Crystal Structure of the Human Centromere Protein B (CENP-B) Dimerization Domain at 1.65-Å Resolution

The human centromere protein B (CENP-B), a centromeric heterochromatin component, forms a homodimer that specifically binds to a distinct DNA sequence (the CENP-B box), which appears within every other α-satellite repeat. Previously, we determined the structure of the human CENP-B DNA-binding domain, CENP-B-(1-129), complexed with the CENP-B box DNA. In the present study, we determined the crystal structure of its dimerization domain (CENP-B-(540-599)), another functional domain of CENP-B, at 1.65-Å resolution. CENP-B-(540-599) contains two α-helices, which are folded into an antiparallel configuration. The CENP-B-(540-599) dimer formed a symmetrical, antiparallel, four-helix bundle structure with a large hydrophobic patch in which 23 residues of one monomer form van der Waals contacts with the other monomer. In the CENP-B-(540-599) dimer, the N-terminal ends of CENP-B-(540-599) are oriented on opposite sides of the dimer. This CENP-B dimer configuration may be suitable for capturing two distant CENP-B boxes during centromeric heterochromatin formation. The human centromere protein B (CENP-B), a centromeric heterochromatin component, forms a homodimer that specifically binds to a distinct DNA sequence (the CENP-B box), which appears within every other α-satellite repeat. Previously, we determined the structure of the human CENP-B DNA-binding domain, CENP-B-(1-129), complexed with the CENP-B box DNA. In the present study, we determined the crystal structure of its dimerization domain (CENP-B-(540-599)), another functional domain of CENP-B, at 1.65-Å resolution. CENP-B-(540-599) contains two α-helices, which are folded into an antiparallel configuration. The CENP-B-(540-599) dimer formed a symmetrical, antiparallel, four-helix bundle structure with a large hydrophobic patch in which 23 residues of one monomer form van der Waals contacts with the other monomer. In the CENP-B-(540-599) dimer, the N-terminal ends of CENP-B-(540-599) are oriented on opposite sides of the dimer. This CENP-B dimer configuration may be suitable for capturing two distant CENP-B boxes during centromeric heterochromatin formation. The centromere, an essential chromosomal region required for the proper segregation of chromosomes during cell division, is embedded within heterochromatin in most eukaryotes (1Sullivan B. Karpen G. J. Cell Biol. 2001; 154: 683-690Crossref PubMed Scopus (67) Google Scholar, 2Choo K.H.A. Dev. Cell. 2001; 1: 165-177Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 3Cleveland D.W. Mao Y. Sullivan K.F. Cell. 2003; 112: 407-421Abstract Full Text Full Text PDF PubMed Scopus (825) Google Scholar). This centromere-specific heterochromatin contains specific DNA-binding proteins that target the centromeric region of chromosomes. In humans, the centromere-specific DNA-binding proteins CENP-A, 1The abbreviations used are: CENP-A/-B/-Ccentromere protein A, B, or CNi-NTAnickel-nitrilotriacetic acidCHES2-(cyclohexylamino)ethanesulfonic acid. CENP-B, and CENP-C were identified as antigens for autoimmune sera from scleroderma patients (4Earnshaw W.C. Rothfield N. Chromosoma. 1985; 91: 313-321Crossref PubMed Scopus (652) Google Scholar, 5Valdivia M.M. Brinkley B.R. J. Cell Biol. 1985; 101: 1124-1134Crossref PubMed Scopus (63) Google Scholar). centromere protein A, B, or C nickel-nitrilotriacetic acid 2-(cyclohexylamino)ethanesulfonic acid. CENP-A is the centromere-specific histone H3 variant (6Sullivan K.F. Hechenberger M. Masri K. J. Cell Biol. 1994; 127: 581-592Crossref PubMed Scopus (365) Google Scholar), and CENP-C is a fundamental component of the inner kinetochore plate (7Saitoh H. Tomkiel J. Cooke C.A. Ratrie III, H. Maurer M. Rothfield N.F. Earnshaw W.C. Cell. 1992; 10: 115-125Abstract Full Text PDF Scopus (318) Google Scholar, 8Tomkiel J. Cooke C.A. Saitoh H. Bernat R.L. Earnshaw W.C. J. Cell Biol. 1994; 125: 531-545Crossref PubMed Scopus (176) Google Scholar, 9Fukagawa T. Brown W.R. Hum. Mol. Genet. 1997; 6: 2301-2308Crossref PubMed Scopus (85) Google Scholar, 10Kalitsis P. Fowler K.J. Earle E. Hill J. Choo K.H.