The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex
2006; Elsevier BV; Volume: 281; Issue: 27 Linguagem: Inglês
10.1074/jbc.m513667200
ISSN1083-351X
AutoresJames R. Thompson, Zachary C. Ryan, Jeffrey L. Salisbury, Rajiv Kumar,
Tópico(s)Enzyme Structure and Function
ResumoHuman centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered α-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an α-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin. Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered α-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an α-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin. Human centrin-2 (HsCen-2) 2The abbreviations used are: HsCen-2, human centrin-2; HsXPC, human xeroderma pigmentosum complementation group C protein; XPC, xeroderma pigmentosum complementation group C protein; SeMet, selenomethionine. is a Ca2+-binding protein of the calmodulin-parvalbumin EF-hand superfamily (2Kawasaki H. Nakayama S. Kretsinger R.H. Biometals. 1998; 11: 277-295Crossref PubMed Scopus (302) Google Scholar, 3Nakayama S. Kretsinger R.H. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 473-507Crossref PubMed Scopus (182) Google Scholar). HsCen-2 and two other human centrins (4Gavet O. Alvarez C. Gaspar P. Bornens M. Mol. Biol. Cell. 2003; 14: 1818-1834Crossref PubMed Scopus (60) Google Scholar) are best known for functions outside the nucleus. Centrins have essential roles in the duplication and segregation of microtubule organizing centers (5Middendorp S. Kuntziger T. Abraham Y. Holmes S. Bordes N. Paintrand M. Paoletti A. Bornens M. J. Cell Biol. 2000; 148: 405-416Crossref PubMed Scopus (101) Google Scholar, 6Salisbury J.L. Suino K.M. Busby R. Springett M. Curr. Biol. 2002; 12: 1287-1292Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Much research has focused on these functions, because abnormal centrosome duplication may lead to chromosomal instability and then cancer, an idea supported by discovery of supernumerary abnormal centrosomes in different human tumor cells (7Lingle W.L. Barrett S.L. Negron V.C. D'Assoro A.B. Boeneman K. Liu W. Whitehead C.M. Reynolds C. Salisbury J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1978-1983Crossref PubMed Scopus (478) Google Scholar, 8Lingle W.L. Lutz W.H. Ingle J.N. Maihle N.J. Salisbury J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2950-2955Crossref PubMed Scopus (435) Google Scholar, 9Lingle W.L. Salisbury J.L. Am. J. Pathol. 1999; 155: 1941-1951Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 10Lingle W.L. Salisbury J.L. Curr. Top. Dev. Biol. 2000; 49: 313-329Crossref PubMed Google Scholar). In addition to its role in centrosome function, HsCen-2 is found as a stabilizing component of xeroderma pigmentosum complement protein C (XPC) and HRad23B complexes (11Popescu A. Miron S. Blouquit Y. Duchambon P. Christova P. Craescu C.T. J. Biol. Chem. 2003; 278: 40252-40261Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Araki M. Masutani C. Takemura M. Uchida A. Sugasawa K. Kondoh J. Ohkuma Y. Hanaoka F. J. Biol. Chem. 2001; 276: 18665-18672Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The XPC-containing heterotrimer is involved in recognition of DNA lesions and initiation of global genome nucleotide excision repair. Global genome nucleotide excision repair is an important DNA repair pathway for damage caused by UV radiation, carcinogens, and chemotherapeutic agents, and impairment of XPC function is associated with the genetic disorder xeroderma pigmentosum. HsCen-2 appears to promote DNA binding by XPC both in vivo and in vitro and increases the specificity of the heterotrimer for damaged DNA (12Araki M. Masutani C. Takemura M. Uchida A. Sugasawa K. Kondoh J. Ohkuma Y. Hanaoka F. J. Biol. Chem. 2001; 276: 18665-18672Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 13Nishi R. Okuda Y. Watanabe E. Mori T. Iwai S. Masutani C. Sugasawa K. Hanaoka F. Mol. Cell. Biol. 2005; 25: 5664-5674Crossref PubMed Scopus (203) Google Scholar). The mechanism by which HsCen-2 binds to XPC is not understood. Centrins are also involved in cilia function (14Keller L.C. Romijn E.P. Zamora I. Yates J.R. III Marshall W.F. Curr. Biol. 2005; 15: 1090-1098Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Moreover, Ca2+-triggered assembly of HsCen-1 and transducin appears to regulate transducin translocation through the connecting cilium of vertebrate photoreceptors (15Giessl A. Pulvermuller A. Trojan P. Park J.H. Choe H.W. Ernst O.P. Hofmann K.P. Wolfrum