HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Research Data Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Atreya, H. S. Articles by Luchinat, C. Search for Related Content PubMed PubMed Citation Articles by Atreya, H. S. Articles by Luchinat, C. Social Bookmarking                   What's this?

Structural basis for sequential displacement of Ca²⁺ by Yb³⁺ in a protozoan EF-hand calcium binding protein

¹ Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai-400005 India
² Centro Risonanze Magnetiche, University of Florence, Florence, Italy

Reprint requests to: K.V.R. Chary, Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai-400005, India; e-mail: chary{at}tifr.res.in; fax: 0091 (22) 2152110.

(RECEIVED July 26, 2002; FINAL REVISION November 14, 2002; ACCEPTED November 18, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0225603.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
We have studied the displacement of Ca²⁺by the trivalent lanthanide ions (Yb³⁺) in a protozoan (Entamoeba histolytica) Ca²⁺-binding protein (EhCaBP), by NMR and thermodynamics. We have demonstrated, for the first time, how one can use in a combined fashion the utility of NMR and thermodynamics to have an insight to the relative binding specificities/affinity between Ca²⁺ and Yb³⁺. As revealed by the titration experiments, Yb³⁺ displaces Ca²⁺ from the four metal binding sites present in EhCaBP in a sequential manner. The study provides a structural origin for such a sequential Ca²⁺ displacement by Yb³⁺ in EhCaBP.

Keywords: EF-hand protein; calmodulin; EhCaBP; pseudocontact shifts; chemical shifts; ytterbium

Abbreviations: NMR, nuclear magnetic resonance • ITC, isothernmal calorimetry • HSQC, heteronuclear single quantum coherence • EhCaBP, Entamoeba histolytica calcium binding protein • CaM, calmodulin • TnC, Troponin C

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
EF-hand proteins belong to a growing family of calcium (Ca²⁺) binding proteins. More than 1000 distinct primary sequences in this class of proteins are known and cataloged into 66 different subfamilies (Kawasaki et al. 1998). These proteins have been a subject of great interest for structural biologists, resulting in the availability of more than 100 three-dimensional (3D) structures, as of today (Nelson and Chazin 1998). They bind cooperatively to Ca²⁺ in the subnanomolar to millimolar range and function as signal transducers or modulators (Lewit-Bentley and Réty 2000). A few EF-hand proteins have also been proposed to be involved in extracellular functions such as cell migration, differentiation, and association (Hohenester et al. 1997). The canonical Ca²⁺-binding motif (hereafter referred to as the Ca²⁺binding loop) in these proteins consists of a contiguous 12-residue loop flanked by two helices, forming the so-called "EF-hand motif" (Kretsinger and Nockholds 1973; Strynadka and James 1989). Within the Ca²⁺-binding loop, residues that are involved in coordination with Ca²⁺ are 1, 3, 5, 7, 9, and 12, forming a pentagonal bi-pyramidal geometry (Strynadka and James 1989), as shown below (highly conserved residues are shown and ±X, ±Y, ±Z refer to the vertices of the pentagonal bi-pyramid):

Out of these, residues at 1, 3, 5, and 12 position coordinate directly to Ca²⁺ via their side-chain carboxylate groups, with position 1 and 12 being an invariant Asp and Glu, respectively (Strynadka and James 1989; Falke et al. 1994). Residue at position 7 coordinate to Ca²⁺ via its backbone carbonyl group, whereas residue at position 9 coordinates to Ca²⁺ indirectly through an intervening water molecule (Strynadka and James 1989).