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1136-1141Crossref PubMed Scopus (116) Google Scholar, 11Fukagawa. T. Pendon. C. Morris J. Brown W. EMBO J. 1999; 18: 4196-4209Crossref PubMed Scopus (89) Google Scholar). CENP-A and CENP-C do not seem to have any obvious sequence specificity in DNA binding. In contrast, CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears in every other 171-base pair α-satellite repeat in human centromeres (12Masumoto H. Masukata H. Muro Y. Nozaki N. Okazaki T. J. Cell Biol. 1989; 109: 1963-1973Crossref PubMed Scopus (554) Google Scholar), and induces nucleosome positioning in the vicinity of the CENP-B box (13Yoda K. Ando S. Okuda A. Kikuchi A. Okazaki T. Genes Cells. 1998; 3: 533-548Crossref PubMed Scopus (58) Google Scholar). These results suggest that CENP-B may function as a transacting factor, which induces the formation of the centromere-specific heterochromatin. Analyses with cultured human cells revealed that the existence of the CENP-B box within the α-satellite sequence is required for the formation of a functional centromere containing CENP-B in minichromosomes in vivo (14Ikeno M. Grimes B. Okazaki T. Nakano M. Saitoh K. Hoshino H. McGill N.I. Cooke H. Masumoto H. Nat. Biotechnol. 1998; 16: 431-439Crossref PubMed Scopus (360) Google Scholar, 15Ohzeki J.-I. Nakano M. Okada T. Masumoto H. J. Cell Biol. 2002; 159: 765-775Crossref PubMed Scopus (223) Google Scholar). Biochemical and genetic studies also showed that CENP-A, CENP-B, and CENP-C cooperatively constitute functional centromeres in mammalian cells (16Howman E.V. Fowler K.J. Nelson A.J. Redward S. MacDonald A.C. Kalitsis P. Choo K.H.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1148-1153Crossref PubMed Scopus (340) Google Scholar, 17Ando S. Yang H. Nozaki N. Okazaki T. Yoda K. Mol. Cell. Biol. 2002; 22: 2229-2241Crossref PubMed Scopus (141) Google Scholar). However, CENP-B null mice appeared to be normal (18Hudson D.F. Fowler K.J. Earle E. Saffery R. Kalitsis P. Trowell H. Hill J. Wreford N.G. de Kretser D.M. Cancilla M.R. Howman E. Hii L. Cutts S.M. Irvine D.V. Choo K.H.A. J. Cell Biol. 1998; 141: 309-319Crossref PubMed Scopus (196) Google Scholar, 19Perez-Castro A.V. Shamanski F.L. Meneses J.J. Lovato T.L. Vogel K.G. Moyzis R.K. Pedersen R. Dev. Biol. 1998; 201: 135-143Crossref PubMed Scopus (112) Google Scholar, 20Fowler K.J. Hudson D.F. Salamonsen L.A. Edmondson S.R. Earle E. Sibson M.C. Choo K.H.A. Genome Res. 2000; 10: 30-41PubMed Google Scholar). This discrepancy about the CENP-B requirement for centromere formation may be explained by presuming the existence of functional homologue(s) of CENP-B. In fact, CENP-B-like proteins of unknown function have been identified in humans, such as the jerky-like protein (21Toth M. Grimsby J. Buzsaki G. Donovan G.P. Nat. Genet. 1995; 11: 71-75Crossref PubMed Scopus (79) Google Scholar, 22Liu W. Seto J. Sibille E. Toth M. Mol. Cell. Biol. 2003; 23: 4083-4093Crossref PubMed Scopus (23) Google Scholar) and the transposases encoded by the human Tigger1 and Tigger2 transposable elements (23Smit A.F. Riggs A.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1443-1448Crossref PubMed Scopus (362) Google Scholar, 24Kipling D. Warburton P.E. Trends Genet. 1997; 13: 141-145Abstract Full Text PDF PubMed Scopus (168) Google Scholar). The functional redundancy of CENP-B homologues has also been found in the fission yeast Schizosaccharomyces pombe. Three fission yeast proteins, Abp1, Cbh1, and Cbh2, are homologues of CENP-B (25Murakami Y. Huberman J.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1996; 9: 502-507Crossref Scopus (48) Google Scholar, 26Lee J.K. Huberman J.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8427-8432Crossref PubMed Scopus (38) Google Scholar, 27Irelan J.T. Gutkin G.I. Clarke L. Genetics. 2001; 157: 1191-1203PubMed Google Scholar) and associate with centromeric heterochromatin (28Halverson D. Baum M. Stryker J. Carbon J. Clarke L. J. Cell Biol. 1997; 136: 487-500Crossref PubMed Scopus (47) Google Scholar, 29Nakagawa H. Lee J.K. Hurwitz J. Allshire R.C. Nakayama J. Grewal S.I. Tanaka K. Murakami Y. Genes Dev. 2002; 16: 1766-1778Crossref PubMed Scopus (84) Google Scholar). None of these proteins is essential for S. pombe viability like human CENP-B (30Baum M. Clarke L. Mol. Cell. Biol. 2000; 20: 2852-2864Crossref PubMed Scopus (48) Google Scholar). However, the double disruptants of Abp1 and Cbh1 showed a synergistic reduction of centromere function in fission yeast (29Nakagawa H. Lee J.K. Hurwitz J. Allshire R.C. Nakayama J. Grewal S.I. Tanaka K. Murakami Y. Genes Dev. 2002; 16: 1766-1778Crossref PubMed Scopus (84) Google Scholar, 30Baum M. Clarke L. Mol. Cell. Biol. 2000; 20: 2852-2864Crossref PubMed Scopus (48) Google Scholar). Human CENP-B is a dimeric protein composed of 80-kDa subunits (31Earnshaw W.C. Machlin P.S. Bordwell B.J. Rothfield N.F. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4979-4983Crossref PubMed Scopus (92) Google Scholar). It contains DNA-binding domains and a dimerization domain at the N terminus and the C terminus, respectively (32Kitagawa K. Masumoto H. Ikeda M. Okazaki T. Mol. Cell. Biol. 1995; 15: 1602-1612Crossref PubMed Google Scholar). Electron microscopic observations showed that CENP-B bundles the two distant CENP-B boxes by its dimer formation and DNA binding activities (13Yoda K. Ando S. Okuda A. Kikuchi A. Okazaki T. Genes Cells. 1998; 3: 533-548Crossref PubMed Scopus (58) Google Scholar). The bundling activity suggests that, in the centromere, CENP-B may function in the higher ordered chromatin formation, in addition to the centromere-specific nucleosome assembly. To reveal the molecular mechanism of the CENP-B functions in centromeric chromatin formation, we determined previously the crystal structure of the CENP-B N-terminal DNA-binding domain complexed with the CENP-B box DNA (33Tanaka Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (74) Google Scholar). In the present study, we determined the crystal structure of another CENP-B functional domain, the dimerization domain, at 1.65-Å resolution. The structure provides new insights into CENP-B dimerization that may play an essential role in the centromere-specific heterochromatin formation. Purification of the Dimerization Domain of CENP-B—A DNA fragment containing the CENP-B-(540-599) portion of the human CENP-B gene was amplified by polymerase chain reaction and cloned into the NdeI site of the pET15b vector. Selenomethionine-labeled CENP-B-(540-599) was overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RP-X cells under the control of the T7 promoter as a His6-tagged protein. The E. coli strains carrying the CENP-B-(540-599) expression vector were grown at 30 °C, and isopropyl-β-d-thiogalactopyranoside (IPTG; 80 μm) was added at an OD600 of 0.4 to induce protein expression. After overnight cultivation at 18 °C, the cells were harvested and disrupted by sonication in 50 mm Tris-HCl buffer (pH 8.0) containing 500 mm NaCl. The cell debris was removed by centrifugation for 20 min at 30,000 × g, and the lysate was mixed gently by the batch method with nickel-nitrilotriacetic acid (Ni-NTA)-agarose beads (Qiagen) at 4 °C for 1 h. The CENP-B-(540-599)-bound Ni-NTA-agarose beads were then packed into an Econo-Column (Bio-Rad) and washed with 30-column volumes of 50 mm Tris-HCl buffer (pH 8) containing 500 mm NaCl, 5% glycerol, 1 mm phenylmethylsulfonyl fluoride, and 5 mm imidazole, at a flow rate of about 0.3 ml/min. The His6-tagged CENPB-(540-599) was eluted in a 20-column volume linear gradient from 5 to 300 mm imidazole in 50 mm Tris-HCl buffer (pH 8) containing 500 mm NaCl, 5% glycerol, and 1 mm phenylmethylsulfonyl fluoride. The protein was dialyzed against 50 mm Tris-HCl buffer (pH 8) containing 50 mm NaCl, 5% glycerol, and 1 mm phenylmethylsulfonyl fluoride, and the His6 tag was uncoupled from CENP-B-(540-599) by a digestion with 10 units of thrombin protease (Amersham Biosciences) per milligram of CENP-B-(540-599). Then, the fractions containing CENP-B-(540-599) were mixed with 5 ml of heparin-Sepharose (Amersham Biosciences) at 4 °C for 2 h and then packed into an Econo column (Bio-Rad). The heparin-Sepharose beads with CENP-B-(540-599) were washed with 6-column volumes of 50 mm Tris-HCl buffer (pH 8) containing 50 mm NaCl, and CENP-B-(540-599) was eluted in a 12-column volume linear gradient from 50 to 800 mm NaCl in this buffer. The CENP-B-(540-599) protein was concentrated and further purified by HiLoad 26/60 Superdex 75 gel filtration chromatography (Amersham Biosciences). Crystallization and Structure Determination—The purified CENP-B-(540-599) was concentrated up to 12 mg of protein per milliliter, and crystals were obtained by the hanging drop method after mixing an equal volume of 12 mg/ml CENP-B-(540-599) with a reservoir solution of 50 mm CHES buffer (pH 9.5) containing 0.5 m sodium citrate. The CENP-B-(540-599) crystals were soaked in a cryo-protectant solution