ABSTRACT The conformational rearrangements that take place after calcium binding in chicken annexin A5 and a mutant lacking residues 3-10 were analyzed, in parallel with human annexin A5, by circular dichroism (CD), infrared spectroscopy (IR), and differential scanning calorimetry. Human and chicken annexins present a slightly different shape in the far-UV CD and IR spectra, but the main secondary-structure features are quite similar (70-80% alpha-helix). However, thermal stability of human annexin is significantly lower than its chicken counterpart (~8 deg C) and equivalent to the chicken N-terminally truncated form. The N-terminal extension contributes greatly to stabilize the overall annexin A5 structure. Infrared spectroscopy reveals the presence of two populations of alpha-helical structures, the canonical alpha-helices (~1650 cm^sup -1^) and another, at a lower wavenumber (~1634 cm^sup -1^), probably arising from helix-helix interactions or solvated a-helices. Saturation with calcium induces: alterations in the environment of the unique tryptophan residue of the recombinant proteins, as detected by near-UV CD spectroscopy; more compact tertiary structures that could account for the higher thermal stabilities (8 to 12 deg C), this effect being higher for human annexin; and an increase in canonical alpha-helix percentage by a rearrangement of nonperiodical structure or 3^sub 10^ helices together with a variation in helix-helix interactions, as shown by amide I curve-fitting and 2D-IR.
INTRODUCTION
Annexins are a widely distributed multigene family of structurally related calcium binding proteins (for review, see Raynal and Pollard, 1994; Swairjo and Seaton, 1994; Gerke and Moss, 2002). Their main characteristic is the ability to reversibly bind to acid phospholipid-rich membranes in the presence of calcium. Several in vitro functions, including anticoagulatory and antiinflammatory activities, involvement in signal transduction, in membrane fusion, endo and exocytosis, and in calcium channel regulation have been described for these proteins, but little is known about their in vivo role (Raynal and Pollard, 1994; Gerke and Moss, 2002). However, some specific diseases, known as annexinopathies, have been described associated with abnormal expression of annexins A2 and A5; their study may contribute to a better understanding of the physiological role of these proteins (Rand, 1999). Some of these functions are specific for particular annexins, even though there is a high structural homology among them. Moreover, tissue-specific activities and alternatively spliced forms have been described for some particular annexins (Bohm et al., 1994; Sable and Riches, 1999). All members of this family of proteins present a highly conserved core structure composed of four (eight in annexin A6) homologous domains of -70 amino acids showing a similar three-dimensional structure (Liemann and Huber, 1997). The main structural differences are located in their variable N-terminal region that differs greatly in length and amino acid sequence (Raynal and Pollard, 1994; Gerke and Moss, 2002). Annexin A5 crystal structure was the first one resolved (Huber et al., 1990); since then, several other annexins have been crystallized and all of them present an almost identical three-dimensional arrangement in the protein core. The molecules display a slightly bent disk shape where the four repeated domains, each of them comprising a four alpha-helix bundle (helices A, B, D, and E), are organized in a cylindrical way and are capped by a fifth alpha-helix (C).
The arrangement of the four domains allows the appearance of a central hydrophilic pore, which could be responsible for the voltage-dependent calcium channel activity reported for several annexins (A1, A2, A5-A7, B12) (Liemann et al., 1996; Hofmann et al., 1997; Matsuda et al., 1997). The interaction with membranes takes place on the convex side of the molecule where the main calcium-binding sites (one per domain) are located. The calcium ion binds to carbonyl oxygens in the loop connecting the A and B helices, and to a bidentate carboxyl group from a glutamic or aspartic acid residue located around 40 residues downstream in the loop connecting helices D and E. The Nterminal region is located in the opposite concave region of the annexin molecule binding together domains I and IV (Huber et al., 1990), at least in annexins with a short N-terminal domain, as annexin A5.
Taking into account that the main structural differences among annexins appear in the N-terminal extension, the search for specific functions of each annexin has been focused in this region. Annexin AS presents the shortest N-terminal tail among all annexins described so far, only about 15 residues. In fact, it has been described that the truncation of 14 residues of human annexin AS (hA5) induces the loss of the calcium channel activity, suggesting the involvement of this region in the regulation of this channel (Berendes et al., 1993). On this idea, we have obtained and characterized a mutant chicken annexin AS (dnt-cA5) lacking amino acid residues 3-10, being the secondary structure of this mutant almost identical to that of the wild-type protein (Turnay et al., 1995; Arboledas et al., 1997).
