Crystal Structure of the N-terminal Domain of Anaphase-promoting Complex Subunit 7
2008; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês
10.1074/jbc.m804887200
ISSN1083-351X
AutoresDohyun Han, Kyunggon Kim, Yeonjung Kim, Yup Kang, Ji Yoon Lee, Youngsoo Kim,
Tópico(s)Cancer-related Molecular Pathways
ResumoAnaphase-promoting complex or cyclosome (APC/C) is an unusual E3 ubiquitin ligase and an essential protein that controls mitotic progression. APC/C includes at least 13 subunits, but no structure has been determined for any tetratricopeptide repeat (TPR)-containing subunit (Apc3 and -6-8) in the TPR subcomplex of APC/C. Apc7 is a TPR-containing subunit that exists only in vertebrate APC/C. Here we report the crystal structure of quad mutant of nApc7 (N-terminal fragment, residues 1-147) of human Apc7 at a resolution of 2.5 Å. The structure of nApc7 adopts a TPR-like motif and has a unique dimerization interface, although the protein does not contain the conserved TPR sequence. Based on the structure of nApc7, in addition to previous experimental findings, we proposed a putative homodimeric structure for full-length Apc7. This model suggests that TPR-containing subunits self-associate and bind to adaptors and substrates via an IR peptide in TPR-containing subunits of APC/C. Anaphase-promoting complex or cyclosome (APC/C) is an unusual E3 ubiquitin ligase and an essential protein that controls mitotic progression. APC/C includes at least 13 subunits, but no structure has been determined for any tetratricopeptide repeat (TPR)-containing subunit (Apc3 and -6-8) in the TPR subcomplex of APC/C. Apc7 is a TPR-containing subunit that exists only in vertebrate APC/C. Here we report the crystal structure of quad mutant of nApc7 (N-terminal fragment, residues 1-147) of human Apc7 at a resolution of 2.5 Å. The structure of nApc7 adopts a TPR-like motif and has a unique dimerization interface, although the protein does not contain the conserved TPR sequence. Based on the structure of nApc7, in addition to previous experimental findings, we proposed a putative homodimeric structure for full-length Apc7. This model suggests that TPR-containing subunits self-associate and bind to adaptors and substrates via an IR peptide in TPR-containing subunits of APC/C. Anaphase-promoting complex/cyclosome (APC/C) 2The abbreviations used are: APC/C, anaphase-promoting complex/cyclosome; TPR, tetratricopeptide repeat; CTPR3, consensus TPR number of repeats 3; r.m.s.d., root-mean-squared deviation; TLS, translation, libration, and screw-rotation; Apc, anaphase-promoting complex subunit; OGT, O-linked GlcNAc transferase; Sel-Met, selenomethionine; WT, wild type. is an E3 ubiquitin ligase that controls mitotic progression (1Peters J.M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). APC/C is an ∼1.7-MDa protein complex that is composed of at least 13 subunits, and it contains a cullin homolog (Apc2), a ring-H2 finger domain (Apc11), and a tetratricopeptide repeat (TPR)-containing subunit (TPR subunit; Apc3 and -6-8) (2Yoon H.J. Feoktistova A. Wolfe B.A. Jennings J.L. Link A.J. Gould K.L. Curr. Biol. 2002; 12: 2048-2054Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Most TPR subunits are essential and evolutionarily conserved in eukaryotes (3Harper J.W. Burton J.L. Solomon M.J. Genes Dev. 2002; 16: 2179-2206Crossref PubMed Scopus (424) Google Scholar). APC/C requires two adaptors that contain a C-terminal WD40 domain, Cdc20 and Cdh1, to recruit and select various substrates at different stages of the cell cycle. Moreover, both adaptors and specific APC/C subunits contribute to substrate recognition (4Visintin R. Prinz S. Amon A. Science. 