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• W2001501463 abstract "Cul7 is a member of the Cullin Ring Ligase (CRL) family and is required for normal mouse development and cellular proliferation. Recently, a region of Cul7 that is highly conserved in the p53-associated, Parkin-like cytoplasmic protein PARC, was shown to bind p53 directly. Here we identify the CPH domains (conserved domain within Cul7, PARC, and HERC2 proteins) of both Cul7 and PARC as p53 interaction domains using size exclusion chromatography and NMR spectroscopy. We present the first structure of the evolutionarily conserved CPH domain and provide novel insight into the Cul7-p53 interaction. The NMR structure of the Cul7-CPH domain reveals a fold similar to peptide interaction modules such as the SH3, Tudor, and KOW domains. The p53 interaction surface of both Cul7 and PARC CPH domains was mapped to a conserved surface distinct from the analogous peptide-binding regions of SH3, KOW, and Tudor domains, suggesting a novel mode of interaction. The CPH domain interaction surface of p53 resides in the tetramerization domain and is formed by residues contributed by at least two subunits. Cul7 is a member of the Cullin Ring Ligase (CRL) family and is required for normal mouse development and cellular proliferation. Recently, a region of Cul7 that is highly conserved in the p53-associated, Parkin-like cytoplasmic protein PARC, was shown to bind p53 directly. Here we identify the CPH domains (conserved domain within Cul7, PARC, and HERC2 proteins) of both Cul7 and PARC as p53 interaction domains using size exclusion chromatography and NMR spectroscopy. We present the first structure of the evolutionarily conserved CPH domain and provide novel insight into the Cul7-p53 interaction. The NMR structure of the Cul7-CPH domain reveals a fold similar to peptide interaction modules such as the SH3, Tudor, and KOW domains. The p53 interaction surface of both Cul7 and PARC CPH domains was mapped to a conserved surface distinct from the analogous peptide-binding regions of SH3, KOW, and Tudor domains, suggesting a novel mode of interaction. The CPH domain interaction surface of p53 resides in the tetramerization domain and is formed by residues contributed by at least two subunits. The ubiquitin-proteosome system plays an important role in controlling diverse biological processes, ranging from signal transduction to cell cycle control (1Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (775) Google Scholar, 2Cardozo T. Pagano M. Nat. Rev. Mol. Cell Biol. 2004; 5: 739-751Crossref PubMed Scopus (860) Google Scholar). These complex processes are controlled via specific degradation of individual or groups of proteins. Protein degradation via the ubiquitin path-way involves two successive steps: tagging of the substrate by covalent attachment of multiple ubiquitin molecules (ubiquitylation) and degradation of the tagged protein by 26 S proteo-some complex with release of free and reusable ubiquitin (3Schwartz A.L. Ciechanover A. Annu. Rev. Med. 1999; 50: 57-74Crossref PubMed Scopus (372) Google Scholar). Ubiquitylation is the ultimate result of coordinated activity of an enzymatic cascade, which includes a ubiquitin-activating enzyme (E1), 3The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-ligating enzyme; TD, tetramerization domain; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride; NOE, nuclear Over-hauser effect; NOESY, NOE spectroscopy; MQLR, M340Q/L344R; HSQC, heteronuclear correlated spectroscopy; TOCSY, total correlated spectroscopy. 3The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-ligating enzyme; TD, tetramerization domain; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride; NOE, nuclear Over-hauser effect; NOESY, NOE spectroscopy; MQLR, M340Q/L344R; HSQC, heteronuclear correlated spectroscopy; TOCSY, total correlated spectroscopy. a ubiquitin-conjugating enzyme (E2), and ubiquitin-ligating (E3) enzymes. The E3 ligases are the “brain” of this process and determine substrate specificity (4Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1635) Google Scholar, 5Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). Cul7, is a recently identified member of the Cullin family of ubiquitin E3 ligases, localizes predominantly in the cytoplasm (6Andrews P. He Y.J. Xiong Y. Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (79) Google Scholar), and forms a unique Skp1-Cul7-Fbx29-like complex with FBXW8, a WD40 containing F-box protein (7Dias D.C. Dolios G. Wang R. Pan Z.Q. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16601-16606Crossref PubMed Scopus (137) Google Scholar, 8Arai T. Kasper J.S. Skaar J.R. Ali S.H. Takahashi C. DeCaprio J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9855-9860Crossref PubMed Scopus (117) Google Scholar, 9Tsunematsu R. Nishiyama M. Kotoshiba S. Saiga T. Kamura T. Nakayama K.I. Mol. Cell Biol. 2006; 26: 6157-6169Crossref PubMed Scopus (47) Google Scholar). Although a target substrate for FBXW8 has not been yet identified, Cul7 recruits RBX1 to form a Skp1-Cul7-Fbx29-like E3 ubiquitin ligase complex (7Dias D.C. Dolios G. Wang R. Pan Z.Q. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16601-16606Crossref PubMed Scopus (137) Google Scholar, 8Arai T. Kasper J.S. Skaar J.R. Ali S.H. Takahashi C. DeCaprio J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9855-9860Crossref PubMed Scopus (117) Google Scholar). The biological function of Cul7 is unclear. However, Cul7 appears to play an important role in development (8Arai T. Kasper J.S. Skaar J.R. Ali S.H. Takahashi C. DeCaprio J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9855-9860Crossref PubMed Scopus (117) Google Scholar, 9Tsunematsu R. Nishiyama M. Kotoshiba S. Saiga T. Kamura T. Nakayama K.I. Mol. Cell Biol. 2006; 26: 6157-6169Crossref PubMed Scopus (47) Google Scholar), and overexpression of Cul7 accelerates the rate of cell proliferation (6Andrews P. He Y.J. Xiong Y. Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (79) Google Scholar). Cul7 has significant sequence similarity (see Fig. 1) with the p53-associated, Parkin-like cytoplasmic protein, PARC (10Nikolaev A.Y. Li M. Puskas N. Qin J. Gu W. Cell. 2003; 112: 29-40Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Both proteins contain CPH (domain that is conserved in Cul7, PARC, and HERC2 proteins) (11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar), DOC (DOC1/APC10), and Cullin homology domains (see Fig. 1A) that are linked with E3 ligase function, suggesting that PARC and Cul7 may both function as E3 ubiquitin ligases. PARC has been shown to sequester p53 in the cytoplasm via interaction between the N terminus of PARC and the C terminus of p53 (10Nikolaev A.Y. Li M. Puskas N. Qin J. Gu W. Cell. 2003; 112: 29-40Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Cul7 may perform functions similar to those of PARC given their degree of sequence similarity and have also recently been shown to interact directly with p53 via its N terminus (6Andrews P. He Y.J. Xiong Y. Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (79) Google Scholar, 11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar). Recently published data identified a domain within Cul7 that is necessary and sufficient for p53 binding (6Andrews P. He Y.J. Xiong Y. Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (79) Google Scholar, 11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar). This domain (the CPH domain) also contributes to the cytoplasmic localization of Cul7 (6Andrews P. He Y.J. Xiong Y. Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (79) Google Scholar, 11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar). This, taken together with its similarity to PARC, another p53 interacting protein and putative E3 ligase, argues for more detailed investigation of these two proteins with respect to their interactions with p53. Understanding the structure, function, and interactions of Cul7/PARC domains with p53 will help to elucidate their involvement in ubiquitylation pathways and the circumstances through which they impinge on the p53 pathway. Inactivation of p53 is considered an important step in the development of many human cancers. It is therefore important to determine how p53 levels are regulated