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J. Biol. Chem., Vol. 275, Issue 33, 25508-25515, August 18, 2000

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Calcium Affinity, Cooperativity, and Domain Interactions of Extracellular EF-hands Present in BM-40^*

Elisabeth Busch, Erhard Hohenester^§¶, Rupert Timpl, Mats Paulsson, and Patrik Maurer^**

From the  Institute for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany, the ^§ Biophysics Section, Blackett Laboratory and the Division of Medicine, Imperial College, London, SW7 2BZ, United Kingdom, and the  Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany

Received for publication, March 3, 2000, and in revised form, April 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The structure and function of cytosolic Ca²⁺-binding proteins containing EF-hands are well understood. Recently, the presence of EF-hands in an extracellular protein was for the first time proven by the structure determination of the EC domain of BM-40 (SPARC (for secreted protein acidic and rich in cysteine)/osteonectin) (Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., and Engel, J. (1996) Nat. Struct. Biol. 3, 67-73). The structure revealed a pair of EF-hands with two bound Ca²⁺ ions. Two unusual features were noted that distinguish the extracellular EF-hands of BM-40 from their cytosolic counterparts. An insertion of one amino acid into the loop of the first EF-hand causes a variant Ca²⁺ coordination, and a disulfide bond connects the helices of the second EF-hand. Here we show that the extracellular EF-hands in the BM-40 EC domain bind Ca²⁺ cooperatively and with high affinity. The EC domain is thus in the Ca²⁺-saturated form in the extracellular matrix, and the EF-hands play a structural rather than a regulatory role. Deletion mutants demonstrate a strong interaction between the EC domain and the neighboring FS domain, which contributes about 10 kJ/mol to the free energy of binding and influences cooperativity. This interaction is mainly between the FS domain and the variant EF-hand 1. Certain mutations of Ca²⁺-coordinating residues changed affinity and cooperativity, but others inhibited folding and secretion of the EC domain in a mammalian cell line. This points to a function of EF-hands in extracellular proteins during biosynthesis and processing in the endoplasmic reticulum or Golgi apparatus.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The EF-hand is a highly conserved Ca²⁺-binding motif found in a large number of intracellular proteins. EF-hands, as present in calmodulin, troponin C, and calcineurin, are responsible for signal transduction by the second messenger Ca²⁺, leading to the activation or inactivation of numerous and diverse target proteins and thus influencing a wide range of cellular processes (1-4). The term EF-hand was introduced by Kretsinger and Nockolds (5) in describing the structure of parvalbumin. Two helices (E and F in parvalbumin) flank a loop of 12 amino acids in which the Ca²⁺ ion is 7-fold coordinated in a pentagonal-bipyramidal arrangement. The Ca²⁺ ion is coordinated by side chain oxygen atoms at the X, Y, and Z vertices, by a backbone carbonyl at Y, by a water molecule at X, and by a carboxylate (mostly glutamate) at Z, which makes a crucial bidentate interaction with the metal ion (see Fig. 1). This canonical EF-hand structure was subsequently found in a number of proteins of the Ca²⁺ second messenger system. Usually, EF-hands occur in pairs, although in some cases one or even both EF-hands in a pair may no longer be functional because of mutations of Ca²⁺-coordinating residues (for reviews see Refs. 6-8).

Several variations of the canonical Ca²⁺-binding mode are known. In the S100 subfamily of EF-hand proteins, two amino acids are inserted in the N-terminal EF-hand loop, while the C-terminal EF-hand is of the canonical type. The insertions lead to a rearrangement in the loop, such that the Ca²⁺ ion is coordinated by backbone carbonyl oxygens at positions X, Y, Z and -Y, with the bidentate ligand at -Z providing the only side chain oxygen atoms (9). Despite the unusual coordination of this site, the affinities of the two EF-hands for Ca²⁺, e.g. in calbindin D9k, are nearly identical (10). A further variation of the canonical EF-hand was found in myosin essential light chain. The loop in the first EF-hand of the EF-hand pair also contains two inserted amino acids but at different positions compared with S100 proteins. In essential light chain the insertions result in a more pronounced rearrangement of Ca²⁺ ligands, with several residues coordinating Ca²⁺ with both their backbone and side chain oxygens (11, 12). Finally, in the Ca²⁺-binding domain of calpain, the first of the five EF-hands binds Ca²⁺ noncanonically. This EF-hand lacks the typical side chains at positions X and Y and uses a carbonyl oxygen and a second water molecule instead (13, 14).

BM-40 (SPARC (for secreted protein acidic and rich in cysteine)/osteonectin) is the only example of an extracellular EF-hand protein that is secreted by virtue of a classic signal peptide and for which a structure determination has proved the presence of EF-hands (15, 16). BM-40 is an abundant glycoprotein and has been suggested to participate in the modulation of cell-matrix interactions, bone mineralization, wound repair, and angiogenesis (17). It is highly expressed in some malignant tumors and has been reported to play a crucial role in the tumorigenicity of human melanomas (18). Developmental anomalies are induced by overexpressing or suppressing BM-40 in nematodes (19, 20). Furthermore, microinjection of BM-40 RNA, protein, peptides, or antibodies against BM-40 interfere with Xenopus embryonic development (21, 22). In contrast, deletion of the BM-40 gene in mice resulted in a relatively mild phenoptype, namely late onset cataract formation in the eye, whereas development proceeded normally (23, 24). However, further defects, such as severe osteopenia, are under investigation (25). It is suspected that homologous proteins, such as SC1, QR1, TSC-36, testican-1, or testican-2 (26-30) may compensate for several of the reported functions of BM-40 during murine embryonic development.