Calcium is essential for one of the main properties of annexins, their ability to bind to specific cellular membranes. Calcium requirements for half-maximal binding to phospholipid bilayers is highly variable among this family of proteins, ranging from submicromolar in annexin A2 to 10-100 (mu)M in annexin A5 (Raynal and Pollard, 1994; Gerke and Moss, 2002). These calcium concentrations may be reached intracellularly under certain physiological conditions; however, calcium binding in the absence of phospholipids requires much higher concentrations of this cation (Raynal and Pollard, 1994; Sopkova et al., 1994). The crystal structure of domain III of annexin A5 reveals significant conformational changes upon calcium binding in the absence of phospholipids. Thus, the aim of this study is to analyze the effect of calcium binding, in the absence of phospholipids, in the stability and structure of annexin A5 and to get further insights into the role of the N-terminus in the maintenance of the overall structure of this protein. We have studied different structural and thermodynamic parameters of chicken annexin A5 (cA5) and dnt-cA5, comparing them with those of hA5.
MATERIALS AND METHODS
Protein preparation
Recombinant cA5 and its mutant dnt-cA5 have been produced and purified as previously reported (Turnay et al., 1995; Arboledas et al., 1997). hA5 cDNA was kindly provided by Dr. Pilar FernAndez (University of Oviedo, Spain) and was subcloned as described for the chicken cDNA. Briefly, cDNAs were cloned into the pTrc99A prokaryotic expression vector (Amersham Pharmacia Biotech, Buckinghamshire, UK) and introduced into JA221 Escherichia coli strain cells. Expression was induced by addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 16 h after the initial cultures reached 0.5 optical density at 550 nm. Recombinant proteins were purified from bacterial homogenates in the presence of 2.5 mM EGTA and using their ability to interact reversibly with phosphatidylserine-enriched liposomes (prepared from bovine brain extract, Folch fraction III, from Sigma, Alcobendas, Spain) in the presence of 2 mM calcium. A final DEAF-cellulose chromatography in 50 mM Tris, pH 7.4, containing I mM EGTA, was performed to further purify the protein preparations and to eliminate lipids. Pure annexin fractions were pooled and dialyzed against 20 mM Hepes, pH 7.4, containing 0.1 M NaCl and 1 mM EGTA, filtered through 0.22 (mu)m membranes, and stored at 4 deg C until used. Before use, protein samples were dialyzed to equilibrium against buffer with or without calcium.
Circular dichroism measurements
Circular dichroism (CD) spectra were recorded in a Jasco J-715 spectropolarimeter at 25 deg C (Neslab RTE- 111 thermostat). The far-UV CD spectra were monitored between 200 and 250 nm and near-UV CD spectra between 250 and 310 nm using 0.01 or 0.05 cm and 0.5 cm optical pathlength cuvettes for far- and near-UV, respectively. Melting curves were determined monitoring ellipticity changes at 222 nm between 25 and 75 deg C and increasing temperature at 60 deg C/h. Monitoring of ellipticity changes upon cooling from 75 to 25 deg C was also performed at 60 deg C/h. Spectra in the absence of calcium were recorded in 20 mM Hepes, pH 7.4, containing 0.1 M NaCl and 1 mM EGTA; titration of calcium influence in the near-UV was performed by sequential addition of a 0.5 M CaCl2 stock solution (in 20 mM Hepes, pH 7.4, containing 0.1 M NaCI) and correcting the spectra for dilution. Checks were made to ensure that equilibrium was reached after each addition of calcium to the protein preparation. The influence of calcium concentration on the melting temperature was analyzed by preparing different protein samples from the same stock equilibrated at increasing calcium chloride concentrations. Samples with equivalent ionic strength obtained by addition of NaCl were used as controls. All spectra were obtained averaged over five scans (eight at low protein concentration) and were corrected by subtracting buffer contribution from parallel spectra in the absence of protein. The calcium-dependent variation in ellipticity at 292 nm, and in the melting curves recorded at 222 nm, was analyzed using a hyperbolic or logistic nonlinear regression fitting using SigmaPlot software (SPSS, Chicago, IL). Prediction of secondary structure from the far-UV CD spectra was performed using the convex constraint algorithm described by Perczel et al. (1992).