and how this regulation is altered in cancer. Transcriptionally active p53 protein is tetrameric, and in this conformation it binds with high affinity to DNA or interacts more efficiently with various other proteins (12Hainaut P. Hollstein M. Adv. Cancer Res. 2000; 77: 81-137Crossref PubMed Scopus (833) Google Scholar, 13Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2279) Google Scholar). The tetramerization domain is therefore important for p53 function because it ensures that the protein is endowed with its correct conformation. The oligomerization state of p53 is also thought to contribute to its subcellular localization by virtue of a cryptic nuclear export sequence that is only exposed in nontetrameric forms of the protein (14Stommel J.M. Marchenko N.D. Jimenez G.S. Moll U.M. Hope T.J. Wahl G.M. EMBO J. 1999; 18: 1660-1672Crossref PubMed Scopus (597) Google Scholar). Here we show that the conserved CPH domain of Cul7 interacts with the tetramerization domain (TD) of p53. To gain insight into Cul7 and its interaction with p53, we have solved the three-dimensional structure of the CPH domain of human Cul7 by NMR spectroscopy. The structure reveals a small domain with a fold similar to SH3, KOW, and Tudor domains, suggesting that it may function as a peptide-binding module. Chemical shift perturbation studies map the p53-TD interaction to regions distinct from the analogous peptide-binding surface of Tudor, KOW, and SH3 domains. Our NMR data also suggest that the Cul7-p53 interaction depends on the p53 oligomerization status. The structures and interactions were compared with the homologous region of PARC, confirming a similar interaction with the CPH domain of PARC. Cloning, Expression, and Purification—The coding regions for Cul7 360-460 and PARC 366-465 (CPH-containing domains) and the p53 TD (residues 310-360) were PCR-amplified from human Cul7 cDNA and human p53 cDNA, respectively, and subcloned into the pET15b expression vector (Novagen) at the 5′-NdeI site and 3′-BamHI site. The p53 fragment was expressed in Escherichia coli BL21 (DE3)-pLysS cells (Stratagene), whereas Cul7 and PARC constructs were expressed in E. coli BL21 (DE3) Rosetta cells (Novagen). For large scale production, the cells were grown at 37 °C until A600 nm of ∼1.0, and then the cultures were induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 5 h at room temperature before harvesting. The bacteria were grown in LB medium for nonlabeled proteins and in M9-defined medium supplemented with [15N]ammonium chloride (0.8 g/liter) and d-glucose. For the 15N/13C-labeled samples, d-glucose was replaced by 13C6-d-glucose (4 g/liter). These highly expressed proteins were purified by Talon (BD) affinity chromatography under native conditions and eluted with buffer containing 500 mm imidazole. The proteins were treated with thrombin and further purified by size exclusion chromatography using a HiLoad 26/60 Super-dex-75 column (GE Healthcare). Gel Filtration Studies—A calibrated Superdex 75 column was equilibrated with 25 mm Tris, pH 7.5, 250 mm NaCl, 2 mm benzamidine, 0.5 mm TCEP, 0.5 mm phenylmethylsulfonyl fluoride. Cul7-CPH and p53-TD were combined in 1:1 and 1:3 molar ratios and subjected to gel filtration chromatography (see Fig. 4A, shown in blue and black, respectively). Peak fractions were pooled and run on SDS-PAGE. NMR Spectroscopy—All of the spectra were recorded at 25 °C on Varian INOVA 600 MHz and Bruker Avance 500 MHz spectrometers equipped with triple resonance 1H/13C/15N cold and cryoprobes, respectively. All of the NMR samples were prepared at pH 7.5 with 25 mm Tris, 250 mm NaCl, 2 mm benzamidine hydrochloride, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm TCEP. The final NMR samples contained 90% H2O, 10% D2O with a protein concentration ranging between 0.5 and 0.7 mm. Two-dimensional, gradient-enhanced 1H/15N HSQC root mean square deviation spectra were acquired on uniformly 15N- or 13C/15N-labeled Cul7- and PARC-CPH domains and 15N-labeled p53-TD. The spectra were processed with NMRPipe software (15Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11280) Google Scholar) and analyzed with the SPARKY program (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco). Linear prediction in the 13C and 15N dimensions was used to improve the digital resolution. The assignment of 94.8% of 1H, 15N, and 13C resonances of Cul7-CPH domain was obtained with the automated ABACUS approach (16Grishaev A. Steren C.A. Wu B. Pineda-Lucena A. Arrowsmith C. Llinas M. Proteins. 