BM-40 is a modular protein built from three domains. A short N-terminal region (domain I), rich in glutamic acid residues, binds several Ca²⁺ ions with low affinity (K_d = 5-10 mM) (31). This is followed by a region homologous to follistatin (FS domain) and an autonomously folding C-terminal Ca²⁺-binding EC domain. A single EF-hand was predicted in the EC domain of BM-40 (32), and Ca²⁺ binding was interpreted as being to a single site (31, 33, 34). However, the structures of the EC domain and the pair of FS and EC domains revealed a pair of EF-hands in the EC domain, each with a bound Ca²⁺ ion (15, 16). Both EF-hands in BM-40 are of unusual structure; a one-residue insertion into the Ca²⁺-coordinating loop of the first EF-hand is accommodated by a cis-peptide bond, which allows a peptide carbonyl to substitute for a Ca²⁺-binding side chain. This novel variation explained why even sophisticated search algorithms failed to detect both EF-hands in BM-40. The second EF-hand has a canonical pattern of Ca²⁺-coordinating residues but is circularized by a disulfide bond between the flanking helices. A collagen-binding site in the EC domain of BM-40, which is only active in the presence of Ca²⁺, has been characterized by an alanine mutagenesis screen (35).

One objective of the present investigation was to determine the Ca²⁺ binding properties of the EF-hands of BM-40. Both the variant and the canonical EF-hand were found to bind Ca²⁺ with high affinity, and there is positive cooperativity between the two EF-hands. In addition, interactions with the neighboring FS domain strongly influence the Ca²⁺ affinity of the EC domain. Some mutations in the EF-hands designed to abolish Ca²⁺ binding interfered with protein secretion, indicating an essential role of Ca²⁺ binding for folding and secretion.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nomenclature of Proteins and Mutants-- The sequence numbering of human BM-40 (36) used starts at the N terminus obtained after cleavage of the signal peptide. Secreted recombinant full-length human BM-40 thus corresponds to amino acid residues 1-286. The designation FS-EC (residues 51-286) is used for the fragment encompassing the FS and EC domains but lacking the N-terminal acidic domain. The EC domain encompasses residues 136-286. The sequence of both EF-hand loops within the EC domain and the point mutations introduced are shown in Fig. 1. The nomenclature for the point mutants includes the domain context, the corresponding EF-hand, and the substitution. For instance, EC-EF1-E234K describes a point mutation introduced in the EC domain in which Glu²³⁴ in EF-hand 1 (EF1) occupying position Z of the pentagonal bipyramid is changed to lysine. The fragment FS-EC-C lacks helix C (residues 196-203) of the EC domain (35).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Amino acid sequences of EF-hand motifs in BM-40 and point mutants. Residues coordinating Ca2+ with side chain oxygen atoms are boxed in black, whereas residues using backbone carbonyl oxygens are boxed in gray (16). The Ca2+ coordination of canonical EF-hands is indicated by letters denoting the position of the coordinating residues in the pentagonal-bipyramid. The glutamic acid at position Z chelates Ca2+ with both oxygens of its side chain. Note that EF1 is a variant of the canonical EF-hand motif. The insertion of His224 induces the coordination of Ca2+ via the backbone carbonyl oxygen of Pro225.

Construction of BM-40 Mutants-- The point mutant EC-EF1-E234K was produced by the method of Deng and Nickoloff (37) using the transformer site-directed mutagenesis kit (CLONTECH) and the cDNA encoding the EC domain of human BM-40 (34). The mutation primer 5'-CCCACACCAAGCTGGCGCCACTGCGT contained the change of the GAG codon for Glu²³⁴ to the AAG codon of lysine. An additional silent T to G mutation resulted in a novel NarI site which was used to identify mutated clones. Mutagenesis was performed as described by the manufacturer (CLONTECH, CA). Production and purification of full-length BM-40, deletion mutants FS-EC and EC, and mutants BM-40-EF2-D257K and BM-40-EF2-E268K was done as described previously (33, 34). cDNA fragments (269 base pairs) containing the mutations in EF2 were obtained by KpnI/XhoI restriction cleavages of BM-40-EF2-D257K and BM-40-EF2-E268K (33) and were cloned into the cDNA of the EC domain resulting in EC-EF2-D257K and EC-EF2-E268K. The double mutant EC-EF1,2-E234K,E268K was produced by subcloning the 136-base pair NcoI/XhoI fragment of EC-EF2-E268K into EC-EF1-E234K fragment. NheI/XhoI fragments of EC-EF1-E234K, EC-EF2-D257K, EC-EF2-E268K, and EC-EF1,2-E234K,E268K were excised and ligated with the NheI/XhoI restricted episomal expression vector pCEP-Pu (38). This vector contains the signal peptide of BM-40 and enables the secretion of recombinant protein into the cell culture media. Correct mutagenesis and cloning was verified by cycle sequencing using the Alfexpress DNA sequencer (Amersham Pharmacia Biotech).