Infrared spectroscopy
Infrared spectroscopy (IR) spectroscopy measurements were performed on a Nicolet Magna II 550 spectrometer (Nicolet Instrument Corp., Madison, WI) equipped with a MCT detector, using a demountable liquid cell (Harrick Scientific, Ossining, NY) with CaF2 windows and 50 (mu)m spacers. A tungsten-copper thermocouple was placed directly onto the window and the cell placed in a thermostatted cell mount. Proteins were concentrated by ultrafiltration using Amicon Centriplus YM-10 membranes (10 kDa cutoff; Millipore, Bedford, MA) up to 20 mg/ml in 20 mM Hepes, pH 7.4, 0.1 M NaCI. Equilibration in D2O buffer was achieved by protein lyophilization in the presence of buffer and reconstitution in D2O with 99.8% isotopic enrichment (Merck, Darmstadt, Germany). Stock calcium solutions and buffer were also lyophilized and reconstituted in D2O.
Thermal analyses were performed by beating continuously from 25 to 85 deg C at a rate of 60 deg C/h. Spectra were taken using a Rapid Scan software under OMNIC (Nicolet). For each degree of temperature interval, 305 interferograms were averaged, Fourier-transformed, and ratioed against a background, obtaining the spectra with a nominal resolution better than 2 cm^sup -1^. Data treatment, band decomposition, and thermal analysis of the original amide I bands were performed as previously described (Arrondo et al., 1993; Arrondo and Goni, 1999). After integrating each component, the corresponding percentages were obtained assuming that the molar absorption coefficients for the different protein structures were the same.
To obtain the 2D-IR maps, heating was used as the perturbation to induce time-dependent spectral fluctuations and to detect dynamical spectral variations on the secondary structure of annexin. Two-dimensional synchronous spectra have been obtained as described elsewhere (Contreras et al., 2001; Paquet et al., 2001).
Differential scanning calorimetry
We are grateful to Dr. M. P. Fernandez (University of Oviedo, Spain), for kindly providing a cDNA clone for human annexin A5, and to Dr J. Villalain (University Miguel Hernandez, Spain) and Dr. M. Pezolet (University Laval, Canada), for providing us the 2D algorithm.
This work was supported by Grants PM98-0083 from the DGES (Spain), 603/98 from the University of Basque Country, and PI 1998-33 from the Basque Government.
[Reference]
REFERENCES
[Reference]
Arboledas, D., N. Olmo, M. A. Lizarbe, and J. Turnay. 1997. Role of the N-terminus in the structure and stability of chicken annexin V. FEBS Lett. 416:217-220.
Arrondo, J. L. R., and F. M. Goni. 1999. Structure and dynamics of membrane proteins as studied by infrared spectroscopy. Prog. Biophys. Mol. Biol. 72:367-405.
Arrondo, J. L. R., A. Muga, J. Castresana, and F. M. Goni. 1993. Quantitative studies of the structure of proteins in solution by Fourier-- transform infrared spectroscopy. Prog. Biophys. Mol. Biol. 59:23-56.
Berendes, R.. A. Burger, D. Voges, P. Demange, and R. Huber. 1993. Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2. FEBS Lett. 317:131-134.
Bewley, M. C., C. M. Boustead, J. H. Walker, and R. Huber. 1993. Structure of chicken annexin V at 2.25A resolution. Biochemistry. 32: 3923-3928.
[Reference]
Bohm, B. B., B. Wilbrink, K. E. Kuettner, and J. Mollenhauer. 1994. Structural and functional comparison of anchorin CII (Cartilage Annexin V) and muscle annexin V. Arch. Biochem. Biophys. 314:64-74.
Byler, D. M., and H. Susi. 1986. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers. 25:469-487. Concha, N. O., J. F. Head, M. A. Kaetzel, J. R. Dedman, and B. A. Seaton.
1993. Rat annexin V crystal structure: Ca(2+)-induced conformational changes. Science. 261:1321-1324.
Contreras, L. M., F. J. Aranda, F. Gavilanes, J. M. Gonzalez-Ros, and J. Villalain. 2001. Structure and interaction with membrane model systems of a peptide derived from the major epitope region of HIV protein gp4l: implications on viral fusion mechanism. Biochemistry. 40:3196-3207.