In recent years, there has been a growing interest with regard to the factors that govern Ca²⁺-binding affinities in EF-hand proteins (Linse and Forsén 1995). This is primarily due to the fact that a wide range of Ca²⁺ binding affinities/specificities has been found among these proteins despite the strong conservation in their primary sequence of Ca²⁺-binding loops (Linse and Forsén 1995). Several studies (Moeschler et al. 1980; Imaizumi et al. 1990; Linse and Forsén 1995; Gopal et al. 1997; Gilli et al. 1998) have concentrated on understanding Ca²⁺ binding affinity/specificity in these proteins against Mg²⁺, which is the only other competitor for Ca²⁺ inside the living cell. Models based on the primary sequence of the Ca²⁺-binding loop have been proposed to explain the origin of differences in Ca²⁺ binding affinities/specificities (Mardsen et al. 1988; Waltersson et al. 1993; Wu and Reid 1997). For example, in one of the studies, the presence of neighboring Asp residues at either the +X and +Y, the +Y and +Z, or the +X, +Y and +Z metal ion coordinating positions have been shown to lower metal binding affinity due to dentate–dentate repulsion (Mardsen et al. 1988). In another study, the side chain of the ligating residue at position +Y in the Ca²⁺-binding loop has been shown to be important (Waltersson et al. 1993). In yet another study, EF-helices that flank the Ca²⁺-binding loops have also been proposed to modulate the Ca²⁺-binding affinity in this class of proteins (Sharma et al. 1997).

In the majority of these studies, the target protein/peptide has been taken in the Ca²⁺-free (apo) form. Very few studies have focussed on the displacement of Ca²⁺ in a Ca²⁺-saturated (holo) protein by other metal ions. Such studies yield information on the relative specificity and strength with which Ca²⁺ is bound to the protein. Tri-positive lanthanide ions (Ln³⁺) serve as ideal candidates for the displacement of Ca²⁺ in a protein owing to their similar ionic radii as Ca²⁺ (Shannon 1976). Such a study yields valuable information on the relative binding affinities of Ca²⁺ and Ln³⁺ with the protein under investigation. Lanthanides such as Tb³⁺ and Eu³⁺ possess optical properties and facilitate structural studies in substituted proteins using fluorescence and UV-Vis absorption spectroscopy (Horrocks and Sudnick 1981; Wu et al. 1996). Another favorable property of paramagnetic Ln³⁺ is their large anisotropic magnetic susceptibility, which has been harnessed in NMR studies of Ln³⁺-substituted proteins (Bertini et al. 2001a). The large pseudocontact shifts, observed for residues as far as 40 Å from the metal center, have been used for 3D structure refinement from NMR data (Allegrozzi et al. 2000). On the other hand, residual dipolar couplings that arise due to the partial alignment of proteins containing Ln³⁺ in high magnetic field have been used as further refinement tool (Bertini et al. 2001b) and to assess domain alignment in multidomain EF-hand proteins such as calmodulin, or CaM (Biekotsky et al. 1999). However, there have been very few studies that have aimed at understanding the binding affinity/ specificity of Ln³⁺ to these proteins. To characterize the factors that govern binding of Ln³⁺ to EF-hand proteins and harness their favorable magnetic properties for structural studies by NMR, we have initiated Ln³⁺-substitution studies in an EF-hand Ca²⁺-binding protein from Entamoeba histolytica (hereafter referred to as EhCaBP). A high-resolution 3D structure of this protein in solution has been recently deposited in the Protein Data Bank (Atreya et al. 2001).

EhCaBP is a 15-kD (134 amino acid residues) monomeric protein containing four canonical EF-hand Ca²⁺binding loops. The overall structural topology of EhCaBP resembles that of CaM and Troponin C (TnC) with two globular domains (the N- and C-terminal) connected by a flexible eight amino acid linker (Sahu et al. 1999a; Atreya et al. 2001). In this article, we present the NMR and thermodynamics study of Ca²⁺ displacement by Yb³⁺ in EhCaBP. Yb³⁺ has been chosen as the paramagnetic probe owing to its large pseudocontact shift to Curie linebroadening ratio (Bertini et al. 2001d). We have used isothermal titration calorimetry (ITC) to obtain binding affinities and free energy changes upon substitution of Ca²⁺ by Yb³⁺. To the best of our knowledge, this is the first study involving a combined use of NMR and thermodynamics to characterize lanthanide binding in EF-hand proteins.