Human embryonic kidney cells (EBNA-293) were transfected with 20 µg of pCEP-Pu vectors using LipofectAMINE (Life Technologies, Inc.) or electroporation. Cells were selected with 0.5 µg/ml puromycin in Dulbecco's modified Eagle's medium/Ham's F-12 medium with 10% fetal calf serum for 14 days as described (38). For purification of proteins from the cell culture media, cells were maintained under serum-free conditions and culture medium was collected every 2 days.

Reverse Transcription-Polymerase Chain Reaction of Transfected Cellular mRNA-- Transfected and nontransfected EBNA-293 cells were trypsinized from confluent plates, and 10⁷ cells were used for isolation of mRNA performed according to the manufacturer's protocol (Amersham Pharmacia Biotech). mRNA concentrations were determined using UV spectroscopy. RT-PCR¹ was performed in a one-tube reaction by addition of 1 unit of reverse transcriptase and 1 unit of Taq polymerase in RT-PCR buffer (0.1 M Tris/HCl, pH 8.4, 0.5 M KCl, 25 mM MgCl₂, 1 mM dithiothreitol, 0.2% gelatin, 6.25% W1, 2.5 mM of each dNTP). After addition of 100 ng of mRNA, 50 pmol of sense primer 5'-GTTCCCAGCACCATGAGGG and 50 pmol of antisense primer 5'-GATCCTCGAGT-TAGATCACAAGATCC reverse transcription was allowed to proceed for 25 min at 37 °C. Five cycles of polymerase chain reaction were then performed at an annealing temperature of 50 °C followed by 30 cycles at an annealing temperature of 65 °C. Denaturation and elongation temperatures were 95 and 72 °C, respectively. Half of the reaction product was subjected to electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. In controls reverse transcriptase was omitted.

Immunoblot of Transfected EBNA-293 Cells-- Transfected EBNA-293 cells of confluent plates (diameter, 10 cm) were washed with phosphate-buffered saline and lysed with 1 ml of 0.5% Triton X-100. Aliquots of serum-free culture medium (1 ml) were precipitated with trichloroacetic acid. The pellets were resuspended in reducing sample buffer. Resuspended pellets and cell lysates were subjected to electrophoresis in 15% SDS-PAGE gels according to Laemmli (39). Proteins were transferred onto nitrocellulose, and blocking was done with 5% milk powder solution. A rabbit polyclonal antiserum against human BM-40 (40) was used as a primary antibody, and detection was performed with a goat antibody against rabbit IgG coupled to peroxidase (DAKO, Denmark) and the enhanced chemoluminescence kit (Amersham Pharmacia Biotech).

Purification of EC-EF1-E234K and EC-EF2-D257K-- Serum-free cell culture media (about 2.5 liters) were dialyzed against 50 mM Tris/HCl, pH 8.6, and subjected to anion exchange chromatography on DEAE-Sepharose. Elution was performed with a linear salt gradient from 0 to 1 M NaCl. After dialysis of pooled fractions, recombinant EC domains were separated on MonoQ (Amersham Pharmacia Biotech) equilibrated in the same buffer. Elution was done with a linear salt gradient (0- 0.5 M NaCl, containing 2 M urea). EC domains eluted in a broad peak between 0.15 and 0.4 M NaCl. Fractions containing the recombinant proteins were pooled and applied to a Sephadex G-75 column equilibrated in 50 mM Tris/HCl, pH 8.6, 150 mM NaCl. Final concentration was achieved on a Resource Q column followed by extensive dialysis against the appropriate buffer.

Circular Dichroism-- Proteins were dialyzed against 5 mM Tris/HCl, pH 7.4, or 50 mM Tris/HCl, pH 7.4, 150 mM NaCl (TBS). Protein concentrations were calculated from the absorption at 280 nm as described (34). For the EC domain and EC domain mutants, an extinction coefficient of 1.39 ml mg¹ cm¹ was used (41). CD spectra in the far UV region were recorded in a thermostated quartz cell of 1-mm optical path length in a Jasco 715 CD spectropolarimeter. Spectra were measured with the protein dissolved in the appropriate dialysis buffer, after adding 2 mM CaCl₂, and after subsequent addition of 3 mM EDTA. Molar ellipticities [] (expressed in degrees cm² dmol¹) were calculated on the basis of a mean residue molecular mass of 110 Da. Ten spectra were accumulated to improve the signal/noise ratio. Spectra of buffers were subtracted. The percentage of change in circular dichroism at 222 nm was calculated as []₂₂₂ = 100 × ([]_Ca  []_EDTA)/[]_Ca with []_Ca representing the signal at Ca²⁺ saturation and []_EDTA representing the CD signal in the presence of excess EDTA.