2005; 61: 36-43Crossref PubMed Scopus (27) Google Scholar) combined with manual analysis using data from HNCO, CBCA(CO)NH, HBHA(CO)NH, CC(CO)NH-TOCSY, HC(CO)NH-TOCSY HC(C)H-TOCSY, and (H)CCH-TOCSY experiments. The assignment of ∼80% of 1H, 15N, and 13C resonances of the PARC-CPH domain were based on the following experiments: HNCACB, CBCA(CO)NH, HNCO, HNCA, and (H)CCH-TOCSY. The backbone resonances (1HN, 15N) of Cul7- and PARC-CPH domains obtained upon the addition of p53-TD were assigned assuming minimal change in chemical shifts relative to the unbound proteins. Structure Calculations—Distance restraints for structure calculations were obtained from 13C-edited NOESY-HSQC (τm = 120 ms) and 15N-edited NOESY-HSQC (τm = 150 ms) experiments. NOESY spectra were analyzed with the SPARKY program. The NOE-derived distance restraints were classified into four categories: 1.8-2.8, 1.8-3.5, 1.8-5.0, and 1.8-6.0Å, corresponding to strong, medium, weak, and very weak NOEs, respectively. The restraints for backbone φ and ψ torsion angles were derived from chemical shifts of backbone atoms using TALOS (17Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2727) Google Scholar). No hydrogen bond constraints were used. Structure calculations were carried out using CNS version 1.1 (18Brunger 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 (16919) Google Scholar) with its standard annealing protocol. Five iterative cycles of automated structure calculation and NOE assignment were applied. Automated NOE assignment was performed using the BACUS procedure (19Grishaev A. Llinas M. J. Biomol. NMR. 2004; 28: 1-10Crossref PubMed Scopus (24) Google Scholar). 15N-1H residual dipolar coupling constraints were incorporated in structure calculations on cycles 2-5 using floating alignment tensor (SANI (20Tjandra N. Omichinski J.G. Gronenborn A.M. Clore G.M. Bax A. Nat. Struct. Biol. 1997; 4: 732-738Crossref PubMed Scopus (468) Google Scholar) energy term with force constant of 2.0 kcal/mol·Hz2). On each cycle, the best 20 of 100 generated structures were selected for 1) filtering prior NOE assignments followed by a BACUS search for new identities and 2) estimating the magnitude of the axial and rhombic components of the alignment tensor using the PALES program (21Zweckstetter M. Bax A. J. Am. Chem. Soc. 2001; 123: 9490-9491Crossref PubMed Scopus (62) Google Scholar). The 20 lowest energy structures were refined using CNS by performing a short constrained molecular dynamics simulation in explicit solvent (22Linge J.P. Williams M.A. Spronk C.A. Bonvin A.M. Nilges M. Proteins. 2003; 50: 496-506Crossref PubMed Scopus (538) Google Scholar). The resulting structures were analyzed using MOLMOL (23Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14 (51-55): 29-32Crossref Scopus (6454) Google Scholar), PROCHEK-NMR (24Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4285) Google Scholar), and PSVS validation software (25Bhattacharya A. Tejero R. Montelione G.T. Proteins. 2006; 66: 778-795Crossref Scopus (538) Google Scholar). A homology model of PARC-CPH was generated from the NMR structure of the Cul7-CPH using YASARA (www.yasara.com). NMR Titration Experiments—Aliquots of 15N-labeled p53-TD and Cul7-CPH proteins were titrated into the unlabeled Cul7-CPH and p53-TD in molar ratios of 1:1, 1:2, 1:3, 1:4, and 1:5, respectively, until no further changes in chemical shifts were detected in the 1H-15N HSQC spectrum. HSQC spectra were acquired and analyzed after each addition of ligand. 15N-PARC-CPH domain was titrated with unlabeled p53-TD. The backbone resonances (1HN, 15N) of the bound form of Cul7- and PARC-CPH domains obtained upon the addition of p53-TD were assigned assuming a minimal change in chemical shifts compared with the unbound forms. 