	Results

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
NMR experiments
The titration of uniformly ¹⁵N-labeled EhCaBP (hereafter refered to as [u-¹⁵N] sample) and Alanine, Leucine, and Lysine selectively ¹⁵N-labeled EhCaBP (hereafter referred to as [ALK]-¹⁵N sample) was carried out with Yb³⁺ and the changes in 2D [¹⁵N-¹H] heteronuclear single quantum correlation (HSQC) spectrum were monitored. During the course of titration, a number of changes were noticed in the HSQC spectrum. Several new signals appeared all over the HSQC spectrum. Several of the original peaks were broadened due to the enhancement of relaxation rates induced by the paramagnetic ions (Yb³⁺), while several others remained unaffected in their line widths during the initial steps of titration. It should be noted that when Yb³⁺ is introduced in a protein, the cross peak of any specific residue undergoes a pseudocontact shift if it lies in a distance range of 10–25 Å of Yb³⁺, while it is expected to completely broaden out if it is located within10 Å of Yb³⁺ (Allegrozzi et al. 2000).

The amide (¹H^N) protons of Gly residues present at the sixth position of the four internally highly homologous Ca²⁺ binding polypeptide stretches in EhCaBP (Scheme 1), exhibit a characteristic downfield shift in the Ca²⁺-bound state (holo) of the protein (Akerfeldt et al. 1996). This is the result of their involvement in hydrogen bonding with the side-chain carboxyl oxygen atom (C'O) of an invariant Asp at the first position of respective Ca²⁺ binding loops (shown as bold letters in Scheme 1 with numbering shown on top).

View larger version (8K): [in this window] [in a new window]   Scheme 1

Up to a Yb³⁺:EhCaBP ratio of 1.0
During the initial stages of titration, [Yb³⁺]/[EhCaBP] ratio of (hereafter referred to as the metal:protein ratio) of 0.0–1.0, the most important observation is the gradual disappearance of cross peaks belonging to Sites III and IV of EhCaBP and the gradual appearance of new pseudocontact shifted cross peaks, whereas cross peaks arising from residues at Sites I and II, are almost unaffected. As an illustrative example of this observation, Figures 1A and 1B, and 2 show selected regions of the 2D [¹⁵N-¹H] HSQC spectrum. Assignment of pseudocontact shifted peaks for residues in Site IV was obtained based on the combined use of the 2D [¹⁵N-¹H] HSQC spectra recorded on both [u-¹⁵N] and [AKL]-¹⁵N EhCaBP samples as shown in Figure 3. As evident from Figure 3A, selective labeling of Ala, Leu, and Lys resulted in a dramatic simplification of the spectrum, which aided in unambiguous resonance assignments of the pseudocontact shifted peaks of residues belonging to Site IV. As an illustrative example, Figure 3B shows the assignment of pseudocontact shifted cross peaks of I107 and L126. No pseudocontact shifted peaks were observed for any of the residues belonging to Site III. To have an insight of these observations more vividly, Figure 4 shows the normalized volumes of cross peaks plotted for a few amino acid residues (both original and pseudocontact shifted peaks) in all the four metal binding sites of EhCaBP. These plots indicate the percentage of various species present up to a metal:protein ratio of 1.0. These observations taken together reveal that Yb³⁺ ions bind first to the C-terminal domain and particularly to the Site III of EhCaBP, resulting in complete broadening of cross peaks for residues belonging to this site. Simultaneously, pseudocontact shifts are observed for residues in Site IV, which lie within 10–25 Å of Site III. Thus, as the titration approaches a metal:protein ratio of 1.0, the third binding site is almost completely filled by the Yb³⁺ ions and a majority of (Ca²⁺)₄-EhCaBP transforms into (Ca²⁺)(I)-(Ca²⁺)(II)-(Yb³⁺)(III)-(Ca²⁺)(IV)-EhCaBP.

View larger version (27K): [in this window] [in a new window]   Figure 1. (A–F) Selected region of 2D [15N-1H] HSQC spectrum of EhCaBP showing G15, G51, G90, T98, and G122 peaks at different metal:protein ratios. The metal:protein ratio is indicated at top left in (A–F). The arrow mark in (B) and (C) points to the appearance of pseudocontact shifted peaks for G122 and G15, respectively.