Fluorimetry and Ca²⁺ Titrations-- Fluorescence was measured with a Perkin-Elmer LS50B spectrophotometer in 10-mm pathlength rectangular cells at 25 °C. Intrinsic fluorescence was excited at 280 nm for all recombinant proteins. Emission maxima were between 333 and 337 nm. Spectra were recorded in TBS in the presence of 2 mM CaCl₂ and after subsequent addition of 4 mM EDTA. The percentage of change in fluorescence intensity at 337 nm was calculated as F₃₃₇ = 100 × (F_Ca²⁺  F_EDTA)/F_Ca²⁺ with F_Ca²⁺ representing the signal at Ca²⁺ saturation and F_EDTA representing the fluorescence signal in the presence of excess EDTA.

Ca²⁺ titrations were performed with the EGTA/CaEGTA buffering system as described by Tsien and Pozzan (42). 100 mM EGTA and 100 mM CaEGTA stock solutions were purchased from Molecular Probes. An equilibrium dissociation constant K_d = 35 nM for the CaEGTA complex was used for the titrations in TBS. Proteins (about 1 µM) were dissolved in buffer containing 10 mM EGTA, and fluorescence intensity was measured. Defined aliquots of the solution were removed and substituted by the same volume of protein dissolved at the same concentration in 10 mM CaEGTA, thus increasing the free Ca²⁺ concentration while keeping the protein concentration constant. This was repeated until finally the fluorescence intensity was measured for protein in 10 mM CaEGTA. Alternatively, proteins were dissolved in buffer containing 10 mM EGTA, and small aliquots of 100 mM CaEGTA stock solutions were added. In this instance, fluorescence intensity was corrected for dilution. Recombinant BM-40 mutants with low affinity for Ca²⁺ (K_d>20 µM) were directly titrated with CaCl₂ in the absence of EGTA. The degree of saturation Y was calculated from the signal S as Y = (S  S₀)/(S_Ca  S₀), where S_Ca and S₀ represent the signals at Ca²⁺ saturation and zero Ca²⁺, respectively.

Data Evaluation-- Two potential mechanisms for binding of Ca²⁺ to BM-40 and its mutants were considered. The single-site model describes the change of the measured signal as a function of the binding of a single Ca²⁺ ion to the protein as shown in the following equation.

(Eq. 1)

The degree of saturation Y is then described by Y = [Ca]/(K_d + [Ca]), where [Ca] represents the free Ca²⁺ concentration and K_d represents the equilibrium dissociation constant. For a protein containing two binding sites, which may be either independent (no cooperativity present) or interacting (cooperativity present), four microscopic equilibrium dissociation constants are needed to fully describe the situation. However, the spectroscopic methods used do not allow for discrimination of Ca²⁺ binding to the individual sites, and thus the four dissociation constants cannot be resolved (44). We therefore used a macroscopic two-site model with two Ca²⁺-binding sites, in which binding of one Ca²⁺ is described by the macroscopic equilibrium dissociation constant K_D1 followed by binding of a second Ca²⁺ with K_D2.

(Eq. 2)

This model also allows detection of cooperativity between the two sites. If positive cooperativity is present, K_D2 will be less than 4 × K_D1, because the second Ca²⁺ ion binds with higher affinity when the first site is occupied than when it is empty. If there is no positive cooperativity, K_D2 will be equal or greater than 4 × K_D1. The degree of saturation can be described by Y = ([PCa] + [PCa₂])/[P_tot] with [PCa] and [PCa₂] representing the concentrations of the corresponding Ca²⁺-bound protein species and [P_tot] representing the total protein concentration. Assuming that contributions to the fluorescence signal are identical for PCa and PCa₂ this equation can be transformed into the following equation.

(Eq. 3)

The program COSY (43) or Grafit (Erithacus Software) were used to fit both models to the experimental data with nonlinear least square fit procedures.

Cooperativity in a two-site system can be characterized by G, which gives the difference in free binding energies between the binding to a given site when the other site is occupied and when the other site is empty. For calculation of this free energy of interaction, G, knowledge of the microscopic equilibrium dissociation constants is required. However, these are not directly accessible from Ca²⁺ titrations. A lower limit of the free energy of interaction G can be calculated from the macroscopic K_D values with G₌₁ = RT ln (4·K_D1/K_D2) (44). G₌₁ is identical to G if the microscopic affinity of both sites is identical. If microscopic affinities are not identical, G is always smaller than G₌₁.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

BM-40 Binds Ca²⁺ with High Affinity and Cooperativity-- The affinity of EF-hands for Ca²⁺ varies for the different cytosolic members of the EF-hand family with dissociation constants (K_d) ranging from nM to µM values. Our first aim was to determine the affinities of the EF-hands of BM-40. We were unable to obtain BM-40 in a sufficiently Ca²⁺-free form either by dialysis against EDTA or Chelex-100 or by chromatography on an EDTA-agarose column (data not shown). We therefore used an EGTA/CaEGTA system (42) to follow Ca²⁺ binding. Briefly, this method relies on the use of two stock solutions of 100 mM EGTA and 100 mM CaEGTA, respectively. The latter is exactly titrated using a pH-metric method. Using different ratios of EGTA and CaEGTA, the free Ca²⁺ concentrations in a solution can be adjusted with high precision to [Ca²⁺]_free = K_d × [CaEGTA/EGTA]. Knowledge of the concentrations of residual Ca²⁺ from buffer solutions and bound to proteins (usually between 1-20 µM) is not necessary because this is buffered by the excess EGTA (normally 10 mM) in the system. For a residual Ca²⁺ concentration of 20 µM the error in the calculated free Ca²⁺ concentration is less than 2%.