15N-p53-TD chemical shifts were those previously reported (20Tjandra N. Omichinski J.G. Gronenborn A.M. Clore G.M. Bax A. Nat. Struct. Biol. 1997; 4: 732-738Crossref PubMed Scopus (468) Google Scholar). The weighted chemical shift displacements were calculated using the following formula: Δppm = [(δnh)2 + (δn/5)2]1/2. Given that Cul7 and PARC have high sequence identity in their N-terminal domains and both interact with p53 via their N-terminal domains, we sought to identify a stable domain common to both proteins that would interact with p53. In screening for stable N-terminal domain constructs of Cul7, a protease-resistant fragment (residues 360-460) was identified from limited trypsin digestion (data not shown). Cul7(360-460) contains a CPH domain, named after a homologous sequence found within the Cul7, PARC, and HERC2 proteins (11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar, 26Hochrainer K. Mayer H. Baranyi U. Binder B. Lipp J. Kroismayr R. Genomics. 2005; 85: 153-164Crossref PubMed Scopus (57) Google Scholar) (Fig. 1). Solution Structure of the Cul7-CPH Domain—The solution structure of the Cul7-CPH was determined by heteronuclear multidimensional NMR spectroscopy. The recombinant protein used for the structural studies comprised residues 360-460 of human Cul7. However, only residues 360-440 adopt a well defined tertiary structure. The additional C-terminal residues are disordered as indicated by few 1H NOEs and small heteronuclear {1H}-15N NOE values (data not shown). The protein is monomeric in solution as determined by size exclusion chromatography (see Fig. 4A, peak P3). The structure was determined using 2144 distance constraints and 144 dihedral angle constraints from three-dimensional 15N and 13C-edited NOE spectra and 51 orientational constraints derived from residual dipolar couplings (Fig. 2A and Table 1).TABLE 1Structure calculation statistics of the CUL7-CPH domainNMR restraintsDistance NOE restraints2144Intraresidue850Sequential (|i - j| = 1)510Medium range (1 < |i - j| < 5)246Long range (|i - j| ≥ 5)538Dihedral angle restraints104φ angles52ψ angles52Residual dipolar coupling restraintsH-N51Characteristics of the structure ensembleaEnsemble of 20 lowest energy structures out of 100 calculated.NMR restraints violationsDistance restraints (>0.5 Å)0Bond angles (>5 degrees)0.50 ± 0.67Root mean square deviation from NMR restraintsDistance restraints (Å)0.015 ± 0.001Bond angles (deg.)0.82 ± 0.32Q factor for residual dipolar coupling restraints0.154 ± 0.005Deviation from ideal covalent geometryBond lengths (Å)0.007 ± 0.0001Bond angles (deg.)0.68 ± 0.02Root mean square deviation from mean structurebThe values were calculated for residues 10-78.Backbone atoms0.75 ± 0.17All heavy atoms1.23 ± 0.12Energies (kcal/mol)cEnergy is calculated using the PARALLHDG5.3 parameters set (1).Total−3077 ± 100Van der Waals'−380 ± 22Electrostatic−3616 ± 101Ramachandran plot (%)aEnsemble of 20 lowest energy structures out of 100 calculated.Most favored regions90.6Additionally allowed regions9.4Generously allowed regions0.0Disallowed regions0.0a Ensemble of 20 lowest energy structures out of 100 calculated.b The values were calculated for residues 10-78.c Energy is calculated using the PARALLHDG5.3 parameters set (1Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (775) Google Scholar). Open table in a new tab The CPH Domain Resembles SH3/Tudor/KOW Domains—The CPH domain has a β-barrel architecture consisting of five antiparallel β-strands, three loops, and an α-helix (Fig. 2B). Strands β1-β2 are connected by a long turn, β2-β3 and β3-β4 are connected by a short turn, whereas strands β4 and β5 are linked by a helical turn. The structure exhibits an overall negatively charged surface. Conserved residues Tyr389 (loop2), Gly406 (loop3), Val407 (loop3), Pro408 (loop3), Pro409 (loop3), Phe413 (β3), Thr420 (β4), and Trp422 (β4) are exposed and form a hydrophobic cluster that may be involved in ligand interactions (Fig. 2C). Other conserved, charged residues Glu391 (β2), Glu399 (β2), Asn404 (loop3), Gln411 (β3), Ser416 (loop4), and Arg419 (β4) could form an additional