U. J. Biol. Chem. 2004; 279: 51472-51481Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 16Wolfrum U. Giessl A. Pulvermuller A. Adv. Exp. Med. Biol. 2002; 514: 155-178Crossref PubMed Scopus (36) Google Scholar, 17Pulvermuller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar, 18Wolfrum U. Salisbury J.L. Exp. Cell Res. 1998; 242: 10-17Crossref PubMed Scopus (60) Google Scholar). Whereas the first centrin, also known as caltractin (Cdc31p), was found in fibers linking the flagellar apparatus to the nuclei of Tetraselmis striata green alga (19Salisbury J.L. Baron A.T. Sanders M.A. J. Cell Biol. 1988; 107: 635-641Crossref PubMed Scopus (191) Google Scholar), homologs have since been identified in many organisms, including fungi, plants, and higher eukaryotes. Functional similarities between centrins from various species seem likely. For example, Ca2+-induced contractions of a fiber formed by caltractin and Sfi1p are thought to play a role in microtubule severing in yeast (20Sanders M.A. Salisbury J.L. J. Cell Biol. 1989; 108: 1751-1760Crossref PubMed Scopus (158) Google Scholar), and a human SfiI homolog binds multiple HsCen-2 molecules (21Salisbury J.L. Curr. Biol. 2004; 14: R27-R29Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 22Kilmartin J.V. J. Cell Biol. 2003; 162: 1211-1221Crossref PubMed Scopus (159) Google Scholar). HsCen-2 shares about 50% amino acid identity with human calmodulin. Like calmodulin, the HsCen-2 primary structure contains four EF-hands, a prototypical helix-loop-helix evolved to bind bivalent metals. Most amino acids unique to HsCen-2 from this comparison are highly conserved among centrins across diverse species. An exception is a stretch of 20-25 amino-terminal residues, which are nonconserved among centrins and which are absent in calmodulin and other Ca2+-binding proteins. The carboxyl-terminal sequence 167KKTSLY172, of HsCen-2 is also missing in HsCen-3 and many other centrins of lower eukaryotes lacking centrioles. The aromatic Tyr172 is not found among calmodulins. Despite high sequence similarity, centrins recognize target proteins distinct from those that partner with calmodulins and other EF-hand family members. Little is known about the structural basis of centrin interactions. Some proteins thought to interact with centrins reveal no clear binding site(s) from amino acid analysis, either by visual inspection or by querying a data base of known sequences and properties of calmodulin targets (23Yap K.L. Kim J. Truong K. Sherman M. Yuan T. Ikura M. J. Struct. Funct. Genomics. 2000; 1: 8-14Crossref PubMed Scopus (468) Google Scholar). Residues 847-863 of the human XPC (HsXPC) sequence shown underlined in Fig. 1A, however, were identified as the HsCen-2 binding site after using the calmodulin target data base to suggest sites (11Popescu A. Miron S. Blouquit Y. Duchambon P. Christova P. Craescu C.T. J. Biol. Chem. 2003; 278: 40252-40261Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). There is no information on the structure of full-length centrin either alone or in association with other proteins. Structural details of the C-terminal domain of Chlamydomonas reinhardtii caltractin and a 19-residue peptide from Kar1p protein, a component of spindle pole bodies required for cell integrity in yeast, have been elucidated using NMR methods (24Hu H. Chazin W.J. J. Mol. Biol. 2003; 330: 473-484Crossref PubMed Scopus (50) Google Scholar). The formation of this complex is dependent on Ca2+ binding (25Hu H. Sheehan J.H. Chazin W.J. J. Biol. Chem. 2004; 279: 50895-50903Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Another solution structure of a HsCen-2 fragment (26Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (67) Google Scholar), including residues Met84-Trp172 shows that a helical portion of the N-terminal domain lies within a hydrophobic binding cavity of the C-terminal domain. A calcium-free solution structure of the HsCen-2 N-terminal domain was just published with a closed conformation (27Yang A. Miron S. Duchambon P. Assairi L. Blouquit Y. Craescu C.T. Biochemistry. 