The reversible change in intrinsic fluorescence of full-length BM-40 (36) was used to monitor Ca²⁺ binding. A simple model of one Ca²⁺ ion binding to BM-40 could not sufficiently describe the measured values, which evidently follow a curve with a steeper slope around the midpoint than predicted from the single-site model. An appropriate description was achieved with the two-site model, which enabled the determination of two macroscopic K_D values of K_D1 = 490 nM and K_D2 = 57 nM, respectively (Fig. 2A). The fact that K_D2 is much smaller than 4 × K_D1 clearly demonstrates that positive cooperativity is present because of interaction between the sites.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Ca2+ binding of BM-40 and its fragments. Ca2+ binding was monitored by intrinsic fluorescence with excitation at 280 nm and emission at 337 nm. The fraction Y of the total change of flurorescence intensity is plotted against the free Ca2+ concentration adjusted with the EGTA/CaEGTA system. A, for full-length BM-40 two macroscopic constants KD1 = 490 nM and KD2 = 57 nM were obtained from the best fit of the model with two binding sites to the experimental data (solid line). Fitting a model with only one binding site was insufficient (dashed line). B, comparison of Ca2+ titrations for full-length BM-40 (), the FS-EC domain pair (), and the EC domain (). Best fit curves are shown as solid lines with KD1 = 470 nM and KD2 = 26 nM for FS-EC domain pair and KD1 = 11750 nM and KD2 = 56 nM for the EC domain. In addition, the curve calculated with Kd = 870 nM for a model with one binding site in the EC domain is shown as a dashed line.

Domain Interactions with the FS Domain Influence Ca²⁺ Affinity-- The change in fluorescence of the deletion mutant FS-EC was also investigated in the EGTA/CaEGTA system. Cooperative high affinity binding of Ca²⁺ to FS-EC was observed (Fig. 2B). The affinity of FS-EC was very similar to that of full-length BM-40 with macroscopic K_D values of 470 and 26 nM. This indicates that the acidic domain I does not significantly influence Ca²⁺ binding to the EC domain. In contrast, removal of both domain I and the FS domain, resulting in the isolated EC domain, strongly decreased Ca²⁺ affinity. Macroscopic K_D values of 11750 and 56 nM were obtained as best fit parameters for the EC domain (Fig. 2B). Ca²⁺ affinity is on average 6-fold lower if one compares (K_D1 K_D2)^0.5, which corresponds to a loss in total free binding energy of 9.9 kJ/mol (Table I).

Point Mutations in EF2 Disturb Folding and Secretion of the EC Domain-- To investigate the influence of the individual EF-hands on Ca²⁺ affinity and cooperativity we mutated the conserved glutamic acid at position Z to lysine, a mutation recently shown to strongly decrease affinity of EF-hands (45-48). Because the two disulfides of the EC domain are essential for proper folding, all proteins were expressed in a eukaryotic expression system. When analyzing cell culture supernatants of transfected EBNA-293 cells recombinant EC domains with mutations EC-EF1-E234K and EC-EF2-D257K were readily seen in an immunoblot (Fig. 3). However, EC-EF2-E268K as well as the double mutant EC-EF1,2-E234K,E268K could not be detected in the medium (Fig. 3). The presence of similar amounts of endogenous BM-40 shows that the absence of the two mutants was not caused by cell death or a general inhibition of the secretory pathway. To prove successful transfection and selection of the EBNA-293 cells, we isolated mRNA from transfected cells and performed RT-PCR with primers specific for the recombinant EC domain. For all transfected cell lines the 540-base pair cDNA fragment could be amplified demonstrating the presence of the respective mRNAs (Fig. 4A). mRNA of endogenous BM-40 was not amplified as the reverse primer partially spanned noncoding region of the vector sequence. Next we prepared cell lysates of the nonsecreting EC-EF2-E268K and EC-EF1,2-E234,268K cells. In an immunoblot a faint band for both proteins could be detected (Fig. 4B). The intensity of the bands was comparable with that of the endogenous BM-40 and presumably represents the small amount of protein that is translated and processed in the endoplasmic reticulum and/or Golgi apparatus.