Echabe, I., U. Domberger, A. Prado, F. M. Goni, and J. L. R. Arrondo. 1998. Topology of sarcoplasmic reticulum Caz+-ATPase: an infrared study of thermal denaturation and limited proteolysis. Protein Sci. 7:1172-1179.
[Reference]
Follenius-Wund, A., E. Piemont, J. M. Freyssinet, D. Gerard, and C. Pigault. 1997. Conformational adaptation of annexin V upon binding to liposomes: a time-resolved fluorescence study. Biochem. Biophys. Res. Commun. 234:111-116.
Gerke, V., and S. E. Moss. 2002. Annexins: from structure to function. Physiol Rev. 82:331-371.
Gilmanshin, R., S. Williams, R. H. Callender, W. H. Woodruff, and R. B. Dyer. 1997. Fast events in protein folding: relaxation dynamics and structure of the I form of apomyoglobin. Biochemistry. 36: 15006-15012.
Hofmann, A., J. Benz, S. Liemann, and R. Huber. 1997. Voltage dependent binding of annexin V, annexin VI and annexin VII-core to acidic phospholipid membranes. Biochim. Biophys. Acta. 1330:254-264.
[Reference]
Huber, R., R. Berendes, A. Burger, M. Schneider, A. Karshikov, H. Luecke, J. Romisch, and E. Paques. 1992. Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins. J. Mol. Biol. 223:683-704.
Huber, R., M. Schenider, I. Mayr, J. Romisch, and E. P. Paques. 1990. The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. FEES Lett. 275:15-21.
Koradi, R., M. Billeter, and K. Wuthrich. 1996. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14: 51-55.
[Reference]
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-685.
Liemann, S., J. Benz, A. Burger, D. Voges, A. Hofmann, R. Huber, and P. Gottig. 1996. Structural and functional characterisation of the voltage sensor in the ion channel human annexin V. J. Mol. Biol. 258:555-561.
Liemann, S., and R. Huber. 1997. Three-dimensional structure of annexins. Cell. Mol. Life Sci. 53:516-521.
Lopez Mayorga, O., and E. Freire. 1987. Dynamic analysis of differential scanning calorimetry data. Biophys. Chem. 27:87-96.
Matsuda, R., N. Kaneko, and Y. Horikawa. 1997. Presence and comparison of Ca 21 transport activity of annexins I, 11, V, and VI in large unilamellar vesicles. Biochem. Biophys. Res. Commun. 237:499-503.
Meets, P. 1990. Location of tryptophans in membrane-bound annexins. Biochemistry. 29:3325-3330.
Meers, P., and T. Mealy. 1993. Calcium-dependent annexin V binding to phospholipids: stoichiometry, specificity, and the role of negative charge. Biochemistry. 32:11711-11721.
Muga, A., H. H. Mantsch, and W. K. Surewicz. 1991. Membrane binding induces destabilization of cytochrome c structure. Biochemistry. 30: 7219-7224.
[Reference]
Paquet, M. J., M. Laviolette, M. Pezolet, and M. Auger. 2001. Twodimensional infrared correlation spectroscopy study of the aggregation of cytochrome c in the presence of dimyristoylphosphatidylglycerol. Biophys. J. 81:305-312.
Perczel, A., K. Park, and G. D. Fasman. 1992. Analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: a practical guide. Anal. Biochem. 203:83-93.
Pigault, C., A. Follenius-Wund, and M. Chabbert. 1999. Role of Trp-187 in the annexin V-membrane interaction: a molecular mechanics analysis. Biochem. Biophys. Res. Common. 254:484-489.
Rand, J. H. 1999. "Annexinopathies": a new class of diseases. N. Engl. J. Med. 340:1035-1036.
Raynal, P., and H. B. Pollard. 1994. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim. Biophys. Acta. 1197:63-93.
[Reference]
Reisdorf, W. C., and S. Krimm. 1996. Infrared amide I' band of the coiled coil. Biochemistry. 35:1383-1386.