2006; 45: 880-889Crossref PubMed Scopus (62) Google Scholar). The binding affinity of HsCen-2 for the XPC-derived peptide is increased 28-fold with saturating calcium present (11Popescu A. Miron S. Blouquit Y. Duchambon P. Christova P. Craescu C.T. J. Biol. Chem. 2003; 278: 40252-40261Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This region of XPC shown underlined in Fig. 1A binds HsCen-2 in vivo (13Nishi R. Okuda Y. Watanabe E. Mori T. Iwai S. Masutani C. Sugasawa K. Hanaoka F. Mol. Cell. Biol. 2005; 25: 5664-5674Crossref PubMed Scopus (203) Google Scholar). Of note, a XPC mutant that fails to bind HsCen-2 also reduces binding affinity of XPC for DNA and reduces the specificity for repair of DNA damage (13Nishi R. Okuda Y. Watanabe E. Mori T. Iwai S. Masutani C. Sugasawa K. Hanaoka F. Mol. Cell. Biol. 2005; 25: 5664-5674Crossref PubMed Scopus (203) Google Scholar). We performed structural characterization of the HsCen-2 and XPC complex, because knowledge of the interactions between the proteins would enhance our understanding of the mechanisms of global genome nucleotide excision repair and the mechanisms underlying the target specificity of HsCen-2. We describe herein the crystallographic structure of full-length HsCen-2 in complex with a 17-residue synthetic peptide derived from human XPC in the presence of Ca2+. Our structure reveals that 1) only the C-terminal domain binds XPC peptide and calcium atoms, 2) the N-terminal lobe exists in a closed conformation with distortions in the loops of its two EF-hands that probably abolish high affinity Ca2+ binding, 3) two XPC peptides bound to separate HsCen-2 molecules interact to form an α-helical coiled-coil conformation, 4) the protein-peptide interface is best characterized as nonpolar, since there are very few hydrogen bonds and only one weak ionic interaction, and 5) the majority of the interface with peptide can be described by analysis of the molecular surfaces surrounding the positions of just three hydrophobic HsXPC residues. General—Research grade reagents were from Sigma unless otherwise indicated. Protein concentration was determined conventionally by absorbance at 277 nm using an Emm = 1.46 mm-1 cm-1 and after purification by the Bio-Rad (Bradford) method (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Native HsCen-2—HsCen-2 was expressed and purified as described earlier (29Craig T.A. Benson L.M. Venyaminov S.Y. Ryan Z.C. Sperry J. Thompson J.R. Salisbury J.L. Bergen H.R. Gross M.L. Kumar R. J. Am. Soc. Mass Spectrom. 2006; (in press)PubMed Google Scholar). Crystallization trials were conducted using hanging drop vapor diffusion methods with over 3000 crystallization solutions, which were then optimized for the production of large single crystals. Similar conditions were used for the biosynthesis of selenomethionine (SeMet)-substituted HsCen-2 and are reported below. SeMet Human Centrin-2—SeMet-substituted HsCen-2 was synthesized in Escherichia coli BL21(DE3) (Invitrogen) grown in minimal M9 medium inoculated from starter culture at 37 °C as published (30Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1091) Google Scholar). After bacterial cultures attained an A600 of ∼1, methionine biosynthesis was suppressed by the addition of leucine, isoleucine, and valine (50 mg/liter each) and lysine, threonine, and phenylalanine (100 mg/liter each). Selenomethionine was added (25 mg/liter), and protein synthesis was induced 15 min later by the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were grown for 12-16 h at 20 °C, collected by centrifugation, and stored frozen at -80 °C some days before further processing. As described for native HsCen-2 (29Craig T.A. Benson L.M. Venyaminov S.Y. Ryan Z.C. Sperry J. Thompson J.R. Salisbury J.L. Bergen H.R. Gross M.L. Kumar R. J. Am. Soc. Mass Spectrom. 2006; (in press)PubMed Google Scholar), the purification involves protease cleavage of a glutathione S-transferase domain fusion. The protein composition and the incorporation of 10 SeMet residues/molecule were confirmed by SDS-PAGE and native gels and also electrospray mass spectrometry analysis. It is to be noted that expression of the glutathione S-transferase-tagged fusion protein results in the addition of 5 extra amino acids (GPLGS) to the amino terminus of the protein following protease treatment of the fusion protein. Peptide Synthesis—The human xeroderma pigmentosa group C peptide (XPC; NWKLLAKGLLIRERLKR), Asn847-Arg863 (11Popescu A. Miron S. Blouquit Y. Duchambon P. Christova P. Craescu C.T. J. Biol. Chem. 2003; 278: 40252-40261Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and solid phase peptide synthesis in the Mayo Proteomics Research Center Peptide Synthesis Facility. The molecular mass of the XPC fragment was verified by microelectrospray ionization mass spectrometry. Preparation of HsCen-2-XPC Complex—A stock solution of 20 mg/ml SeMet-centrin 2 in 50 mm Tris-HCl, 1 mm dithiothreitol, 1 mm EDTA, pH 7.4, was