of 50 mm CHES buffer (pH 9.5) containing 5% glycerol for a few minutes. Then, the crystals were transferred to 50 mm CHES buffer (pH 9.5) containing 10% glycerol for a few minutes and transferred again to 50 mm CHES buffer (pH 9.5) containing 20% glycerol. The crystals were flash-frozen in a stream of N2 gas (100 K). The crystals belong to the orthorhombic space group P212121, with unit cell constants of a = 43.7 Å, b = 49.0 Å, and c = 100.7 Å, and contain two dimers per asymmetric unit. High resolution diffraction data were obtained using the synchrotron radiation source at the RIKEN beam line BL44B2 station (RIKEN Structural Biology Beamline II; Ref. 34Adachi S. Oguchi T. Tanida H. Park S.-Y. Shimizu H. Miyatake H. Kamiya N. Shiro Y. Inoue Y. Ueki T. Iizka T. Nucl. Intr. Meth. Phys. Res. Sect. A. 2001; 467/468: 711-714Crossref Scopus (59) Google Scholar) of SPring-8, Harima, Japan. Intensity data were collected with a MAR CCD detector. The structure of CENP-B-(540-599) was initially solved to 1.8-Å resolution by the multiple wavelength anomalous dispersion (MAD) method. Diffraction data were integrated and scaled with HKL2000 and SCALEPACK (35Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). General handling of the scaled data was carried out with programs from the CCP4 suite (36Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The positions of the selenium atoms were determined using the program SOLVE (37Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The magnitudes of the anomalous differences at the peak wavelength were normalized, and the density modification was performed with RESOLVE (37Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The resulting electron density map was sufficiently clear to build an initial model of the structure. The model was built with the program TURBO-FRODO (38Roussel A. Cambillau C. Silicon Graphics Geometry Partner Directory. Silicon Graphics, Mountain View, CA1989: 77-78Google Scholar). Structural refinement was performed using ARPwARP (39Perrakis A. Morris R. Lamizin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar), XPLOR version 3.851 (40Brunger A.T. X-PLOR Version 3.851 Manual. Yale University Press, New Haven, CT1996Google Scholar), and REFMAC. Solvent molecules were placed at the positions where spherical electron density peaks were found above 1.3 σ in the 2Fo - Fc map and above 3 σ in the Fo - Fc map and where the stereochemically reasonable hydrogen bonds were allowed. Structural evaluations of the final models using PROCHECK (41Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: Google Scholar) that of the residues are in the most of the with residues in the of the data and refinement is in The of CENP-B-(540-599) have been for Structural of the cell constants c = = and are for data and for the resolution The resolution for the most column to the one are and Å, and are for data and for the resolution The resolution for the most column to the one are and Å, after RESOLVE is of resolution and are for data and for the resolution The resolution for the most column to the one are and Å, and are for data and for the resolution The resolution for the most column to the one are and Å, where is of an in the for that and the are where Fo and Fc are the and structure The was with 5% of the data from the is in most in and are for data and for the resolution The resolution for the most column to the one are and Å, is of where is of an in the for that and the are where Fo and Fc are the and structure The was with 5% of the data from the is in a new Structure composed of acid residues was as a His6-tagged protein in E. coli cells and purified by chromatography on a column (Qiagen) The His6 tag was uncoupled with thrombin protease (Amersham Biosciences) from the CENP-B-(540-599) which the His6-tagged protein gel Then, CENP-B-(540-599) was further purified by heparin-Sepharose column chromatography (Amersham by HiLoad 26/60 Superdex 75 gel filtration column chromatography (Amersham Biosciences) filtration revealed that the molecular of the purified CENP-B-(540-599) is which to that of two molecules of CENP-B-(540-599) for two CENP-B-(540-599) we that the purified CENP-B-(540-599) formed in Then, the CENP-B-(540-599) dimer was concentrated up to 12 mg of protein per milliliter, and the crystals were obtained by the hanging drop method. The crystals belong to the