Xeroderma Pigmentosum Group C Protein Possesses a High Affinity Binding Site to Human Centrin 2 and Calmodulin
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m302546200
ISSN1083-351X
AutoresAurel Popescu, Simona Miron, Y. Blouquit, Patricia Duchambon, P. Christova, Constantin T. Craescu,
Tópico(s)Mitochondrial Function and Pathology
ResumoHuman centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca2+-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca2+ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway. Analysis of the XPC sequence (940 residues), using a calmodulin target recognition software, enabled us to predict two putative binding sites. The binding properties of the two corresponding peptides were investigated by isothermal titration calorimetry. Only one of the peptides (P1-XPC) interacts strongly (K a = 2.2 × 108m-1, stoichiometry 1:1) with HsCen2 in a Ca2+-dependent manner. This peptide also binds, with a similar affinity (K a = 1.1 × 108m-1) to a C-terminal construct of HsCen2, indicating that the interaction with the integral protein is mainly the result of the contribution of the C-terminal half. The second peptide (P2-XPC) failed to show any detectable binding either to HsCen2 or to its C-terminal lobe. The two peptides interact with different affinities and mechanisms with calmodulin. Circular dichroism and nuclear magnetic resonance were used to structurally characterize the complex formed by the C-terminal domain of HsCen2 with P1-XPC. Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca2+-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca2+ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway. Analysis of the XPC sequence (940 residues), using a calmodulin target recognition software, enabled us to predict two putative binding sites. The binding properties of the two corresponding peptides were investigated by isothermal titration calorimetry. Only one of the peptides (P1-XPC) interacts strongly (K a = 2.2 × 108m-1, stoichiometry 1:1) with HsCen2 in a Ca2+-dependent manner. This peptide also binds, with a similar affinity (K a = 1.1 × 108m-1) to a C-terminal construct of HsCen2, indicating that the interaction with the integral protein is mainly the result of the contribution of the C-terminal half. The second peptide (P2-XPC) failed to show any detectable binding either to HsCen2 or to its C-terminal lobe. The two peptides interact with different affinities and mechanisms with calmodulin. Circular dichroism and nuclear magnetic resonance were used to structurally characterize the complex formed by the C-terminal domain of HsCen2 with P1-XPC. Centrin (also called caltractin) is a Ca2+-binding protein highly conserved in diverse evolutionary lineages, including algal, higher plant, invertebrate, and mammalian cells (1Schiebel E. Bornens M. Trends Cell Biol. 1995; 5: 197-201Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 2Salisbury J.L. Curr. Opin. Cell Biol. 1995; 7: 39-45Crossref PubMed Scopus (321) Google Scholar). It is an acidic protein of 19.5 kDa belonging to the highly conserved EF-hand calmodulin (CaM) 1The abbreviations used are: CaM, calmodulin; CD, circular dichroism; HsCen2, human centrin 2; SC-HsCen2, a short C-terminal fragment of human centrin2 (Thr94-Tyr172); ITC, isothermal titration calorimetry; NER, nuclear excision repair; P1-XPC, Asn847-Arg863 fragment of xeroderma pigmentosum protein; XPC, xeroderma pigmentosum complementing group C protein; P2-XPC, Pro703-Ala720 fragment of XPC; SPB, spindle pole body; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HSQC, heteronuclear single-quantum correlation. superfamily. Comparative sequence analysis suggests that centrins consist of two structural domains, each containing two putative Ca2+-binding EF-hand motifs. In humans, three centrin isoforms (HsCen1 to HsCen3) have been identified so far (3Errabolu R. Sanders M.A. Salisbury J.L. J. Cell Sci. 1994; 107: 9-16Crossref PubMed Google Scholar, 4Lee V.D. Huang B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11039-11043Crossref PubMed Scopus (118) Google Scholar, 5Middendorp S. Paoletti A. Schiebel E. Bornens M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9141-9146Crossref PubMed Scopus (127) Google Scholar) with variable sequences and different tissue and cell distributions. HsCen1 and HsCen2 are highly similar to each other (sequence identity 84%) and to the algae centrin (68 and 71%, respectively), whereas HsCen3, discovered lately (5Middendorp S. Paoletti A. Schiebel E. Bornens M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9141-9146Crossref PubMed Scopus (127) Google Scholar), has a more distant sequence; it shows only 54% identity with both HsCen1 and HsCen2, and is