Abstract
We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (μESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. Δ25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary structure.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Baron, A. T.; Salisbury, J. L. Identification and localization of a novel, cytoskeletal, centrosome-associated protein in PtK2 cells. J. Cell. Biol. 1988, 107(6 Pt 2), 2669–2678.
Sanders, M. A.; Salisbury, J. L. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii. J. Cell. Biol. 1989, 108(5), 1751–1760.
Coling, D. E.; Salisbury, J. L. Characterization of the calcium-binding contractile protein centrin from Tetraselmis striata (Pleurastrophyceae). J. Protozool. 1992, 39(3), 385–391.
Salisbury, J. L. Centrin, centrosomes, and mitotic spindle poles. Curr. Opin. Cell Biol. 1995, 7(1), 39–45.
Salisbury, J. L.; Suino, K. M.; Busby, R.; Springett, M. Centrin-2 is required for centriole duplication in mammalian cells. Curr. Biol. 2002, 12(15), 1287–1292.
Salisbury, J. L.; Baron, A.; Surek, B.; Melkonian, M. Striated flagellar roots: Isolation and partial characterization of a calcium-modulated contractile organelle. J. Cell Biol. 1984, 99(3), 962–970.
Huang, B.; Mengersen, A.; Lee, V. D. Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+ binding protein: Homology in its protein sequence with calmodulin and the yeast CDC31 gene product. J. Cell Biol. 1988, 107(1), 133–140.
Gonda, K.; Yoshida, A.; Oami, K.; Takahashi, M. Centrin is essential for the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels. Biochem. Biophys. Res. Commun. 2004, 323(3), 891–897.
Keller, L. C.; Romijn, E. P.; Zamora, I.; Yates, J. R. III; Marshall, W. F. Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes. Curr. Biol. 2005, 15(12), 1090–1098.
Giessl, A.; Pulvermuller, A.; Trojan, P.; Park, J. H.; Choe, H. W.; Ernst, O. P.; Hofmann, K. P.; Wolfrum, U. Differential expression and interaction with the visual G-protein transducin of centrin isoforms in mammalian photoreceptor cells. J. Biol. Chem. 2004, 279(49), 51472–51481.
Wolfrum, U.; Giessl, A.; Pulvermuller, A. Centrins, a novel group of Ca2+ binding proteins in vertebrate photoreceptor cells. Adv. Exp. Med. Biol. 2002, 514, 155–178.
Wolfrum, U.; Salisbury, J. L. Expression of centrin isoforms in the mammalian retina. Exp. Cell Res. 1998, 242(1), 10–17.
Kilmartin, J. V. Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication. J. Cell Biol. 2003, 162(7), 1211–1221.
Salisbury, J. L. Centrosomes: Sfi1p and centrin unravel a structural riddle. Curr. Biol. 2004, 14(1), R27-R29.
Araki, M.; Masutani, C.; Takemura, M.; Uchida, A.; Sugasawa, K.; Kondoh, J.; Ohkuma, Y.; Hanaoka, F. Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair. J. Biol. Chem. 2001, 276(22), 18665–18672.
Popescu, A.; Miron, S.; Blouquit, Y.; Duchambon, P.; Christova, P.; Craescu, C. T. Xeroderma pigmentosum group C protein possesses a high affinity binding site to human centrin 2 and calmodulin. J. Biol. Chem. 2003, 278(41), 40252–40261.
Baron, A. T.; Greenwood, T. M.; Bazinet, C. W.; Salisbury, J. L. Centrin is a component of the pericentriolar lattice. Biol. Cell. 1992, 76(3), 383–388.
Paoletti, A.; Moudjou, M.; Paintrand, M.; Salisbury, J. L.; Bornens, M. Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles. J. Cell Sci. 1996, 109(Pt 13), 3089–3102.
Durussel, I.; Blouquit, Y.; Middendorp, S.; Craescu, C. T.; Cox, J. A. Cation- and peptide-binding properties of human centrin 2. FEBS Lett. 2000, 472(2/3), 208–212.
Matei, E.; Miron, S.; Blouquit, Y.; Duchambon, P.; Durussel, I.; Cox, J. A.; Craescu, C. T. C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain. Biochemistry. 2003, 42(6), 1439–1450.
Tourbez, M.; Firanescu, C.; Yang, A.; Unipan, L.; Duchambon, P.; Blouquit, Y.; Craescu, C. T. Calcium-dependent self-assembly of human centrin 2. J. Biol. Chem. 2004, 279(46), 47672–47680.
