.2No. of protein atoms/B-factors (Å2)146/42.9Root mean square deviationsBond length (Å)0.033Bond angle (degrees)2.4a Values in parentheses are from the highest resolution shellb Rmerge = Σ|I - 〈I〉|/Σ〈I〉c Rfree = ΣT||Fo| - |Fc||/ΣTb|Fo| (where T is a test data set of 5% for Bre5 of the total reflections randomly chosen and set aside prior to refinement)d Rworking = Σ||Fo| - |Fc||/Σ|Fo| Open table in a new tab Mapping and Characterization of the Bre5 Binding Site in Ubp3—We employed a GST-Bre5 fusion protein harboring the NTF2-like domain of Bre5 ((GST-Bre5-(1–146)) and His6-tagged Ubp3 fusion proteins to map the Bre5 binding site in Ubp3. Initial pull-down experiments (data not shown) revealed that GST-Bre5-(1–146) did not show detectable interaction with the C-terminal catalytic domain of Ubp3 (residues 389–912). Based on this, we tested whether the N-terminal region of Ubp3 mediated Bre5 interaction. To address this possibility, we prepared a series of N-terminal Ubp3 deletion constructs (deleting about 50 residues at a time) for GST pull-down experiments. Specifically, we prepared His6 fusion proteins containing Ubp3-(1–912), -(50–912), -(150–912), -(189–912), and -(260–912) (Fig. 1a). As can be seen in Fig. 1b, each of the Ubp3 deletion constructs, except for Ubp3-(260–912), were pulled down with GST-Bre5-(1–146) at levels comparable with the intact protein. The His-Ubp3-(260–916) deletion construct did not show any pull-down with GST-Bre5-(1–146). These experiments revealed that a region within residues 189–260 of Ubp3 is necessary for interaction with the Bre5 NTF2-like domain. A secondary structure prediction of Ubp3 using PSIpred reveals two contiguous regions of high secondary structure, residues 280–912, roughly corresponding to the Ubp3 catalytic domain, and residues 186–272, roughly corresponding to the region of Ubp3 that we identified is involved in Bre5 interaction. Taken together, our studies suggested that residues 186–272 of Ubp3 constitute a folded protein domain that mediates Bre5 interaction. In order to directly test this hypothesis, we prepared two new recombinant Ubp3 constructs in which GST was fused to either residues 181–260 (GST-Ubp3-(181–260)) or residues 181–282 (GST-Ubp3-(181–282)). These GST fusion proteins were overexpressed in E. coli and purified by GST affinity, followed by thrombin cleavage to remove the GST component. In agreement with secondary structure predictions, a CD spectrum of these Ubp3 domains was consistent with the presence of a mainly helical protein domain (Fig. 1c). To determine whether recombinant Ubp3-(181–282) was sufficient for interaction with the NTF2-like domain of Bre5, we carried out a series of additional biochemical experiments. We first prepared a 2:1 stoichiometric complex of Bre5-(1–146) and Ubp3-(181–282) (since we expected an NTF2-like domain dimer to bind one Ubp3 subunit) and subjected the complex to gel filtration analysis. As can be seen from Fig. 1d, the two protein fragments coelute as a homogeneous complex. GST-pull-down studies employing GST-Bre5-(1–146) and untagged Ubp3-(181–260) and Ubp3-(181–282) also showed that the Ubp3 N-terminal domains bound to Bre5 as efficiently as full-length Ubp3 (Fig. 1b). These studies suggest that a region within residues 181–260 of Ubp3 is necessary and sufficient for interaction with the NTF2-like domain of Bre5. We recently reported that Bre5 and Ubp3 forms an active deubiquitination complex that cleaves off ubiquitin from Sec23, a COPII subunit essential for the transport between the endoplasmic reticulum and the Golgi apparatus (22Cohen M. Stutz F. Dargemont C. J. Biol. Chem. 2003; 278: 51989-51992Abstract Full Text Full Text PDF PubMed Scopus (64) Google" @default.
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