slightly closer to Cdc31, the centrin equivalent in yeast Saccharomyces cerevisiae (59% sequence identity). A large part of the sequence diversity among the centrins occurs within the first 20 residues of the N-terminal domain, that have no counterpart in the "standard" Ca2+-binding protein, CaM. The centrins are usually found in association with the microtubule organizing centers (centrosomes in animal cells, and spindle pole bodies in yeast) that are cytoplasmic organelles encountered in almost all eukaryotic cells, with an important role in microtubule structural and temporal organization (1Schiebel E. Bornens M. Trends Cell Biol. 1995; 5: 197-201Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 2Salisbury J.L. Curr. Opin. Cell Biol. 1995; 7: 39-45Crossref PubMed Scopus (321) Google Scholar). HsCen2 is ubiquitously expressed but was first discovered in the distal lumen of centrioles, where its presence is required for normal centriole duplication during the cell cycle (6Salisbury J.L. Suino K.M. Busby R. Springett M. Curr. Biol. 2002; 12: 1287-1292Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). A large fraction of the cellular centrin is not permanently associated with the centrosome (7Paoletti A. Moudjou M. Paintrand M. Salisbury J.L. Bornens M. J. Cell Sci. 1996; 109: 3089-3102Crossref PubMed Google Scholar), but fractionates with the cytoplasm and nuclei in human cells. The precise function of these pools is not well understood and constitutes a subject of intense investigation. For instance, the presence of HsCen2 in the nuclear fractions is thought to play a role in coordinating the nuclear and cytoplasm events during the division cycles. Recently, studies conducted in the Hanaoka group (8Araki 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 (287) Google Scholar) shed a new light on the possible role of nuclear HsCen2 fraction in the nucleotide excision repair (NER) process. Nuclear excision repair is a major pathway for recognition and removal of bulky DNA lesions such as the UV photoproducts, or carcinogen adducts. Its dysfunction produces severe disorders in humans such as xeroderma pigmentosum, a hereditary disease characterized by a high photosensitivity and a large incidence of sunlight-induced cancer. One of the molecular component involved in several xeroderma pigmentosum forms is the XPC complex, a heterodimer composed of the XPC gene product (XPC) and HR23B, the human homologue of yeast Rad23 B. XPC complex plays a key role in the initial phase of NER and is involved in the recognition of the DNA damage. NER is of great importance for the maintenance of the genomic integrity, but the molecular mechanism of the NER pathway involving damage recognition, excision, gap-filling, and ligation steps has not been elucidated (9Benhamou S. Sarasin A. Mutat. Res. 2000; 462: 149-158Crossref PubMed Scopus (114) Google Scholar). According to the recent work of Araki et al. (8Araki 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 (287) Google Scholar), the XPC complex may contain an additional component, HsCen2, which, together with HR23B, stabilizes in a cooperative manner XPC, and thus stimulates the NER activity in vitro. This observation opens a new field of investigation of the centrin cellular functions, with the possibility that centrin may fill the gap between the DNA nuclear repair process and the functions of the mitotic spindle apparatus. Recent biochemical and biophysical studies performed in our and other laboratories have provided a wealth of physicochemical data on the structure and Ca2+ binding properties of centrins of various origins. From this, in agreement with the sequence alignment analysis, it appears that the integral centrins are composed of two independent domains, each containing two putative EF-hand motifs (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar, 11Veeraraghavan S. Fagan P.A. Hu H. Lee V. Harper J.F. Bessie H. Chazin W.J. J. Biol. Chem. 2002; 277: 28564-28571Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In contrast to the Chlamydomonas centrin (11Veeraraghavan S. Fagan P.A. Hu H. Lee V. Harper J.F. Bessie H. Chazin W.J. J. Biol. Chem. 2002; 277: 28564-28571Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), which is able to bind four Ca2+ ions with significant affinity, HsCen2 exhibits one strong and one weak binding site, localized in the C-terminal domain (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar, 12Durussel I. Blouquit Y. Middendorp S. Craescu C.T. Cox J.A. FEBS Lett. 2000; 472: 208-212Crossref PubMed Scopus (75) Google Scholar). Structural NMR studies provided evidence that the C-terminal domain of HsCen2 is conformationally sensitive to Ca2+ binding, and folds into