mixed with enough CaCl2 to allow the binding of 2 molofCa2+/mol of protein. Then XPC peptide was added to a 1.3 molar ratio of peptide to protein. The XPC peptide is not completely soluble; complex formation was allowed to take place overnight, at which point precipitate was visible. The pellet consisted of HsCen-2 and XPC peptide. The concentration of the remaining Ca2+-HsCen-2 and XPC peptide complex in solution varied from 6.8 to 12 mg/ml. Crystallization/X-ray Data Collection—The best diffracting crystals resulted from hanging or sitting drop vapor diffusion experiments at 18 or 22 °C. A 2-μl aliquot of SeMet-protein solution was mixed with an equal volume of reservoir containing 2.5-3.2 m (NH4)2SO4 and 0.1-0.4 m Na2HPO4. Streak seeding was routinely utilized to produce crystals in a reproducible manner with microplates that were allowed to equilibrate 1-2 days beforehand. To transfer crystalline micronuclei, a cat whisker was used for serial dilutions of broken up crystals. A novel method called microseed matrix screening was used later to obtain large crystals (31Ireton G.C. Stoddard B.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 601-605Crossref PubMed Scopus (68) Google Scholar). Superior cryoprotection resulted by placing crystals in silicon oil saturated with the crystallization solution prior to flash cooling in liquid N2. Table 1 summarizes the statistics for crystallographic diffraction data collection and structural refinement. The data presented were collected from a flash-cooled crystal (100 K) at beamline X12-C (National Synchrotron Light Source, Brookhaven National Laboratory).TABLE 1Data collection and refinement statisticsParametersValuesLow remoteSelenium peakSelenium edgeSpace groupC21Unit cella = 59.4 Å, b = 59.3 Å, c = 104.7 Å, β = 94.3°Wavelength (Å)0.885600.979800.97952Resolution (Å)40.0 to 2.3540.0 to 2.3540 to 2.35Unique reflections15,09515,16114,999Redundancy1Shown in parenthesis are statistics within 2.39 – 2.35 Å resolution bin6.6 (4.8)6.5 (4.7)6.6 (4.7)Completeness (%)99.3 (96.3)99.3 (96.2)99.2 (95.9)Rsym2Rsym = Σj|Ij – 〈I 〉|/ΣjIj0.082 (0.560)0.097 (0.590)0.082 (0.565)〈I/σI 〉3Average intensity signal-to-noise ratio18.0 (1.74)14.4 (1.64)18.2 (1.66)Figure of merit0.60No. of reflections used14,318Root mean square bond distance0.024No. of atoms2899Root mean square angle2.139R4R = Σ||Fo| – |Fc||/Σ|Fo| for observed and calculated structure factors0.192Rfree54% of the reflections were randomly set aside for Rfree calculation0.245a Shown in parenthesis are statistics within 2.39 – 2.35 Å resolution binb Rsym = Σj|Ij – 〈I 〉|/ΣjIjc Average intensity signal-to-noise ratiod R = Σ||Fo| – |Fc||/Σ|Fo| for observed and calculated structure factorse 4% of the reflections were randomly set aside for Rfree calculation Open table in a new tab Structure Determination—The structure was solved by multiwave-length anomalous dispersion using selenomethionine-labeled protein. Diffraction data collected at three different wavelengths were processed with HKL2000 and SCALEPACK (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar) (Table 1). 16 of 20 possible selenium atomic positions were identified by SOLVE (33Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar), and RESOLVE (34Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1174-1182Crossref PubMed Scopus (75) Google Scholar) built 58% of the HsCen-2 and 95% of the XPC peptide initial atomic coordinates into the experimental electron density map, using phases from multiple wavelength anomalous dispersion, without any user intervention. Programs REFMAC5 (35Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) and COOT (36Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar) were used for structure refinement and model building. No preexisting atomic coordinates were obligatory for model building. Diffraction data of the low remote wavelength from SeMet-crystals were used during refinement, although native crystals diffracted to 1.3 Å resolution. Native data always contained multiple crystalline lattices, most apparent above 3 Å resolution, despite numerous efforts to overcome the problem. TLS parameters were used to model anisotropic displacements (37Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1654) Google Scholar) after determining TLS regions, using normal mode analysis with ElNémo (38Suhre K. Sanejouand Y.H. Nucleic Acids Res. 2004; 32: W610-W614Crossref PubMed Scopus (582) Google Scholar) on the asymmetric unit contents. The stereochemistry and the agreement between model and x-ray data were verified by CNS (39Brunger 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 (16979) Google Scholar) simulated annealing omit maps, PROCHECK (40Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), and SFCHECK (41Vaguine A.A. Richelle J. Wodak S.