View larger version (74K): [in this window] [in a new window]   Fig. 3.   Immunoblot of cell culture media. 1 ml of serum-free cell culture medium transfected with EC-EF1-E234K, EC-EF2-E268K, EC-EF2-D257K, EC-EF1,2-E234K,E268K, or from untransfected cells (denoted EBNA 293) were separated by 15% SDS-PAGE under reducing conditions, blotted onto a nitrocellulose membrane, and stained with an anti-BM40 antiserum. Molecular masses of protein standards are given in kDa. The arrowhead marks the endogenous BM-40, and the arrow marks the EC domain.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   RT-PCR of mRNA from untransfected and transfected cells (A) and immunoblot of cell lysates (B). A, mRNA was isolated from untransfected (EBNA 293) cells and cells transfected with EC-EF2-E268K, EC-EF1,2-E234K,E268K, and EC-EF2-D257K. Reverse transcription and PCR were performed with primers specific for the EC domain. Reaction products were separated by agarose gel electrophoresis and stained with ethidium bromide. B, cell lysates of transfected EC-EF2-E268K and EC-EF1,2-E234K,E268K EBNA-293 cells were separated by 15% SDS-PAGE under reducing conditions, blotted onto a nitrocellulose membrane, and stained with an antiserum against BM40. The arrowhead marks the endogenous BM-40, and the arrow marks the EC domain.

Point Mutations in EF-hands Abolish Cooperativity of Ca²⁺ Binding-- Both EC-EF1-E234K and EC-EF2-D257K were produced and could be purified to homogeneity from cell culture media (Fig. 5), even though expression levels were decreased compared with the nonmutated EC domain. The proteins eluted in broad peaks from ion exchange columns, and as a consequence, yields were very low (<25 µg/liter of medium). Nonetheless, circular dichroism spectra demonstrated that the mutated EC domains in the Ca²⁺-free form had similar folds to that of the nonmutated EC domain (Fig. 6). Upon addition of Ca²⁺, the EC domain showed a 39% decrease in molar ellipticity at 222 nm, whereas only a 28% decrease was observed for EC-EF1-E234K. The conformational change was even more compromised in mutant EC-EF2-D257K, where the signal changed by only 10%.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   SDS-PAGE of the purified EC domain mutants. Electrophoresis of the purified proteins EC-EF1-E234K and EC-EF2-D257K was performed under nonreducing conditions. The gel was stained with Coomassie Brillant Blue. Molecular masses of protein standards are given in kDa.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Conformation of mutated EC domains. Far UV circular dichroism spectra of the EC domain and its mutants EC-EF1-E234K and EC-EF2-D257K were recorded in 5 mM Tris/HCl, pH 7.4. Spectra were measured in the presence of 2 mM Ca2+ (solid line) and after addition of 3 mM EDTA (dashed line).

In fluorescence studies, EC, EC-EF1-E234K, and EC-EF2-D257K showed emission maxima at about 340 nm when excited at their excitation maxima at 280 nm (data not shown). Titration of EC-EF1-E234K and EC-EF2-D257K revealed no change in fluorescence at submicromolar Ca²⁺ concentrations. However, changes in intrinsic fluorescence intensities could be observed when Ca²⁺ concentrations were raised significantly (Fig. 7). In contrast to results obtained with the EC domain, analysis of Ca²⁺ titrations of EC-EF1-E234K and EC-EF2-D257K revealed no sign of cooperativity. A model assuming a single binding site with K_d = 45 µM for EC-EF1-E234K and K_d = 205 µM for EC-EF2-D257K, respectively, could adequately describe the data (Fig. 7).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Ca2+ binding to EC domains with mutated EF-hands. Ca2+ binding was monitored by intrinsic fluorescence as described in the legend to Fig. 2. Macroscopic constants KD1 = 11750 nM and KD2 = 56 nM resulted from the best fit of the model with two binding sites to the experimental data of wild type EC domain (). Best fit curves for EC-EF1-E234K () and EC-EF2-D257K () were obtained with a model with one binding site. Corresponding dissociation constants were Kd = 45 µM and Kd = 205 µM, respectively. The loss of cooperativity in the mutants is evident from the reduced slope of the binding curves.

The mutations EF2-D257K and EF2-E268K have been introduced previously into full-length BM-40 (BM-40-EF2-D257K and BM-40-EF2-E268K) (33). To analyze cooperativity in the context of the full-length protein, we reinvestigated Ca²⁺ binding to these mutants. The fluorescence change upon Ca²⁺ addition could be described with a model assuming a single binding site, and a K_d = 0.22 µM for BM-40-EF2-D257K was evaluated (Fig. 8), confirming the earlier results (33). For BM-40-EF2-E268K, the Ca²⁺ affinity was clearly decreased and not cooperative (K_d = 1.2 µM). In the mutant FS-EC-C, the helix C is lacking in the EC domain. This mutation leads to enhanced collagen binding to the EC domain (35). The structure of FS-EC-C was recently solved and showed two Ca²⁺ ions bound to the EF-hands (35). The affinity of FS-EC-C for Ca²⁺ was 10-fold reduced relative to FS-EC when (K_D1K_D2)^0.5 are compared, but the cooperativity between the sites was enhanced by the deletion (Fig. 8 and Table I).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Ca2+ binding to full-length BM-40 with mutated EF-hands. Ca2+ binding was monitored by intrinsic fluorescence as described in the legend to Fig. 2. Dissociation constants KD1 = 490 nM and KD2 = 57 nM resulted from the best fit of the model with two binding sites to the experimental data for full-length BM-40 (). The best fit curve for BM-40-EF2-D257K () assumed one binding site with Kd = 220 nM. Dissociation constants KD1 = 21540 nM and KD2 = 52 nM were obtained for FS-EC-C (). The best fit curve for BM-40-EF2-E268K () assumed one binding site with Kd = 1200 nM. The loss of cooperativity in the mutants EF2-D257K and EF2-E268K is evident from the reduced slope of the binding curves.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytosolic EF-hand proteins are designed to fulfill different functions at low Ca²⁺ concentrations in the resting cell (about 0.1 µM Ca²⁺) and when the cell has been activated (about 10 µM Ca²⁺). Their affinity for Ca²⁺ is accordingly tuned to this concentration range, allowing the proteins to switch from Ca²⁺-free to Ca²⁺-bound conformations upon activation. BM-40 is the only example so far of an extracellular protein containing EF-hands of which the three-dimensional structure is known. The role of Ca²⁺-binding to extracellular EF-hands is rather mysterious, because the extracellular free Ca²⁺ concentration is high (about 1.2 mM) and believed to be constant (49). The experiments presented here were designed to shed some light on the possible function(s) of extracellular EF-hands.