an open conformation with a large exposed hydrophobic surface (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar). The capacity of both proteins to bind amphiphilic peptides, primarily mediated by the C-terminal domain (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar, 11Veeraraghavan S. Fagan P.A. Hu H. Lee V. Harper J.F. Bessie H. Chazin W.J. J. Biol. Chem. 2002; 277: 28564-28571Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), strongly supports the hypothesis of a Ca2+-dependent regulatory role of centrins. The next step in this direction is the search for specific molecular targets and exploration of conformational, energetic, and functional aspects of the corresponding intermolecular interactions. In this work, we focused on a sequence-based identification of the XPC binding site to HsCen2 and on the thermodynamic and structural characterization of the intermolecular interactions. ITC experiments showed that one of the two predicted binding peptides derived from XPC binds with high affinity to HsCen2, the isolated HsCen2 C-terminal domain, and CaM. In all cases, the peptide binding is Ca2+-dependent, but stoichiometry and molecular mechanism seem to be different for centrin and CaM. CD and preliminary NMR experiments enabled us to characterize some structural aspects of the complex between one XPC peptide and the C-terminal domain of HsCen2. Protein Expression and Purification—Recombinant proteins HsCen2 and SC-HsCen2 (Thr94-Tyr172) were overexpressed in Escherichia coli and purified as described previously (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar, 12Durussel I. Blouquit Y. Middendorp S. Craescu C.T. Cox J.A. FEBS Lett. 2000; 472: 208-212Crossref PubMed Scopus (75) Google Scholar). For 15N-labeled samples, we used a culture medium (M9) containing 15NH4Cl (1.5 g/liter) as the sole source of nitrogen, and the induction step with isopropyl-1-thio-β-d-galactopyranoside (0.1 mm) was prolonged to 18 h. Target Peptides—Two peptides from the human XPC protein, encompassing the sequences Asn847-Arg863 (P1-XPC) and Pro703-Ala720 (P2-XPC) were purchased from Biofidal (Vaulx en Velin, France). Purity was greater than 95%, as assessed by high pressure liquid chromatography analysis. CD Spectroscopy—CD experiments were performed on a Jasco 715 CD spectrometer equipped with a Peltier temperature control unit. Far-UV spectra were recorded between 195 and 250 nm at 20 °C using 1-mm quartz cells. Spectra were collected as an average of four scans, with a scan speed of 20 nm/min and a response time of 1 s. Samples (30 μm) were dissolved in Tris-HCl buffer (10 mm) containing 100 mm NaCl and 2 mm CaCl2. Temperature denaturation curves were obtained between 20 and 95 °C, with a temperature increasing rate of 1 °C/min. Isothermal Titration Calorimetry—Thermodynamic parameters of molecular interactions between human centrin or calmodulin and the target peptides at 30 °C were investigated by ITC using a MicroCal MCS instrument (MicroCal Inc., Northampton, MA). The proteins and peptides were equilibrated in the same buffer containing 20 mm Tris (or BisTris), pH 6.5, 100 mm NaCl, and Ca2+ (2 mm) or EDTA (5 mm). In a standard experiment, the protein (7-20 μm) in the 1.337-ml calorimeter cell was titrated by the peptide (generally 10 times more concentrated) by ∼30 successive automatic injections of 7-10 μl each. The first injection of 2-3 μl was ignored in the final data analysis. Integration of the peaks corresponding to each injection and correction for the base line were done using Origin-based software provided by the manufacturer. Fitting of the data to various interaction models results in the stoichiometry (n), equilibrium binding constant (K a), and enthalpy of complex formation (ΔH). The reported thermodynamic parameters represent an average of at least two experiments. Usually, control experiments, consisting of injecting peptide solutions into the buffer, were performed to evaluate the heat of dilution. NMR Spectroscopy—NMR samples (0.7-1.2 mm) were obtained by dissolving the lyophilized protein in deuterated Tris-HCl buffer (20 mm, pH 6.5) containing 100 mm NaCl and 5 mm CaCl2. NMR spectra were recorded on a Varian Unity 500 NMR spectrometer equipped with a triple resonance probe and a Z-field gradient, at 308 K. Homonuclear and heteronuclear [15N-1H]HSQC two-dimensional spectra in 1H2O were performed using standard pulse sequences (13Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York1986Crossref Google Scholar, 14Cavanagh J. Fairbrother W.J. Palmer III, A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego1996Google Scholar). Spectra analysis was carried out using Felix software (Accelrys, San Diego, CA). Prediction of the