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 191-205Crossref PubMed Scopus (861) Google Scholar). Structural Description—We determined the crystallographic structure (Table 1) of Ca2+-bound HsCen-2 in complex with the centrin-2 binding peptide derived from human xeroderma pigmentosum group C protein (HsXPC; 847NWKLLAKGLLIRERLKR863) and calcium. The model is refined to 2.35 Å resolution, with R/Rfree values of 0.192 and 0.245, against diffraction data of the low remote wavelength from SeMet-crystals. Fig. 1B shows the rainbow-colored backbone trace of molecule A of HsCen-2 with its HsXPC peptide and two Ca2+ atoms (green spheres) bound to the C-terminal domain. The overall dumbbell-like fold consists of two domains, N-terminal (blue) and C-terminal (red), that do not interact. There is a linker region, shown here as an ordered helix. No interactions are observed between the N-terminal domain of HsCen-2 and the XPC peptide; only residues from the C-terminal domain are involved in XPC binding. The complex contains two Ca2+ atoms bound to EF-hand sites III and IV of the C-terminal domain of HsCen-2. Two complexes are situated within the asymmetric unit as highlighted in Fig. 1C, and no 2-fold noncrystallographic symmetry is found relating them. Atomic coordinates exist for residues 23-172 for the subunit designated as molecule A, whereas molecule B includes residues 25-172. Atomic coordinates are found for all residues of both XPC peptides and numbered with regard to human XPC protein (Asn847-Arg863). Only the C-terminal two XPC residues for the peptide bound to molecule A and the last four for that bound to molecule B are supported by any broken electron density. The entire HsXPC peptide is inα-helical conformation. The helical dipole is orientated in the same manner as observed in the binding of the Kar1p peptide to the yeast centrin, caltractin (24Hu H. Chazin W.J. J. Mol. Biol. 2003; 330: 473-484Crossref PubMed Scopus (50) Google Scholar). Both independent complexes of HsCen-2, XPC peptide, and calcium are very similar structurally, as shown by superposition in Fig. 1D. The root mean square deviation is 0.39 Å for 144 Cα atoms. However, there are minor differences found at the two peptide binding sites, which appear due to intermolecular interactions between both HsXPC peptides. HsXPC Peptides Form Helical Coiled-coil—There are no direct interactions between the C-terminal domains for the two HsCen-2 subunits in Fig. 1C. Rather the crystal forms in part from hydrophobic interactions between the two XPC peptides. The most significant XPC residues involved in the interaction, based on buried surface area, are Leu850, Ile857, and Leu861. The interface also requires a small side chain like the glycine found at position 854 for complete interdigitation of these hydrophobic side chains. Burial of aliphatic carbons from Lys853, Arg858, and Lys862 play a smaller role. The overall conformation of the XPC peptides taken together is that of an α-helical coiled-coil with a parallel orientation of both helical dipoles. HsCen-2 Interactions with XPC—The buried surfaces at the interface of HsCen-2 and XPC peptide are extensively hydrophobic, and the packing provides for substantial van der Waals interactions. Calculations using the Lee and Richards algorithm (42Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5360) Google Scholar) reveal roughly 732 Å2 of the solvent-accessible surface area of HsCen-2 is buried at the interface with XPC peptide; for the peptide, the value is 1020 Å2. The molecular surface of HsCen-2 at this interface is a highly curved 23 × 22-Å area formed by 30% polar atoms and 70% nonpolar atoms. The complementary surface of the XPC peptide measures 21 × 15 Å, created by 24% polar and 76% nonpolar atoms. Hydrogen bonds play only a small role between 8 and 10 amino acids. The most surprising result was the lack of electrostatic interactions given the basic nature of the HsXPC peptide. Only one ionic interaction, a weak salt bridge, was identified between Arg858 and HsCen-2 Glu132. No bridging solvent molecules mediate peptide binding. Once more, nonpolar interactions predominate. Shown in Fig. 2A is an α-helix portrayal of the XPC peptide backbone, labeled by