1997; 278: 460-463Crossref PubMed Scopus (725) Google Scholar). APC/C specifically ubiquitinates cell cycle regulatory proteins that contain destruction (D) or KEN box motifs (5Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1903) Google Scholar, 6Pfleger C.M. Kirschner M.W. Genes Dev. 2000; 14: 655-665Crossref PubMed Google Scholar, 7Kraft C. Vodermaier H.C. Maurer-Stroh S. Eisenhaber F. Peters J.M. Mol. Cell. 2005; 18: 543-553Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), which target them for destruction by the 26 S proteosome (8Passmore L.A. Barford D. Biochem. J. 2004; 379: 513-525Crossref PubMed Scopus (231) Google Scholar). During the cell cycle, APC/C mediates the metaphase-anaphase transition by ubiquitinating and degrading securin, a separase inhibitor, which participates in the degradation of chromatic cohesion complexes and ubiquitinates B-type cyclin, thereby accelerating transition from the late mitotic phase to G1 (9Peters J.M. Nat. Rev. Mol. Cell Biol. 2006; 7: 644-656Crossref PubMed Scopus (1027) Google Scholar). In addition to its primary role in cell cycle regulation, APC/C participates in postmitotic processes, such as regulation of synaptic size and axon growth (10Konishi Y. Stegmüller J. Matsuda T. Bonni S. Bonni A. Science. 2004; 303: 1026-1030Crossref PubMed Scopus (323) Google Scholar, 11van Roessel P. Elliott D.A. Robinson I.M. Prokop A. Brand A.H. Cell. 2004; 119: 707-718Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). To assess the mechanism that underlies cell cycle regulation by APC/C and the various roles of its subunits, we need to understand how APC/C is organized into higher order structures and the manner in which the subunits assemble. Although little is known regarding the crystal structures of APC/C components, three-dimensional models of APC/C have recently been obtained by cryo-negative staining EM in human, Xenopus laevis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe (12Gieffers C. Dube P. Harris J.R. Stark H. Peters J.M. Mol. Cell. 2001; 7: 907-913Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13Dube P. Herzog F. Gieffers C. Sander B. Riedel D. Müller S.A. Engel A. Peters J.M. Stark H. Mol. Cell. 2005; 20: 867-879Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 14Passmore L.A. Booth C.R. Vénien-Bryan C. Ludtke S.J. Fioretto C. Johnson L.N. Chiu W. Barford D. Mol. Cell. 2005; 20: 855-866Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Ohi M.D. Feoktistova A. Ren L. Yip C. Cheng Y. Chen J.S. Yoon H.J. Wall J.S. Huang Z. Penczek P.A. Gould K.L. Walz T. Mol. Cell. 2007; 28: 871-885Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Several studies have indicated that APC/C assumes an asymmetric triangular shape that is composed of an outer shell and a cavity that extends through its center (12Gieffers C. Dube P. Harris J.R. Stark H. Peters J.M. Mol. Cell. 2001; 7: 907-913Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 14Passmore L.A. Booth C.R. Vénien-Bryan C. Ludtke S.J. Fioretto C. Johnson L.N. Chiu W. Barford D. Mol. Cell. 2005; 20: 855-866Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Furthermore, APC/C includes a catalytic subcomplex (Doc1/Apc10, Apc11, and Apc2), a structural complex (Apc1, Apc4, and Apc5), and a TPR subcomplex (TPR-containing subunits and nonessential subunits) (16Thornton B.R. Ng T.M. Matyskiela M.E. Carroll C.W. Morgan D.O. Toczyski D.P. Genes Dev. 2006; 20: 449-460Crossref PubMed Scopus (126) Google Scholar). A TPR unit consists of a 34-residue repeat motif that adopts a helix-turn-helix conformation, which is associated with protein-protein interactions (17Wilson C.G. Kajander T. Regan L. FEBS J. 2005; 272: 166-179Crossref PubMed Scopus (29) Google Scholar). Multiple copies of TPR-containing subunits are organized into the TPR subcomplex within APC/C, and this subcomplex is functionally important for the recruitment of adaptors and substrates (18Thornton B.R. Toczyski D.P. Genes Dev. 2006; 20: 3069-3078Crossref PubMed Scopus (132) Google Scholar). In fact, adaptors (Cdc20 and Cdh1) and Doc1/Apc10 bind to the C-terminal domain of the TPR-containing subunits Apc3 and Apc7 via the IR peptide tail sequence (7Kraft C. Vodermaier H.C. Maurer-Stroh S. Eisenhaber F. Peters J.M. Mol. Cell. 2005; 18: 543-553Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 16Thornton B.R. Ng T.M. Matyskiela M.E. Carroll C.W. Morgan D.O. Toczyski D.P. Genes Dev. 2006; 20: 449-460Crossref PubMed Scopus (126) Google Scholar, 19Vodermaier H.C. Gieffers C. Maurer-Stroh S. Eisenhaber F. Peters J.M. Curr. Biol. 2003; 13: 1459-1468Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). It is unknown, however, how TPR-containing subunits form homo- and heterosubunit complexes, although studies have