platform for protein-ligand interaction. A DALI search (www.ebi.ac.uk/dali/) for structurally similar proteins yielded a list of ∼90 proteins with Z score of >2.0. The NusG KOW domain (Protein Data Bank code 1M1G) and the SH3 domain of the kinase p56lck (LCK; Protein Data Bank code 1lck) are the two closest matches (Z = 6.7 and 6.6, respectively), although they lack any significant sequence similarity. The next 17 hits are all SH3 domains; the Tudor domains of SMN (Protein Data Bank code 1g5v) and 53BP1 are found in the 19th and 28th positions (Z = 5.4 and 4.3, respectively). This suggests that the CPH domain may be a protein-protein interaction module, and/or a nucleic acid-binding protein, because the KOW domain of NusG (27Steiner T. Kaiser J.T. Marinkovic S. Huber R. Wahl M.C. EMBO J. 2002; 21: 4641-4653Crossref PubMed Scopus (95) Google Scholar) and the Tudor domain of 53BP1 (28Charier G. Couprie J. Alpha-Bazin B. Meyer V. Quemeneur E. Guerois R. Callebaut I. Gilquin B. Zinn-Justin S. Structure. 2004; 12: 1551-1562Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) have been implicated in both these activities. The arrangement of β sheets in the CPH domain is almost identical to that found in SH3, Tudor, and KOW domains with backbone root mean square deviations on the order of 1.5 Å (Fig. 3). Nevertheless, there is significant variability in the length and arrangement of the loops that contribute to peptide binding in each domain. For example, the RT-Src loop of SH3 domain (29Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (240) Google Scholar) consists of 17 residues, whereas there are only 11 residues in the corresponding loop of the Cul7-CPH domain. Furthermore, despite the similar fold, all three domains display variety in the location of functional binding sites. Peptide binding by SH3 domains is mediated by a hydrophobic surface that is rich in aromatic residues and by various polar residues located in the RT-Src and N-Src loops (Fig. 3C) (30Larson S.M. Davidson A.R. Protein Sci. 2000; 9: 2170-2180Crossref PubMed Scopus (135) Google Scholar). The Tudor domain of SMN uses loops 1 and 3 to bind to symmetrically dimethylated arginines of RG-rich sequences (Fig. 3D) (31Selenko P. Sprangers R. Stier G. Buhler D. Fischer U. Sattler M. Nat. Struct. Biol. 2001; 8: 27-31Crossref PubMed Scopus (261) Google Scholar). The NusG KOW domain can bind proteins and nucleic acids at the same time via different surfaces (Fig. 3B) (27Steiner T. Kaiser J.T. Marinkovic S. Huber R. Wahl M.C. EMBO J. 2002; 21: 4641-4653Crossref PubMed Scopus (95) Google Scholar). Thus, each module uses the same fold in a unique manner to interact with partners, making it difficult to predict a substrate-binding surface for Cul7 from domain comparisons. The CPH Domain Interacts with p53-TD—The N-terminal region of Cul7 has been observed by us and others to be a major p53 binding determinant (6Andrews P. He Y.J. Xiong Y. Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (79) Google Scholar, 11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar). Recently Kasper et al. (11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar) reported that residues 268-438 of Cul7 were sufficient for association with p53. Residues 331-437 of Cul7 were shown to co-precipitate with p53 (6Andrews P. He Y.J. Xiong Y. Oncogene. 2006; 25: 4534-4548Crossref PubMed Scopus (79) Google Scholar, 11Kasper J.S. Arai T. Decaprio J.A. Biochem. Biophys. Res. Commun. 2006; 348: 132-138Crossref PubMed Scopus (24) Google Scholar). Because both of these fragments encompass the CPH domain, we further examined whether the latter was responsible for the p53 interaction using size exclusion chromatography and NMR titration experiments. As shown in Fig. 4A, when Cul7-CPH is preincubated with an excess of p53-TD at high protein concentrations (2 mg/ml), Cul7-CPH co-eluted with p53-TD as a Cul7-CPH/p53-TD complex (shown in black). 