orthorhombic space group P212121, with unit cell constants of a = = = Å, and = = = and contain two dimers per asymmetric unit. The crystal structure was solved by the multiple wavelength anomalous dispersion (MAD) method using crystals of the diffraction data was collected at 1.65-Å resolution at BL44B2 in SPring-8, Harima, Japan. the crystal CENP-B-(540-599) is composed of two and which are folded into an antiparallel configuration The human CENP-B-(540-599) region is the mammalian for the CENP-B that the dimerization domain of mammalian are The and are and the two CENP-B-(540-599) which hydrophobic form a dimer with a symmetrical, antiparallel, four-helix bundle structure The residues of CENP-B-(540-599) C terminus, which is the C terminus of the CENP-B, are are not required for dimer formation. This may the of the CENP-B which is in the acid residues in the CENP-B region containing the are in the human Tigger1 which also sequence with CENP-B in the N-terminal DNA-binding and the Tigger1 may be a CENP-B functional which the CENP-B function in the acid residues in the region of CENP-B-(540-599) are also in the S. pombe CENP-B homologues Abp1, Cbh1, and that the dimerization of the human and fission yeast are The crystals used in the present contain two dimers per asymmetric unit in which the CENP-B-(540-599) dimers with other The these CENP-B-(540-599) dimers was for the that these CENP-B-(540-599) dimers The in the two CENPB-(540-599) to form an antiparallel, four-helix This of dimerization is also present in the protein of for the The two of CENP-B-(540-599) are in the two of the monomer are also in the dimeric The in the dimeric forms of CENP-B-(540-599) and are the the N-terminal with of the other monomer. four-helix bundle were also found in the human proteins and L. L. Moss H. de T. D. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) C and However, these are of the large dimerization domains of and the dimerization domains of these proteins from that of CENP-B. The the CENP-B-(540-599) dimer and the dimerization of and were not of and Å, for the and the two of the or monomer within the are (the these are C and these results that the CENP-B-(540-599) dimer structure is The dimerization of CENP-B-(540-599) is a of of the monomer which is large to the dimerization of CENP-B. the other the in the in the asymmetric unit is as as that this is In the functional dimerization 23 residues of one monomer form van der Waals contacts with the other monomer hydrogen bonds were also found in the large hydrophobic the that the CENP-B-(540-599) dimer formation is The chain group of forms a hydrogen with the of the other monomer The group of forms a hydrogen with the of the other monomer These are the of bundles two distant CENP-B boxes its dimer formation and DNA-binding (13Yoda K. Ando S. Okuda A. Kikuchi A. Okazaki T. Genes Cells. 1998; 3: 533-548Crossref PubMed Scopus (58) Google Scholar). This activity be for the centromeric chromatin formation. To reveal the functional of the CENP-B of the DNA-binding and dimerization domains of CENP-B be determined previously the crystal structure of the CENP-B N-terminal DNA-binding domain in a form with the CENP-B box DNA (33Tanaka Y. Nureki O. Kurumizaka H. Fukai S. Kawaguchi S. Ikuta M. Iwahara J. Okazaki T. Yokoyama S. EMBO J. 2001; 20: 6612-6618Crossref PubMed Scopus (74) Google Scholar). In the present study, we determined the crystal structure of another CENP-B functional domain, the dimerization domain, which revealed that the CENP-B dimerization domains form a homodimer with a symmetrical, antiparallel, four-helix bundle structure. hydrophobic residues the and form van der Waals contacts the In fact, CENPB-(540-599) monomer was in solution by the gel filtration the form of the CENP-B dimerization domain in the hydrophobic which to about of the monomer are to the In the crystal the N-terminal of the CENPB-(540-599) dimer were on opposite sides of the dimer. These N-terminal positions of the CENP-B-(540-599) dimer may be suitable for capturing two distant CENP-B boxes with its N-terminal DNA-binding as in the model in the CENP-B box sequence in every other α-satellite repeat CENP-B may a pair of centromeric that contain and a centromere-specific histone H3 CENP-A, two CENP-B boxes by the CENP-B dimer N. Kamiya and S. for data at

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