We have used a Ca²⁺ buffering system that allows precise adjustment of Ca²⁺ concentrations in the nanomolar range to examine Ca²⁺ binding by BM-40. The two EF-hands in BM-40 were found to bind Ca²⁺ with a free energy of 77.3 kJ/mol. We used two models for analyzing the binding data. The first model assumes a single Ca²⁺ binding site and is described by the microscopic equilibrium dissociation constant K_d. The second model assumes two binding sites and is described by two macroscopic dissociation constants, K_D1 and K_D2. The values obtained for K_D1 and K_D2 allow to detect positive cooperativity between the sites. If Ca²⁺ binding to any site is independent from the occupation of the other site (i.e. no cooperativity), K_D2 will be equal or greater than 4 × K_D1. If positive cooperativity is present between the sites, K_D2 is smaller than 4 × K_D1 because the second Ca²⁺ ion binds with higher affinity when one site is already occupied by a Ca²⁺ ion. This model is an alternative to the use of the Hill equation but allows more detailed insight into the binding process and the energetics of binding (44).

The experimental results for calcium binding to BM-40 could only be fitted satisfactorily with the two-site model. The macroscopic dissociation constants, K_D1 = 490 nM and K_D2 = 57 nM, demonstrate that binding of the second Ca²⁺ ion is facilitated by positive cooperativity. A minimum interaction energy of 8.8 kJ/mol can be calculated from these values (Table I). Because a signal specific for EF1 or EF2 cannot be obtained by circular dichroism and fluorescence spectroscopy, it is not possible to know whether the EF-hands become occupied sequentially in a defined order or whether they bind Ca²⁺ in parallel. The macroscopic dissociation constants can therefore not be attributed to the individual EF-hands.

The failure to detect cooperative binding in our previous studies (33, 34) is now seen to have been caused by incomplete removal of Ca²⁺ from BM-40 and buffer solutions. Thus, Ca²⁺ titrations were started with partially saturated proteins, and only the second halves of the saturation curves were recorded. At the time, only one binding site had been predicted for BM-40 (32, 50), and we therefore used a simple single-site model for data evaluation, which happened to describe the experimental data reasonably well. Of course, the data could also be fitted with more complex models including cooperativity, but these models did not produce a significantly better fit. In the present study, high accuracy in the low nanomolar range could be achieved through the use of the EGTA/CaEGTA system, finally allowing the single-site and the two-site model with cooperativity to be clearly distinguished.

BM-40 is half-saturated at a free Ca²⁺ concentration of 170 nM. As already mentioned, free Ca²⁺ concentrations in serum and several other extracellular tissue fluids are much higher (1.2 mM), but local gradients or changes in the extracellular Ca²⁺ concentration down to 0.1 mM have been observed (49, 51, 52). Nevertheless, even at the lowest extracellular Ca²⁺ concentrations reported to date, the EF-hands in BM-40 will be in the fully Ca²⁺-saturated form. This indicates that Ca²⁺ bound to the EF-hands in BM-40 is likely to play a structural rather than a regulatory role. It is known that the structure maintained by Ca²⁺ is essential for the binding of BM-40 to collagens, and the collagen-binding site has indeed been mapped to the EC domain (34, 35).

Nearly identical Ca²⁺ binding profiles were observed for the FS-EC domain pair and for full-length BM-40, showing that the acidic N-terminal domain I does not influence the affinity and cooperativity of Ca²⁺ binding to the EC domain. In marked contrast, when both domain I and the FS domain were deleted the free energy of Ca²⁺ binding was increased by 7.8 kJ/mol to 69.5 kJ/mol, demonstrating a strong interaction between the FS and EC domains. The first macroscopic dissociation constant, K_D1, was increased about 20-fold compared with full-length BM-40, whereas the second constant, K_D2, was not affected. Interestingly, the cooperativity between the EF-hands, as measured by G, is higher in the isolated EC domain relative to FS-EC and full-length BM-40 (Table I). Presumably, the strong interaction between the FS and EC domain in FS-EC restricts the conformational changes in the EF-hands that accompany Ca²⁺ binding, whereas in the isolated EC domain binding of the first equivalent of Ca²⁺ leads to a relatively larger rearrangement. The second equivalent then binds to similar structures in both FS-EC and EC, as evidenced by a similar value of K_D2.