demonstrated that TPR-containing subunits self-associate in vivo and in vitro (15Ohi M.D. Feoktistova A. Ren L. Yip C. Cheng Y. Chen J.S. Yoon H.J. Wall J.S. Huang Z. Penczek P.A. Gould K.L. Walz T. Mol. Cell. 2007; 28: 871-885Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and that they interact with other TPR-containing subunits (20Lamb J.R. Michaud W.A. Sikorski R.S. Hieter P.A. EMBO J. 1994; 13: 4321-4328Crossref PubMed Scopus (214) Google Scholar). Apc7 is found only in vertebrate APC/C and is estimated to contain 9-15 TPR motifs, similar to other TPR-containing subunits (9Peters J.M. Nat. Rev. Mol. Cell Biol. 2006; 7: 644-656Crossref PubMed Scopus (1027) Google Scholar). Apc7 is considered to be a molecular descendant of the same ancestral protein that gave rise to Apc3. Furthermore, the N-terminal domain of Apc7 has been reported to contain cell cycle-regulated phosphorylation sites (21Kraft C. Herzog F. Gieffers C. Mechtler K. Hagting A. Pines J. Peters J.M. EMBO J. 2003; 22: 6598-6609Crossref PubMed Scopus (336) Google Scholar), and the C-terminal TPR domain of Apc7 interacts with Cdh1 and Cdc20 (19Vodermaier H.C. Gieffers C. Maurer-Stroh S. Eisenhaber F. Peters J.M. Curr. Biol. 2003; 13: 1459-1468Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In Drosophila APC/C, the homolog of vertebrate Apc7 participates in synergistic genetic interactions with other TPR-containing subunits (22Pál M. Nagy O. Ménesi D. Udvardy A. Deák P. J. Cell Sci. 2007; 120: 3238-3248Crossref PubMed Scopus (25) Google Scholar). The function of Apc7 within vertebrate APC/C, however, is poorly understood. Moreover, although the C-terminal regions of Apc3 and Apc7 include a tandem of nine TPR motifs, the N-terminal domains of human Apc3 and Apc7 share little homology with the canonical TPR sequence. Therefore, the N-terminal domain of human Apc7 is expected to have a significant function in vertebrate APC/C. In this study, we determined the crystal structure of the N-terminal fragment of human Apc7 (residues 1-147, denoted nApc7), and the homodimeric self-association of nApc7 structure led us to insights into mechanisms of vertebrate APC/C. Cloning, Expression, and Purification—The N-terminal fragment of human Apc7 (residues 1-147, nApc7) was amplified by PCR from a plasmid encoding full-length human Apc7 and subcloned into the NdeI-XhoI site of the pET24(+) and pET28(+) vectors. The pET24(+) vector adds a C-terminal His tag to the expressed recombinant protein, whereas pET28(+) adds an N-terminal His tag and a thrombin cleavage site to the expressed recombinant protein. Apc7 constructs were transformed into Escherichia coli BL21-CodonPlus (DE3)-RIL (Stratagene, Madison, WI). E. coli BL21-CodonPlus (DE3)-RIL containing the pET28a(+)-nApc7 was grown in LB medium containing kanamycin (50 μg/ml) overnight at 37 °C. When the absorbance at 600 nm reached 0.5-0.6, protein expression was induced by adding 0.5 mm isopropyl d-thiogalactopyranoside at 20 °C. After overnight induction, cells were harvested by centrifugation at 5,000 × g for 15 min. Cell pellets were resuspended in ice-cold 20 mm Tris-HCl, pH 7.8, containing 200 mm NaCl, 1 mm β-mercaptoethanol, and a protease inhibitor mixture (Roche Applied Science). Resuspended cells were lysed by ultrasonication. Cell supernatants were obtained by centrifugation at 15,000 × g for 50 min at 4 °C. Selenomethionine (Sel-Met)-substituted nApc7 protein was expressed according to our protocols, as described previously (23Han D. Kim K. Oh J. Park J. Kim Y. Proteins. 2008; 70: 900-914Crossref PubMed Scopus (18) Google Scholar). Additionally, cells were grown at 20 °C for 90 min and then induced with isopropyl d-thiogalactopyranoside (0.5 mm) for 24 h at 20 °C. Sel-Met-substituted protein was purified using nickel-nitrilotriacetic acid resin (Peptron, Daejeon, Chungnam, South Korea) and a Superdex S-200 gel filtration column. Purified protein was dialyzed into storage buffer (20 mm Tris, pH 8.0, 1 mm β-mercaptoethanol, and 0.1 mm phenylmethylsulfonyl fluoride) and concentrated to 10 mg/ml by ultrafiltration using an Amicon filter (Millipore, Billerica, MA). Five millimolar 1,4-dithiothreitol was added to all buffers to prevent Sel-Met