Centrin Binding Site of XPC Protein—The available structural and functional data consistently suggest that HsCen2 is a sensor protein, capable to translate a Ca2+ cellular signal into an activation/inhibition of a target molecule (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar). CaM, the prototype of the Ca2+ sensor proteins, has a large and diverse number of known target proteins, analysis of which revealed some molecular characteristics of the CaM-binding sites, including α-helix propensity and the basic amphiphilic character (15O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (738) Google Scholar, 16Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (754) Google Scholar, 17Yap K.L. Kim J. Truong K. Sherman M. Youan T. Ikura M. J. Struct. Funct. Genomics. 2001; 1: 8-14Crossref Scopus (476) Google Scholar). Recently, a web-based data base containing the known CaM target sequences and their properties, as well as sequence analysis tools permitting to predict putative CaM binding sites within a given protein sequence, has been developed (17Yap K.L. Kim J. Truong K. Sherman M. Youan T. Ikura M. J. Struct. Funct. Genomics. 2001; 1: 8-14Crossref Scopus (476) Google Scholar). Assuming that CaM and HsCen2 binding sites may share common molecular features, we proceeded to an analysis of the XPC sequence using these tools. Sequence comparison failed to find any region in the XPC protein sharing significant sequence homology with known CaM binding sites but did predict two sequences with a good probability as putative CaM binding sites. The two peptides (P1-XPC, Asn847-Arg863; and P2-XPC, Pro703-Ala720), situated in the last quarter of the XPC sequence (Fig. 1A), were associated with the moderate to excellent score of probability (9 and 6 for P1-XPC and P2-XPC, respectively). Sequence comparison with classical CaM binding motifs suggests that the peptides belong to the 1-14 class, including the recognition sites of myosin light chain kinase, CaM kinase IV, calcineurin A, and human death-associated kinase I. P1-XPC is predicted to be the best binding sequence with a pattern of bulky hydrophobic side chains at positions 1, 5, 8, and 14 and a good propensity to form an amphiphilic α-helix (Fig. 1A). It is worth noting that P1-XPC together with Cdc31 binding peptide Kar1 include negatively charged side chains, whereas the CaM-binding peptides are generally positively charged (16Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (754) Google Scholar). Binding to HsCen2 and Its C-terminal Domain—Binding of the P1-XPC and P2-XPC peptides was studied using the ITC method with the protein solution in the cell and the peptides in the syringe. As illustrated in the thermograms of Fig. 2, successive injections of the peptides are accompanied by exothermic heat pulses for which the integral decreases to a stable base line. In the presence of saturating Ca2+ concentrations, the data analysis indicates that the integral protein HsCen2 binds the P1-XPC peptide with a stoichiometry of 1:1 and a high affinity K a = 2.2 (±0.4) × 108m-1 (K d = 4.5 nm). The large reaction enthalpy of approximately -27 kcal/mol largely overcomes the entropic component to the free energy of interaction (Table I). The short C-terminal domain SC-HsCen2 binds the peptide with a similar stoichiometry, affinity, and binding enthalpy, strongly suggesting that it represents the major interacting domain. Indeed, the free energy of P1-XPC binding to SC-HsCen2 represents 97% of the binding energy of the intact protein. In the absence of Ca2+, both the integral protein and the SC-HsCen2 domain are still able to bind exothermally the peptide, but with a considerably decreased affinity (28 and 17 times, respectively) (Fig. 2 and Table I).Table IThermodynamic parameters of the peptide binding to CaM, HsCen2, and its C-terminal constructProteinLigandCa2+K a (±error)ΔGΔH (±error)TΔS10 8 m −1kcal/molkcal/molkcal/molHsCen2P1-XPC+2.2 (0.4)−11.6−27.2 (0.2)−15.6HsCen2P1-XPC−0.08 (0.01)−9.6−35.8 (0.6)−26.2HsCen2P2-XPC±NBaNB, no binding observed in the present conditions.---HsCen2Melittin+0.16 (0.02)−10.0−8.2 (0.01)+1.2SC-HsCen2P1-XPC+1.2 (0.5)−11.2−29.1 (0.1)−17.9SC-HsCen2P1-XPC−0.07 (0.01)−9.5−31.5 (0.1)−22.0SC-HsCen2P2-XPC±NBaNB, no binding observed in the present conditions.---CaMP1-XPC+∼1+0.0059 (0.0007)−8.0−8.32 (0.03)−0.32CaMP1-XPC−0.007 (0.001)−8.0−6.4 (0.2)+1.6CaMP2-XPC+0.19 (0.02)−10.1−10.6 (0.1)−0.5CaMP2-XPC−NBaNB, no binding observed in the present conditions.