The interface between the FS and EC domain is revealed in the structure of the FS-EC domain pair (16). The relatively small domain interface (550 Å²) is largely formed by -strand 5 of the FS domain and -helix E of the EC domain, the second helix of EF1 (Fig. 9). A further contact between the FS domain and EF1 involves His⁶² and His²²⁴, which participates in the unusual cis-peptide bond in EF1. There are no direct contacts between the FS domain and EF2. A comparison of the EF-hand structures in FS-EC domain pair and the isolated EC domain does not reveal any significant differences that could explain the large effect of the FS domain on Ca²⁺ binding. This is perhaps not surprising, because both structures were determined under saturating concentrations of Ca²⁺. The corresponding Ca²⁺-free forms have so far defied a structure determination, but we assume that because of the intimate contacts between the FS domain and EF1, the EF-hands in Ca²⁺-free FS-EC adopt a different structure than in the Ca²⁺-free EC domain. EF1 is presumably predisposed for Ca²⁺ binding in FS-EC, perhaps because the cis-trans equilibrium of the His²²⁴-Pro²²⁵ peptide bond is pushed toward the cis-conformation required for Ca²⁺ binding or because Glu²³⁴ is positioned appropriately. It is thus tempting to associate K_D1 in the interpretation of our binding data with EF1 and K_D2 with EF2, although we stress again that the macroscopic dissociation constants are not necessarily linked to microscopic sequential events.

View larger version (68K): [in this window] [in a new window]   Fig. 9.   Structure of the interface of FS and EC domains. The interface between the FS domain (green) and the EC domain (light blue) is shown, based on the crystal structure of FS-EC (16). The calcium ions are shown as pink spheres. The disulfide bond between Cys256 and Cys272 is in yellow. Residues involved in the domain interface are shown as atomic models, as are the glutamic acids Glu234 and Glu268 at position Z in EF1 and EF2, respectively. The helices in the EF-hands are labeled D-G. This figure was created with BOBSCRIPT (53, 54) and RASTER3D (55).

To analyze the contributions of individual EF-hands to Ca²⁺ binding, we replaced the conserved glutamic acid at position Z in each EF-hand by lysine (Fig. 1). Although the mutant EC-EF1-E234K was readily secreted from the human kidney cells used for recombinant expression, the EC domain with the corresponding mutation in the second EF-hand, EC-EF2-E268K, as well as the double mutant EC-EF1,2-E234K,E268K, could not be detected in the cell culture supernatant. However, the corresponding mRNA as well as small amounts of both proteins were present in cellular extracts (Fig. 4). Apparently, mutation of the bidentate Ca²⁺ ligand Glu²⁶⁸ in EF2 interfered with proper folding in the endoplasmic reticulum, resulting in protein degradation. These results point to a function of EF-hands in the folding and secretion of extracellular EF-hand proteins. It remains to be elucidated whether chaperones are involved in this process. To determine whether Ca²⁺ binding to EF2 is strictly required for folding and secretion of BM-40, we produced mutant EC-EF2-D257K, in which the aspartic acid at position X was replaced by lysine. This mutant was secreted from the cells. No cooperativity could be detected in the Ca²⁺ titrations, and a model assuming a single binding site with a dissociation constant of K_d = 205 µM fitted the data well. In the mutant EC-EF1-E234K, cooperativity was also lost, and a dissociation constant of K_d = 45 µM was obtained. Most probably, the mutations decreased the Ca²⁺ affinity of the affected site to such an extent that binding could no longer be detected at the Ca²⁺ concentrations used. The remaining Ca²⁺ binding thus represents binding to the nonmutated EF-hand when the other hand in the pair is mutated. The dramatic, several 100-fold reduction in Ca²⁺ affinity of the nonmutated EF-hand is additional evidence for a strong coupling between the two EF-hands, which underlies the cooperativity of Ca²⁺ binding. Further insight into the energetics of Ca²⁺ binding can be gained by comparing the effects of mutations in the EC domain with those in full-length BM-40. Although the EF2-D257K mutation strongly decreased the Ca²⁺ affinity when introduced in the EC domain, the same mutation had only a small effect when introduced in full-length BM-40 (Table I), supporting the interpretation that the FS domain increases Ca²⁺ affinity in the EC domain mainly via interactions with EF1. Interestingly, the interaction of the FS and EC domains allows the mutant BM-40-EF2-E268K to be folded and secreted, whereas the same mutation in the isolated EC domain abolished secretion.

In conclusion, our results show that the EF-hands in the extracellular protein BM-40 bind Ca²⁺ cooperatively and with high affinity, just like their counterparts in cytosolic proteins. However, an additional layer of complexity is introduced by the presence of the FS domain, which physically interacts with the variant EF-hand 1 and, therefore, is intimately involved in Ca²⁺ binding to the EC domain.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant Ma 1932/1-1 (to P. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Wellcome Trust Senior Research Fellow.

^** To whom correspondence should be addressed: Inst. for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany.

Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M001770200

	ABBREVIATIONS

The abbreviations used are: RT, reverse transcription; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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