15N HSQC NMR experiments in which the 15N-Cul7-CPH was titrated with p53-TD revealed chemical shift perturbations for several residues (Fig. 4B). The free and bound forms of the protein are in fast exchange on the NMR time scale, suggesting that the affinity between CPH and p53-TD is likely in the micromolar range. An approximate equilibrium dissociation constant (Kd) of the Cul7-CPH/p53-TD complex was determined by least square fitting of the chemical shift changes accompanying ligand binding as a function of the total ligand concentration as described (32Tugarinov V. Kay L.E. J. Mol. Biol. 2003; 327: 1121-1133Crossref PubMed Scopus (83) Google Scholar). Using chemical shift changes of Tyr389, Gln411, and Gly398 upon the addition of p53-TD, a Kd of ∼530 μm was estimated. This result is in agreement with the gel filtration results where the co-elution of two proteins could only be observed when Cul7-CPH is saturated by p53-TD and proteins are preincubated at high concentrations (0.6 and 1.6 mm for Cul7-CPH and p53-TD, respectively). No interaction was observed when proteins were preincubated at 1:1 molar ratio (Fig. 4A, shown in blue). The central p53-TD peptide-binding region (defined as those residues with weighted chemical shift displacements greater than 0.05 ppm) includes Tyr389, Glu391, Asn404, Gly406, Val407, Gln411, Glu415, Ser416, Arg419, and Thr420 and forms a contiguous surface on the protein (Fig. 4B). In addition, the resonance peak for Trp422 completely disappeared and that of Phe413 was significantly broadened. Other residues surrounding this surface (Gly398, Glu399, Val410, and Val412) also showed chemical shift perturbations (between 0.03 and 0.05 ppm; Fig. 4B). These residues form a cluster between loops 1-3 and strands β2-β4 forming an interface for interaction with p53 (Fig. 4C). Significantly, these residues also encompass the conserved residues Tyr389, Glu391, Gly406, Val410, Gln411, Thr420, and Trp422 (Fig. 1B), suggesting that p53 binds to Cul7-CPH through a conserved surface formed by a combination of hydrophobic negatively charged residues. Oligomeric Forms of p53-TD Residues Are Required for Binding Cul7-CPH—To assess the surface of p53-TD that interacts with Cul7-CPH, 1H-15N HSQC titration experiments were performed on uniformly 15N-labeled samples of wild type p53-TD with increasing amounts of unlabeled Cul7-CPH (Fig. 5A). The observed spectral changes that occurred after the addition of Cul7-CPH are characterized by reductions in resonance intensity of the residues Phe328, Arg335, Arg342, and Asn345. Resonances for residues Gly325, Leu330, Ile332, Arg333, and Gly334 became so weak they can no longer be detected (Fig. 5A). Residues Phe328, Leu330, Ile332, and Arg333 belong to the β-strand and are exposed to the surface of p53. In addition, these residues are hydrophobic (Fig. 5, C and D) and have been proposed to be a potential site for interaction with other proteins (33Wagner P. Fuchs A. Gotz C. Nastainczyk W. Montenarh M. Oncogene. 1998; 16: 105-111Crossref PubMed Scopus (27) Google Scholar, 34Wagner P. Fuchs A. Prowald A. Montenarh M. Nastainczyk W. FEBS Lett. 1995; 377: 155-158Crossref PubMed Scopus (11) Google Scholar). Residues Gly334 and Arg335 are also surface-exposed, and they belong to the turn that links the α-helices to the β-sheets. An additional, six residues in our p53-TD spectrum had reduced intensities but could not be assigned because of spectral overlap (35Lee W. Harvey T.S. Yin Y. Yau P. Litchfield D. Arrowsmith C.H. Nat. Struct. Biol. 1994; 1: 877-890Crossref PubMed Scopus (234) Google Scholar). Because of the small size of p53-TD (∼30 residues/subunit) and as a result of possible conformational changes in p53-TD, it is likely that residues distinct from the binding surface may also be affected by interaction with Cul7-CPH protein. Furthermore, a 1:1 complex between the symmetric p53 tetramer and the asymmetric Cul7-CPH domain would destroy the symmetry of the p53-TD, further complicating the NMR spectrum of the latter. This may explain why distinct chemical shift changes" @default.
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• W2001501463 title "The Conserved CPH Domains of Cul7 and PARC Are Protein-Protein Interaction Modules That Bind the Tetramerization Domain of p53" @default.
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