amino acid. The most significant interfacial peptide residues, in order of their nonpolar occluded surface, are Trp848, Lys849 molecule A, Leu851, Leu855, Leu856, and Lys849 molecule B. The high amino acid conservation of these specific XPC residues points to their importance in formation of the binding site. Fig. 2B shows a frequency histogram of a multiple sequence alignment that encompasses the XPC HsCen-2 binding site, an alignment that includes homologous sequences to human XPC from 38 species (the amino acid alignment is provided as supplemental data). Trp848 is completely conserved. The leucines at 851, 855, and 856 are highly conserved; only hydrophobic residues are found at positions 851 and 855. Lys849 is also conserved but can be substituted by the similar arginine or small hydrophobic amino acids. Given our finding that these residues are of prime significance in binding HsCen-2, interactions between proteins homologous to XPC and centrin 2 seem likely throughout eukaryotic phyla. We also determined the degree to which nonpolar atoms of HsCen-2 amino acids were occluded by the XPC peptide. The results identify in rank order Leu133, Leu112, Met145, Phe113, Met166, Leu126, Ala109, Glu105, and Val129. These nine nonpolar residues cover nearly every residue of the XPC peptide. The arrows in Fig. 2A point to specific HsCen-2 amino acids interacting with each residue of both XPC-derived peptides in the asymmetric unit. To be identified, amino acids must have substantial buried nonpolar surface within 4 Å of an XPC residue. Only minor differences are found among the interactions in both HsCen-2 binding cavities within the asymmetric unit. Several nonpolar interactions are of seemingly great consequence. 1) Both Met145 and Phe113 form the greater part of a deep pocket shown in Fig. 2C into which the invariant Trp848 of HsXPC is inserted. The Trp848 side chain also forms a weak hydrogen bond to the Met145 main chain. 2) Leu851 of HsXPC fits a shallow pocket formed largely by Leu133 and again Phe113.3) Leu855 of HsXPC associates most with Leu133, Leu112, and Phe113. 4) The packing of the aliphatic side chain of Lys849 of the peptide with Met166 buries substantial nonpolar surface. Closed Conformation for N-terminal Domain—The interhelical angles between α-helices within EF-hand protein structures have been used to classify and measure conformational state. The angular values for HsCen-2 indicate the N-terminal domain without calcium bound resides in a "closed" conformation (Table 2), although differences are found compared with those reported for a solution structure of the HsCen-2 N-terminal domain alone (27Yang A. Miron S. Duchambon P. Assairi L. Blouquit Y. Craescu C.T. Biochemistry. 2006; 45: 880-889Crossref PubMed Scopus (62) Google Scholar). Values for the C-terminal domain unsurprisingly indicate an open state. Data from equivalent computations on other homologous proteins are maintained on an internet Web site called the CaBP Data Library (available on the World Wide Web at structbio.vanderbilt.edu/cabp_database/).TABLE 2Interhelical angles within HsCen-2 Adding/removing 1-2 residues from the helical ends had little effect on these results computed by the Interhlx program. Sign convention is as described (1Drohat A.C. Amburgey J.C. Abildgaard F. Starich M.R. Baldisseri D. Weber D.J. Biochemistry. 1996; 35: 11577-11588Crossref PubMed Scopus (121) Google Scholar).α I ∠α IIα I ∠α IIIα I ∠α IVα II ∠α IIIα II ∠α IVα III ∠α IVN-terminal138–87127116–52143C-terminal93–139124126–4596 Open table in a new tab Fig. 3 depicts an overlay of backbone traces from the N-terminal HsCen-2 domain (blue) on that of the C-terminal domain (red) in a cross-eye stereoimage. The α-helices are numbered I-IV to correspond with past literature on homologous EF-hand folds and with the interhelical angle data in Table 2. The bound peptide (green) and both Ca2+ atoms are presented as well. Looking at the blue ribbon, it is obvious that no helical peptide could bind within the N-terminal domain within its existing structure. Fig. 3 also allows comparison of the more distorted loops of EF-hand sites I and II in the N-terminal and without calcium present to sites III and IV. Site I differs from the more canonical HsCen-2 site IV (or EF-hand I in calmodulin) mainly by the replacement of an aspartate important for Ca2+ binding by Thr45. An analogous comparison suggests two substitutions reduce Ca2+ affinity at site II, Glu79 rat
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