oxidation. The protocol described above was used to prepare the quad mutant of nApc7 (mnApc7). Crystallization and Data Collection—Crystals of wild-type (WT) nApc7 protein were obtained at 21 °C by the sitting-drop vapor diffusion method. The drops consisted of 2.0 μl of protein solution (20 mm Tris, pH 8.0) mixed with 2.0 μl of reservoir solution (0.2 m sodium/potassium tartrate, 15% PEG3350). Droplets were equilibrated against 400 μl of reservoir solution, and protein crystals that were 0.1 × 0.1 × 0.1 mm were obtained after 3 days at 21 °C. WT crystals were only diffracted to a resolution of 3 Å, but we could not obtain any phasing information. To reduce the number of methionine residues in the asymmetric unit, the methionine residues in WT nApc7 were mutated to other hydrophobic amino acids. Eventually, only the quad mutant (denoted mnApc7; containing M29L/M108L/M114V/M140L) generated a new crystal form that had enhanced diffraction characteristics, although we tried to crystallize all mutant constructs, i.e. the constructs that contained one, two, or three mutations. Quad mutant mnApc7 crystals were grown in 200 mm magnesium formate and 20% (w/v) PEG3350 at 21 °C. After crystal conditions were optimized, crystals were grown to 0.2 × 0.2 × 0.1 mm after 1 week. The final crystallization solutions contained 200 mm magnesium formate, 15% (w/v) PEG3350, and 10 mm strontium chloride. For data collection, crystals were transferred to a cryoprotectant solution (200 mm magnesium formate, pH 8.0, 20% (w/v) PEG3350, 10% glycerol) and flash-frozen in liquid nitrogen at 100 K. Diffraction data were collected at three wavelengths (0.97950, 0.97956, and 0.97171 Å) on the 4A beam line at the Pohang Accelerator Laboratory, Republic of Korea. mnApc7 crystals were diffracted to a resolution of 2.5 Å. Crystals were found to belong to the P21 space group, with unit cell dimensions of a = 80.5 Å, b = 64.1 Å, c = 81.6 Å and α = γ = 90°, and β = 95.2°, and contained four molecules in an asymmetric unit with a Matthews coefficient of 2.6 Å Da-1 (solvent content = 55%) (24Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7927) Google Scholar). Data from 50 to 2.5 Å were indexed and scaled using the HKL2000 software package (HKL, Charlottesville, VA). Statistical findings related to data collection and processing are summarized in Table 1.TABLE 1Data collection and refinement statisticsSel-MetmnApc7Data collectionSpace groupP21a = 80.5, b = 64.1, c = 81.6 Å; α = 90°, β = 95.2°, γ = 90°PeakInflectionRemoteWavelength (Å)0.979500.979560.97171Resolution (Å)50~2.5 (2.59-2.50)50~2.5 (2.59-2.50)50~2.5 (2.59-2.50)Rsym (%)6.7 (51.1)6.3 (48.1)6.3 (59.0)I/σI23.6 (1.3)24.3 (1.6)19.2 (1.2)Completeness (%)95.4 (77.2)96.0 (81.9)94.6 (73.7)Redundancy5.35.25.2RefinementResolution41.0-2.5 ÅNo. of reflections24,411Rwork/Rfree21.4/24.1%No. of atomsProtein3997Water48B-factorsProtein26.8 Å2Water52.7 Å2r.m.s.d.Bond lengthBond angle0.822° Open table in a new tab Structure Determination and Refinement—The overall structure of nApc7 was determined using the optimized multiwavelength anomalous diffraction method (25Gonzalez A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1935-1942Crossref PubMed Scopus (27) Google Scholar). To optimize dispersive scattering, we chose a remote energy of ∼500 eV away from the edge energy. Sel-Met positions and phasing were determined using SOLVE/RESOLVE (26Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (434) Google Scholar, 27Terwilliger T. J. Synchrotron Radiat. 2004; 11: 49-52Crossref PubMed Scopus (364) Google Scholar). One molecule contained four selenium sites, and thus a total of 16 selenium sites were present per asymmetric unit. Ten selenium sites were well located by SOLVE. As a result, an auto-traced model was obtained using RESOLVE, in which misconnected loop regions were manually rebuilt with good connectivity. Initial model building was performed using the "O" program (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). After mutation of the four methionine residues to three leucines and one valine, the residues were fitted into an experimental electron density map. Further model building was carried out using COOT (29Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). The final model contained residues 1-146 at a resolution of 2.5 Å, without the 20 N-terminal vector residues, the 20 residues of the flexible linker (residues 77-96), and 1 C-terminal residue (residues 147). Interestingly, the inserted residues of the cloning vector, pET28a(+), were disordered in the experimental electron density map, indicating that the nApc7 model did not contain amino acids that originated from the vector. The model was refined using REFMAC 5 with the Translation, Libration, and Screw-rotation (TLS) option (30Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1654) Google Scholar). TLS refinement was applied to the final models with TLS groups for each chain that was recommended by the TLSMD server (31Painter J. Merritt E.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 439-450Crossref PubMed Scopus (1108) Google Scholar). The optimal TLS groups for each chain were composed of three TLS group segments (residues 1-54, 55-113, and 114-146 in monomer A; residues 1-52, 53-116, and 117-146 in monomer B; residues 2-48, 49-104, and 105-146 in monomer C; and residues 2-21, 22-122, and 123-145 in monomer D). The final model was refined to an R/Rfree of 0.217/0.241, with 68 water molecules per asymmetric unit, and analyzed using PROCHECK in CCP4i (32Winn M.D. Ashton A.W. Briggs P.J. Ballard C.C. Patel P. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1929-1936Crossref PubMed Scopus (42) Google Scholar). The Ramachandran plot of the nApc7 model revealed that 93.1% of the residues were in the most favored region, 6.9% of the residues were in the allowed region, and no residues were in the disallowed region. Refinement statistics are summarized in Table 1. Site-directed Mutagenesis—After analyzing the secondary structure of Apc7 using the GOR4 program (33Garnier J. Gibrat J.F. Robson B. Methods Enzymol. 1996; 266: 540-553Crossref PubMed Google Scholar), the four methionine residues were substituted with three leucines and one valine to enhance crystallization and reduce Sel-Met content in the asymmetric unit of the crystal. Site-specific mutations (M29L, M108L, M114V, and M108L) in nApc7 were introduced by site-directed mutagenesis PCR. Constructs were confirmed by DNA sequencing. Mutants were expressed and purified using the protocol described above for the wild-type protein. Analytical Gel Filtration—Chromatography was performed using a Superdex-200 Tricorn 10/300 column attached to an AKTA FPLC system (Applied Biosystems, Foster City, CA) at a flow rate of 0.5 ml/min using 150 μl of protein at a concentration of 1 mg/ml. Protein elution was monitored at 280 nm using the UNICORN 5.0 program (Applied Biosystems). The column was calibrated with standard proteins, i.e. alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and ribonuclease A (15.7 kDa). Structural Analysis—Structural similarities between nApc7 and known structures were searched for in the SSM server (34Krissinel E. Henrick K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2256-2268Crossref PubMed Scopus (3189) Google Scholar). The PISA server was used to identify the dimer interface of the crystal lattice (35Krissinel E. Henrick K. J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6929) Google Scholar). Residues 169-200 were predicted to be a disordered domain linker by DLP-SVM (36Tanaka T. Yokoyama S. Kuroda Y. Biopolymers. 2006; 84: 161-168Crossref PubMed Scopus (27) Google Scholar). Sequence alignment was carried out using ClustalW (37Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). The representations rendered were generated using PyMOL (DeLano Scientific LLC, South San Francisco, CA). Structure Determination—Human Apc7 has a molecular mass of 62 kDa and is composed of 10 TPR repeats and two primary unassigned regions (residues 1-134 and residues 169-200) (Fig. 1A). To obtain insights into the function of the two unassigned regions, various cloning constructs were examined, and consequently, only construct residues 1-147 (nApc7) were able to be expressed as a soluble protein and used for crystallization. This strategy led to the successful crystallization of nApc7, and its structure was determined at a resolution of 2.5 