Veeraraghavan, S.; Fagan, P. A.; Hu, H.; Lee, V.; Harper, J. F.; Huang, B.; Chazin, W. J. Structural independence of the two EF-hand domains of caltractin. J. Biol. Chem. 2002, 277(32), 28564–28571.
Kumar, R.; Hunziker, W.; Gross, M.; Naylor, S.; Londowski, J. M.; Schaefer, J. The highly efficient production of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) in a bacterial expression system. Arch. Biochem. Biophys. 1994, 308(1), 311–317.
Craig, T. A.; Veenstra, T. D.; Naylor, S.; Tomlinson, A. J.; Johnson, K. L.; Macura, S.; Juranic, N.; Kumar, R. Zinc binding properties of the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor. Biochemistry. 1997, 36(34), 10482–10491.
Veenstra, T. D.; Johnson, K. L.; Tomlinson, A. J.; Naylor, S.; Kumar, R. Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry. Biochemistry. 1997, 36(12), 3535–3542.
Veenstra, T. D.; Tomlinson, A. J.; Benson, L.; Kumar, R.; Naylor, S. Low temperature aqueous electrospray ionization mass spectrometry of noncovalent complexes. J. Am. Soc. Mass Spectrom. 1998, 9(6), 580–584.
Veenstra, T. D.; Johnson, K. L.; Tomlinson, A. J.; Craig, T. A.; Kumar, R.; Naylor, S. Zinc-induced conformational changes in the DNA-binding domain of the vitamin D receptor determined by electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 1998, 9(1), 8–14.
Veenstra, T. D.; Benson, L. M.; Craig, T. A.; Tomlinson, A. J.; Kumar, R.; Naylor, S. Metal mediated sterol receptor-DNA complex association and dissociation determined by electrospray ionization mass spectrometry. Nat. Biotechnol. 1998, 16(3), 262–266.
Craig, T. A.; Benson, L. M.; Tomlinson, A. J.; Veenstra, T. D.; Naylor, S.; Kumar, R. Analysis of transcription complexes and effects of ligands by microelectrospray ionization mass spectrometry. Nat. Biotechnol. 1999, 17(12), 1214–1218.
Johnson, K. L.; Veenstra, T. D.; Londowski, J. M.; Tomlinson, A. J.; Kumar, R.; Naylor, S. On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins. Biomed. Chromatogr. 1999, 13(1), 37–45.
Craig, T. A.; Benson, L. M.; Naylor, S.; Kumar, R. Modulation effects of zinc on the formation of vitamin D receptor and retinoid X receptor α-DNA transcription complexes: Analysis by microelectrospray mass spectrometry. Rapid Commun. Mass Spectrom. 2001, 15(12), 1011–1016.
Craig, T. A.; Benson, L. M.; Venyaminov, S. Y.; Klimtchuk, E. S.; Bajzer, Z.; Prendergast, F. G.; Naylor, S.; Kumar, R. The metal-binding properties of DREAM: evidence for calcium-mediated changes in DREAM structure. J. Biol. Chem. 2002, 277(13), 10955–10966.
Loo, J. A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom. Rev. 1997, 16(1), 1–23.
van Berkel, W. J.; van den Heuvel, R. H.; Versluis, C.; Heck, A. J. Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry. Protein Sci. 2000, 9(3), 435–439.
Zhang, Z.; Krutchinsky, A.; Endicott, S.; Realini, C.; Rechsteiner, M.; Standing, K. G. Proteasome activator 11S REG or PA28: Recombinant REG α/REG β hetero-oligomers are heptamers. Biochemistry. 1999, 38(17), 5651–5658.
Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Detection and selective dissociation of intact ribosomes in a mass spectrometer. Proc. Natl. Acad. Sci. U.S.A. 2000, 97(10), 5185–5190.
Hernandez, H.; Robinson, C. V. Dynamic protein complexes: Insights from mass spectrometry. J. Biol. Chem. 2001, 276(50), 46685–46688.
Tito, M. A.; Miller, J.; Walker, N.; Griffin, K. F.; Williamson, E. D.; Despeyroux-Hill, D.; Titball, R. W.; Robinson, C. W. Probing molecular interactions in intact antibody: Antigen complexes, an electrospray time-of-flight mass spectrometry approach. Biophys. J. 2001, 81(6), 3503–3509.