---a NB, no binding observed in the present conditions. Open table in a new tab In the case of P2-XPC, the heat rate is very small and remains roughly constant during the titration (Fig. 3), failing to show the typical transition phase observed for interacting systems. This observation indicates the absence of binding, or a very weak binding (K a < 103m-1), that cannot be detected in the present conditions. Therefore, in agreement with the knowledge-based prediction, HsCen2/Ca2+, mainly through its C-terminal domain, exhibits a strong binding to one of the two putative CaM binding sites situated in the C-terminal part of the XPC protein. Analysis of the free energy components (Table I) shows that the decrease in binding energy in the absence of Ca2+ is the result of an increase of the unfavorable entropic contribution, only partially compensated by the increase in enthalpy. This trend is characteristic both for the intact protein and the short C-terminal construct, and it may be related to the fact that, in the absence of Ca2+, the C-terminal domain represents an ensemble of highly disordered conformations (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar), stabilization of which upon complex formation gives a negative entropic contribution. Measurement of the temperature dependence of the binding enthalpy allowed us to evaluate the heat capacity changes (ΔC p) caused by protein/peptide interactions. For the complexes of P1-XPC with HsCen2 and SC-HsCen2, the negative binding enthalpy increases significantly when the temperature varies between 20 and 34 °C. The heat capacity, calculated as the correlation coefficient between ΔH and temperature, has large negative values (-995 ± 78 and -366 ± 49 cal/K mol for HsCen2 and SC-HsCen2, respectively) that correspond to the upper range of values observed for CaM/target peptides (18Wintrode P.L. Privalov P.L. J. Mol. Biol. 1997; 266: 1050-1062Crossref PubMed Scopus (84) Google Scholar, 19Brokx R.D. Lopez M.M. Vogel H.J. Makhatadze G.I. J. Biol. Chem. 2001; 276: 14083-14091Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). It was noted (19Brokx R.D. Lopez M.M. Vogel H.J. Makhatadze G.I. J. Biol. Chem. 2001; 276: 14083-14091Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) that, among the CaM binding peptides, the heat capacity changes were more negative for those exhibiting a primary interaction with the C-terminal protein domain and having a bulk aromatic side chain in the peptide N-side, as is the case for P1-XPC. Empirical observations in a number of cases pointed to the existence of a linear correlation between the interaction-induced heat capacity changes and the accompanying decrease in the water-accessible surface area (20Baker B.M. Murphy K.P. J. Mol. Biol. 1997; 268: 557-569Crossref PubMed Scopus (144) Google Scholar, 21Luque I. Todd M.J. Gomez J. Semo N. Ernesto F. Biochemistry. 1998; 37: 5791-5797Crossref PubMed Scopus (79) Google Scholar). The corresponding dehydration results in a positive entropic contribution, essentially caused by the released water molecules. Therefore, the negative heat capacity changes observed in this work should be associated with a major contribution of aliphatic and aromatic side chains to the total decrease of the accessible surface area upon complex formation (22Makhatadze G.I. Privalov P.L. Adv. Protein Chem. 1995; 47: 307-425Crossref PubMed Scopus (1012) Google Scholar). This is in agreement with the large hydrophobic surface exposed by the C-terminal domain of HsCen2 in the presence of Ca2+ ions (10Matei E. Miron S. Blouquit Y. Duchambon P. Durussel I. Cox J.A. Craescu C.T. Biochemistry. 2003; 42: 1439-1450Crossref PubMed Scopus (68) Google Scholar) and is consistent with a largely apolar protein/peptide interface. An additional factor that may account for the large negative values of the heat capacity changes observed upon P1-XPC complex formation is the significant conformational changes of both partners, as was suggested for many protein/DNA interactions (23Spolar R.S. Record Jr., M.T. Science. 1994; 263: 777-784Crossref PubMed Scopus (1379) Google Scholar). We also performed titration calorimetric experiments to measure the interaction between HsCen2 and melittin, a natural peptide extracted from the bee venom often used as a CaM-binding peptide. In the presence of Ca2+, the binding is weaker than for P1-XPC (K a = 1.6 × 107m-1) but is still driven by a negative enthalpy change (ΔH = -8 kcal/mol, at 30 °C). Binding to Calmodulin—The two XPC peptides used in the present work have been selected based on the CaM target consensus sequence, and the hypothesis that proteins from the EF-hand superfamily may share similar targets. On the other hand, CaM and centrins could be observed in the same cellular regions (24Li C.