Å. Crystallized nApc7 diffracted at a resolution of 3.0 Å. Diffraction data for nApc7 crystals indicated that it belongs to the primitive tetragonal space group, with unit-cell parameters a = 98.7, b = 98.7, c = 286.3 Å and α = β = γ = 90°, and has six molecules per asymmetric unit. We observed many methionine residues (n = 48) in the asymmetric unit, which were presumed to interfere with correct phasing. To reduce the methionine count in the asymmetric unit, four methionines (Met-29, Met-108, Met-140, and Met-114) were sequentially replaced in nApc7 with three leucines and one valine. Although we attempted to crystallize all of the mutant constructs (i.e. species that had one, two, three, or four mutations), only the quad mutant (mnApc7; M29L/M108L/M114V/M140L) generated a new crystal form that harbored enhanced diffraction properties. This new mutant crystal belonged to space group P21, with cell dimensions of a = 80.5 Å, b = 64.1 Å, c = 81.6 Å and α = γ = 90°, and β = 95.2°, and had four mnApc7 molecules per asymmetric unit. By mutating the aforementioned four methionines per mnApc7 (for a total of 16 methionines in the asymmetric unit), we overcame the phasing problem. The resultant electron density map from the multiwavelength anomalous diffraction phasing was readily traceable (see supplemental Fig. 1). The final structure was refined to an R value of 21.4% and an Rfree of 24.1% at a resolution of 2.5 Å (Table 1). Overall Structure of the N-terminal Fragment of Apc7—The overall structure of nApc7 contains seven anti-parallel α-helices (A1-A7) and is 41 Å long and 25 Å wide, with a pitch of 30 Å. Each monomer consists of two small domains, comprising residues 1-76 and 97-146, which are joined by a disordered flexible linker (residues 77-96) (Fig. 1, A-C). The asymmetric unit of the crystal lattice contains four molecules of nApc7 (molecules A-D). The superposition of C-α atoms of the four monomers of nApc7 in the asymmetric unit yields an r.m.s.d. of ∼1 Å onto their corresponding C-α positions. Monomer A forms a tightly packed dimer with its counterpart (monomer C′) in the neighboring asymmetric unit, in which these two nApc7 molecules are related by the crystallographic 2-fold axis (data not shown; see Fig. 4A). Using an SSM server, we searched for known proteins that were structurally similar to nApc7 (34Krissinel E. Henrick K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2256-2268Crossref PubMed Scopus (3189) Google Scholar). To this end, the overall folding of nApc7 resembled that of TPR proteins that contained three TPR repeats, although nApc7 does not contain any sequence that is homologous to the canonical TPR motif. CTPR3 (38Main E.R. Xiong Y. Cocco M.J. D'Andrea L. Regan L. Structure (Lond.). 2003; 11: 497-508Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) and NrfG (23Han D. Kim K. Oh J. Park J. Kim Y. Proteins. 2008; 70: 900-914Crossref PubMed Scopus (18) Google Scholar) proteins, which contain three TPR motifs, were superimposed onto nApc7 using r.m.s.d. values of 1.57 and 2.8 Å, respectively, indicating that the TPR-like fold of nApc7 is structurally conserved, despite its lacking the canonical TPR sequence (Trp-4, Leu-7, Gly-8, Tyr-11, Ala-20, Phe-24, Ala-27, and Pro-32 of the 34-residue TPR motif) (Fig. 2, A and B). Interestingly, the insertion of a long flexible linker (residues 77-96) did not disrupt the superhelical shape of the three TPR-like repeats of nApc7 (Fig. 2, A and B). nApc7 Folds into Atypical TPR-like Motifs—To compare the TPR-like folds of nApc7 with the typical TPR motif, nApc7 was superimposed onto the TPR domain of CTPR3 (Protein Data Bank code 1NA0), which was the TPR protein that had the highest Z-score, according to a Dali data base server (39Holm L. Kääriäinen S. Rosenström P. Schenkel A. Bioinformatics. 