Elviri, L.; Zagnoni, I.; Careri, M.; Cavazzini, D.; Rossi, G. L. Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques. Rapid Commun. Mass Spectrom. 2001, 15(22), 2186–2192.
Potier, N.; Rogniaux, H.; Chevreux, G.; Van Dorsselaer, A. Ligand-metal ion binding to proteins: Investigation by ESI mass spectrometry. Methods Enzymol. 2005, 402), 361–389.
Greig, M. J.; Gaus, H.; Cummins, L. L.; Sasmor, H.; Griffey, R. H. Measurement of macromolecular binding using electrospray mass spectrometry. Determination of dissociation constants for oligonucleotide: Serum albumin complexes. J. Am. Chem. Soc. 1995, 117), 10765–10766.
Sannes-Lowry, K. A.; Mei, H. Y.; Loo, J. A. Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry. Int. J. Mass Spectrom. 1999, 193), 115–122.
Loo, J. A.; Edmonds, C. G.; Smith, R. D. Tandem mass spectrometry of very large molecules: serum albumin sequence information from multiply charged ions formed by electrospray ionization. Anal. Chem. 1991, 63(21), 2488–2499.
Chowdhuray, S. K.; Katta, V.; Chait, B. T. Probing conformational changes in proteins by mass spectrometry. J. Am. Chem. Soc. 1990, 112), 9012–9013.
Mirza, U. A.; Cohen, S. L.; Chait, B. T. Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry. Anal. Chem. 1993, 65(1), 1–6.
Mohimen, A.; Dobo, A.; Hoerner, J. K.; Kaltashov, I. A. A chemometric approach to detection and characterization of multiple protein conformers in solution using electrospray ionization mass spectrometry. Anal. Chem. 2003, 75(16), 4139–4147.
Ray, S. S.; Singh, S. K.; Balaram, P. An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins. J. Am. Soc. Mass Spectrom. 2001, 12), 428–438.
Cohen, S. A.; Bidlingmeyer, B. A.; Tarvin, T. L. PITC derivatives in amino acid analysis. Nature. 1986, 320(6064), 769–770.
Bidlingmeyer, B. A.; Cohen, S. A.; Tarvin, T. L. Rapid analysis of amino acids using pre-column derivatization. J. Chromatogr. 1984, 336(1), 93–104.
Hunkapiller, M. W.; Hewick, R. M.; Dreyer, W. J.; Hood, L. E. High-sensitivity sequencing with a gas-phase sequenator. Methods Enzymol. 1983, 91), 399–413.
Hewick, R. M.; Hunkapiller, M. W.; Hood, L. E.; Dreyer, W. J. A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 1981, 256(15), 7990–7997.
Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chain-terminating inhibitors, 1977. Biotechnology. 1992, 24), 104–108.
Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1977, 74(12), 5463–5467.
Caruthers, M. H.; Beaucage, S. L.; Efcavitch, J. W.; Fisher, E. F.; Matteucci, M. D.; Stabinsky, Y. New chemical methods for synthesizing polynucleotides. Nucleic Acids Symp. Ser. 1980, 7), 215–223.
Matteucci, M. D.; Caruthers, M. H. Synthesis of deoxyoligonucleotides on a polymer support, 1981. Biotechnology. 1992, 24), 92–98.
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72), 248–254.
Craig, T. A.; Kumar, R. Synthesis and purification of soluble ligand binding domain of the human vitamin D3 receptor. Biochem. Biophys. Res. Commun. 1996, 218(3), 902–907.
Berndt, T.; Craig, T. A.; Bowe, A. E.; Vassiliadis, J.; Reczek, D.; Finnegan, R.; Jan DeBeur, S. M.; Schiavi, S. C.; Kumar, R. Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent. J. Clin. Invest. 2003, 112(5), 785–794.
Veenstra, T. D.; Gross, M. D.; Hunziker, W.; Kumar, R. Identification of metal-binding sites in rat brain calcium-binding protein. J. Biol. Chem. 1995, 270(51), 30353–30358.
Gross, M. D.; Kumar, R.; Hunziker, W. Expression in Escherichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein. J. Biol. Chem. 1988, 263(28), 14426–14432.
Sreerama, N.; Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 2000, 287(2), 252–260.
Sreerama, N.; Venyaminov, S. Y.; Woody, R. W. Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 1999, 8(2), 370–380.