-J. Heim R. Lu P. Pu Y. Tsien R.Y. Chang D.C. J. Cell Sci. 1999; 112: 1567-1577Crossref PubMed Google Scholar, 25Moser M.J. Flory M.R. Davis T.N. J. Cell Sci. 1997; 110: 1805-1812Crossref PubMed Google Scholar, 26Moisoi N. Erent M. Whyte S. Martin S. Bayley P.M. J. Cell Sci. 2002; 115: 2367-2379PubMed Google Scholar), where proteins possessing CaM-binding motifs (27Flory M.R. Moser M.J. Monnat Jr., R.J. Davis T.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5919-5923Crossref PubMed Scopus (78) Google Scholar, 28Matsumoto Y. Maller J.L. Science. 2002; 295: 499-502Crossref PubMed Scopus (91) Google Scholar) were identified. This raised the question of the possible promiscuity in recognition and binding of the molecular targets by the two EF-hand proteins, and motivated us to investigate the binding of P1-XPC and P2-XPC to CaM in the same physicochemical conditions. In the presence of Ca2+, the thermogram corresponding to the titration of CaM by P1-XPC shows a complex pattern, which could not be fitted using a single-site model (Fig. 4), suggesting that CaM exhibits two binding sites with distinct affinities. When the peptide to protein concentration ratio (R) is changed from 5.4 to 13.5, the thermogram becomes progressively dominated by the low affinity binding contribution, and the corresponding binding isotherm can be fitted confidently to a single-site model (Fig. 4). This allows us to obtain an estimation of the thermodynamic parameters characterizing the weak binding site: K a = 5.9 × 105m-1 and ΔH = -8.3 kcal/mol. Although the thermograms for lower peptide to protein ratios could not be modeled confidently, we assume that the affinity of the strong binding site is of the order of 108m-1. P1-XPC binding to CaM is also Ca2+-sensitive, because in the absence of the divalent cations, a single low affinity binding site (K a = 7 × 105m-1) was observed (Fig. 4). In contrast to HsCen2, the P2-XPC peptide also shows a significant binding to CaM. The binding isotherm could be fitted to a single-binding site model (Fig. 3), with a significant affinity (K a = 1.9 × 107m-1) largely accounted for by the enthalpy contribution (ΔH = -10.6 kcal/mol). In the absence of Ca2+, the binding of P2-XPC is undetectable in the present experimental conditions. Conformational Changes and Structural Stability Induced by the Complex Formation—CD and NMR spectroscopy were used as primary investigation tools to characterize the conformational properties of the HsCen/P1-XPC interaction. Fig. 5 illustrates the far-UV CD experiments on the complex formation of HsCen2 and its C-terminal domain with the P1-XPC in the presence of Ca2+. The peptide alone exhibits a CD spectrum characteristic for a highly disordered structure, as is generally the case for linear polypeptides of this size. In contrast, the Ca2+-bound integral protein and SC-HsCen2 domain exhibit a CD spectrum typical for a well folded protein with a major α-helical content. The spectral characteristics of the helical secondary structure are the negative bands at 222 and 207 nm and the positive band at 195 nm (29Johnson W.C. Proteins Struct. Funct. Genet. 1999; 35: 307-312Crossref PubMed Scopus (633) Google Scholar). Adding of P1-XPC, at a 1:1 molar ratio, to SC-HsCen2/Ca2+ induces a considerable enhancement of the CD signal (by ∼50%) with a rough conservation of the relative intensities of different bands (Fig. 5A). A moderate increase of the negative ellipticity (on the order of 10-15%) could be eventually associated to the conformational rearrangements of the protein, as was suggested in the case of Ca2+ binding to the apo N-terminal domain of troponin C (30Li M.X. Gagné S.M. Tsuda S. Kay C.M. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: 8330-8340Crossref PubMed Scopus (81) Google Scholar, 31Gagné S.M. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (177) Google Scholar). However, structural studies on complexes of EF-hand domains with peptide targets showed that the protein domains undergo only small tertiary changes (32Hoeflich K.P. Ikura M. Cell. 2002; 108: 739-742Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Therefore, the large variation of the negative band intensity at 222 nm, the most characteristic for the α structure, suggests that a fragment of the peptide undergoes a random coil-to-helix structural transition upon binding to the protein, bringing a significant contribution to the α-helix CD band. This conclusion is corroborated by preliminary NMR studies of the SC-HsCen2/P1-XPc complex. In the case of the integral protein (Fig. 5B), addition of the peptide induces a similar (but slightly larger) increase in the spectrum intensity, probably the result of a larger α-helix content in the protein stabilized by the complex formation. Doubling the peptide-to-protein ratio (2:1) is not accompanied by significant CD changes, in agreement w
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