Rosengarth, A., J. Rbsgen, H. J. Hinz, and V. Gerke. 1999. A comparison of the energetics of annexin I and annexin V. J. MoL BioL 288: 1013-1025.
[Reference]
Sable, C. L., and D. W. H. Riches. 1999. Cloning and functional activity of a novel truncated form of annexin IV in mouse macrophages. Biochem. Biophys. Res. Common. 258:162-167.
Sinchez-Ruiz, J. M., J. L. L6pez-Lacomba, M. Cortijo, and P. L. Mateo. 1988. Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin. Biochemistry. 27:1648-1652.
Silvestro, L., and P. H. Axelsen. 1999. Fourier transform infrared linked analysis of conformational changes in annexin V upon membrane binding. Biochemistry. 38:113-121.
Sopkova, J., J. Gallay, M. Vincent, P. Pancoska, and A. Lewit-Bentley. 1994. The dynamic behavior of annexin V as a function of calcium ion binding: a circular dichroism, UV absorption, and steady-state and time-resolved fluorescence study. Biochemistry. 33:4490-4499.
Sopkova, J., M. Renouard, and A. Lewit-Bentley. 1993. The crystal structure of a new high-calcium form of annexin V. J. Mol. Biol. 234: 816-825.
[Reference]
Sopkova, J., M. Vincent, M. Takahashi, A. Lewit-Bentley, and J. Gallay. 1998. Conformational flexibility of domain III of annexin V studied by fluorescence of tryptophan 187 and circular dichroism: the effect of pH. Biochemistry. 37:11962-11970.
Sopkova-de Oliveira Santos, J., S. Fischer, C. Guilbert, A. Lewit-Bentley, and J. C. Smith. 2000. Pathway for large-scale conformational change in annexin V. Biochemistry. 39:14065-14074,
Sopkova-de Oliveira Santos, J., M. Vincent, S. Tabaries, A. Chevalier, D. Kerboeuf, F. Russo-Marie, A. Lewit-Bentley, and J. Gallay. 2001. Annexin AS D226K structure and dynamics: identification of a molecular switch for the large-scale conformational change of domain 111. FEBS Lett. 493:122-128.
Swaiijo, M. A., and B. A. Seaton. 1994. Annexin structure and membrane interactions: a molecular perspective. Annu. Rev. Biophys. Biomol. Struct. 23:193-213.
[Reference]
Torii, H., and M. Tasumi. 1992. Model calculations on the amide-I infrared bands of globular proteins. J. Chem. Phys. 96:3379-3387.
Turnay, J., E. Pfanniiller, M. A. Lizarbe, W. M. Bertling, and K. von der Mark. 1995. Collagen binding activity of recombinant and N-terminally modified annexin V (anchorin CII). J. Cell. Biochem. 58:208-220.
Vogl, T., C. Jatzke, H. J. Hinz, J. Benz, and R. Huber. 1997. Thermodynamic stability of annexin V E17G: equilibrium parameters from an irreversible unfolding reaction. Biochemistry. 36:1657-1668.
Wu, F., C. R. Flach, B. A. Seaton, T. R. Mealy, and R. Mendelsohn. 1999. Stability of annexin V in ternary complexes with Ca" and anionic phospholipids: IR studies on monolayer and bulk phases. Biochemistry. 38:792-799.
[Author Affiliation]
Javier Turnay,* Nieves Olmo,* Maria Gasset,^ Ibon Iloro,^^ Jose Luis R. Arrondo,^^ and M. Antonia Lizarbe*
*Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Quimicas, Universidad Complutense, 28040 Madrid, Spain; ^Instituto Rocasolano de Quimica-Fisica, CSIC, 28006 Madrid, Spain; and ^^Unidad de Biofisica (Centro Mixto CSIC-UPV) y Departamento de Bioquimica, Universidad del Pais Vasco, Apdo. 644, 48080 Bilbao, Spain
[Author Affiliation]
Submitted April 11, 2002, and accepted for publication May 29, 2002. Address reprint requests to Prof. M. A. Lizarbe, Departamento de Bioquimica y Biologia Molecular I, Facultad de Ciencias Quimicas, Universidad Complutense, 28040 Madrid, Spain. Tel.: +34-91-3944148; Fax: +34-91-3944159; E-mail: lizarbe@bbm.l.ucm.es.
Комментариев нет:
Отправить комментарий