Provencher, S. W.; Glockner, J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry. 1981, 20(1), 33–37.
van Stokkum, I. H.; Spoelder, H. J.; Bloemendal, M.; van Grondelle, R.; Groen, F. C. Estimation of protein secondary structure and error analysis from circular dichroism spectra. Anal. Biochem. 1990, 191(1), 110–118.
Johnson, W. C. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins. 1999, 35(3), 307–312.
Venyaminov, S.; Vassilenko, K. S. Determination of protein tertiary structure class from circular dichroism spectra. Anal. Biochem. 1994, 222(1), 176–184.
Sreerama, N.; Venyaminov, S. Y.; Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: Inclusion of denatured proteins with native proteins in the analysis. Anal. Biochem. 2000, 287(2), 243–251.
Mihaly, E. J. Numerical values of the adsorbances of the aromatic amino acids in acid, neutral and alkaline solutions. Chem. Eng. Data. 1968, 13), 179–182.
Gill, S. C.; von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182(2), 319–326.
Mach, H.; Middaugh, C. R.; Lewis, R. V. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal. Biochem. 1992, 200(1), 74–80.
Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995, 4(11), 2411–2423.
Persechini, A.; Moncrief, N. D.; Kretsinger, R. H. The EF-hand family of calcium-modulated proteins. Trends Neurosci. 1989, 12(11), 462–467.
Moncrief, N. D.; Kretsinger, R. H.; Goodman, M. Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J. Mol. Evol. 1990, 30(6), 522–562.
Nakayama, S.; Moncrief, N. D.; Kretsinger, R. H. Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories. J. Mol. Evol. 1992, 34(5), 416–448.
Nakayama, S.; Kretsinger, R. H. Evolution of EF-hand calcium-modulated proteins. III. Exon sequences confirm most dendrograms based on protein sequences: Calmodulin dendrograms show significant lack of parallelism. J. Mol. Evol. 1993, 36(5), 458–476.
Kretsinger, R. H.; Nakayama, S. Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did. not determine the domain compositions of EF-hand proteins. J. Mol. Evol. 1993, 36(5), 477–488.
Nakayama, S.; Kretsinger, R. H. Evolution of the EF-hand family of proteins. Annu. Rev. Biophys. Biomol. Struct. 1994, 23), 473–507.
Kawasaki, H.; Nakayama, S.; Kretsinger, R. H. Classification and evolution of EF-hand proteins. Biometals. 1998, 11(4), 277–295.
Wiech, H.; Geier, B. M.; Paschke, T.; Spang, A.; Grein, K.; Steinkotter, J.; Melkonian, M.; Schiebel, E. Characterization of green alga, yeast, and human centrins. Specific subdomain features determine functional diversity. J. Biol. Chem. 1996, 271(37), 22453–22461.
Hu, H.; Sheehan, J. H.; Chazin, W. J. The mode of action of centrin. Binding of Ca2+ and a peptide fragment of Kar1p to the C-terminal domain. J. Biol. Chem. 2004, 279(49), 50895–50903.
Prakash, H.; Mazumdar, S. Direct correlation of the crystal structure of proteins with the maximum positive and negative charge states of gaseous protein ions produced by electrospray ionization. J. Am. Soc. Mass Spectrom. 2005, 16(9), 1409–1421.
Lafitte, D.; Heck, A. J.; Hill, T. J.; Jumel, K.; Harding, S. E.; Derrick, P. J. Evidence of noncovalent dimerization of calmodulin. Eur. J. Biochem. 1999, 261(1), 337–344.
Hautreux, M.; Hue, N.; Alexis, N.; Du Fou de Kerdaniel, A.; Zahir, A.; Malec, V.; Laprévote, O. Under nondenaturing solvent conditions, the mean charge state of a multiply charged protein ion formed by electrospray is linearly correlated with the macromolecular surface. Int. J. Mass Spectrom. 2004, 231), 131–137.
Sperry, J.; Gross, M.; Kumar, R. Calcium binding properties of human centrin 2. Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics; San Antonio, TX, June, 2005.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online June 5, 2006
Rights and permissions
About this article
Cite this article
Craig, T.A., Benson, L.M., Bergen, H.R. et al. Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy. The official journal of The American Society for Mass Spectrometry 17, 1158–1171 (2006). https://doi.org/10.1016/j.jasms.2006.04.029
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2006.04.029