2008; 24: 2780-2781Crossref PubMed Scopus (849) Google Scholar). Seven anti-parallel α-helices (A1-A7) of nApc7 represented 3.5 tandem TPR-like motifs despite the lack of a conserved canonical sequence (Fig. 2, A and B). Although the overall structure of nApc7 assumed a superhelical TPR-like fold, our structural alignment indicated that the structure and conserved sequences of TPR-like motifs differed from those of the typical TPR motif, except for the presence of hydrophobic residues in the A4 helix (Fig. 2B). In particular, the Trp residue at position 4 of the canonical TPR sequence, which generally stabilizes the turns between helices through hydrophobic interactions, does not exist at the corresponding position in the TPR-like motifs of nApc7. Generally, packing interactions between adjacent TPR motifs are mediated by conserved hydrophobic residues in the typical TPR motif. In contrast, polar interactions, formed primarily by charged amino acids in helices A3 and A5, stabilize packing between TPR-like motifs in nApc7 (Fig. 2C). For example, turn 2 is stabilized by hydrogen bond formation between the side chain of Lys-42 in the A3 helix and the backbone oxygen of Pro-34 in loop 2. Also, packing between TPR-like motif 2 and TPR-like motif 3 is stabilized by polar interactions in both the convex and concave sides; Asn-51 and Lys-107 reinforce packing on the convex side, whereas polar interactions are formed by Arg-60, Lys-74, Glu-101, Lys-105, and Gln-116 on the concave side (Fig. 2C). These charged residues that are involved in packing interactions are highly conserved between other homologous Apc7 subunits (Fig. 3). Consequently, hydrogen bonds maintain the packing angle between the helices of nApc7 despite its unusually sized helices and loops. Thus, we found that nApc7 has secondary structural features that are unlike those of the typical TPR motif but that its overall shape resembles that of the TPR protein, i.e. a helix-loop-helix conformation and a superhelical structure. N-terminal Region of the TPR-like Repeats of nApc7 Mediates Self-dimerization—The crystal structure of nApc7 revealed it to be a homodimer in which two molecules of nApc7 associate along the crystallographic 2-fold screw axis (data not shown; see Fig. 4A). The PISA server was used to identify the dimer interface (35Krissinel E. Henrick K. J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6929) Google Scholar). The solvent-accessible surface that was calculated between the two symmetry mates was 586 Å2, which indicates a relatively small interfacial area. However, the ΔiG and ΔiGp values that were calculated using the PISA server were -13.9 kcal/mol and 0.022, respectively, which implies that a stable interaction underlies formation of the homodimer. Two nApc7 monomers are organized in an anti-parallel fashion to form the dimer. Helices A1 and A2 of one monomer interact with the interdigitating helices (A1 and A2) of the other monomer (Figs. 4A and 1B). Overlooking the 2-fold crystallographic axis, the dimer maintains an elongated shape, with overall dimensions of 25 × 30 × 80 Å (Figs. 4A and 1C). Formation of the nApc7 homodimer is mediated by motif 1 (Figs. 4A and 1C). One face of motif 1 is composed of many hydrophobic side chains that interact with those in the other monomer of the dimer (Fig. 4B). Val-3, His-6, and Met-10 (in the A1 helix); Leu-15 (in loop 1); Leu-21, Leu-22, Leu-25, Leu-26, and Met-29 (in the A2 helix); and Leu-36 and Phe-37 (in loop 2) participate in hydrophobic interactions with the residues of a symmetrically paired molecule (Fig. 4C). Importantly, sequence alignment showed that the hydrophobic residues that are involved in dimerization are highly conserved between homologous Apc7 subunits (Fig. 3), which suggests that Apc7 dimerization is a common feature of these homologs. Analytical gel filtration showed that nApc7 eluted at a molecular mass of ∼34 kDa, corresponding to dimeric nApc7 (Fig. 4D). In this study, we mutated two hydrophobic residues that are involved in forming the dimeric interface to charged residues (L22R and L26K) and performed size-exclusion chromatography for the double mutant protein. Doubly mutated nApc7 eluted at an apparent monomer size of 17 kDa (Fig. 4D), which supports our hypothesis that the Apc7 subunit exists as a dimer in solution. Interestingly, two monomers of dimeric nApc7 stack head-to-head because of strong hydrophobic interactions, resembling the interaction between adjacent TPR repeats in typical TPR proteins. These distinctive properties of dimerization be
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