View larger version (24K): [in this window] [in a new window]   Figure 2. Illustrative examples of pseudocontact shifted cross peaks for I107, A118, G120, G122, and L126 belonging to the Site IV of EhCaBP at a metal:protein ratio of 0.4.

View larger version (21K): [in this window] [in a new window]   Figure 3. (A) Superposition of 2D [15N-1H] HSQC spectra of [u-15N] (blue) and [ALK]-15N EhCaBP (red; see text). (B) An illustrative example of how the ambiguity in the assignment of pseudocontact shifted cross peaks of I107 and L126 belonging to Site IV could be overcome by comparing the 2D [15N-1H] HSQC spectra of [u-15N] labeled (left) and [ALK]-15N labeled (right) EhCaBP.

View larger version (26K): [in this window] [in a new window]   Figure 4. Plot of normalized volumes of original as well as pseudocontact shifted (PCS) cross peaks for three amino acid residues each in (A) Site I, (B) Site II, (C) Site III, and (D) Site IV of EhCaBP as a function of the metal:protein ratio.

View larger version (16K): [in this window] [in a new window]   Figure 5. Plot of normalized volumes of (A) the original and pseudocontact shifted (PCS) cross peaks for G15, and (B) the original cross peak of G51 as a function of the metal:protein ratio. These plots represent, respectively, the intensity profiles of cross peak volumes for residues in Sites I and II. The integral volumes of pseudocontact shifted cross peak of G15 plotted in (A) have been normalized with respect to the volume of its original cross peak at a metal:protein ratio of 0.

View larger version (27K): [in this window] [in a new window]   Figure 6. Plot of normalized volumes of original as well as pseudocontact shifted (PCS) cross peaks for A118 and G122 belonging to Site IV as a function of the metal:protein ratio. The integral volumes of the pseudocontact shifted cross peak of G122 and A118 have been normalized with respect to the volume of their original cross peaks at a metal:protein ratio of 0.

ITC measurements
A typical calorimetric reaction of the addition of aliquots of Yb³⁺ ligand to EhCaBP is shown in Figure 7A along with its Q versus the metal:protein ratio in Figure 7B. Analysis of the data using the ligand-binding model by Wisemen et al. (1989), carried out using ORIGIN software, was made assuming the presence of four binding sites for Yb³⁺ in the protein. The fitting reveals three sites of endothermic in nature, while one site exhibits an exothermic behavior for the Ca²⁺ displacement by Yb³⁺. By varying the initialization parameters used to begin the fitting procedure, it was determined that the fit was stable and no other parameter set could be found that provided a good fit to the data. Binding constants along with enthalpy, entropy, and free energy of binding are given in Table 1. The endothermic nature of Ca²⁺ displacement by Yb³⁺ is obvious considering the fact that the hydration enthalpy of Yb³⁺ is higher than Ca²⁺ (Marcus 1985). This implies that energy released on Ca²⁺ hydration is not sufficient to break the Yb³⁺ hydration shell to bind to the protein, resulting in heat being absorbed from the solution. The binding constants represent the following displacement reaction:

View larger version (26K): [in this window] [in a new window]   Figure 7. (A) A calorimetric titration of 1.2-µL aliquots of 20 mM YbCl3 solution into 0.24 mM Ca2+-loaded EhCaBP at 308 K. (B) Plot of kcal/mole of heat absorbed/released per injection of YbCl3 as a function of the metal:protein ratio at 308 K and the best least-squares fit of the data to binding models described in the text.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
EF-hand proteins maintain specificity for Ca²⁺ against its strong competitor Mg²⁺, which is present in 1000-fold excess inside the cell. In the past, binding affinity of Ca²⁺ in EF-hand proteins has been shown to be largely determined by the amino acid composition of Ca²⁺-binding loops (Mardsen et al. 1988; Waltersson et al. 1993; Wu and Reid 1997), although there have been few instances in which the helices flanking Ca²⁺-binding loops also have been shown to influence the binding affinity (Sharma et al. 1997). The thermodynamics study of Ca²⁺-binding in EhCaBP carried out earlier showed that IV site has the highest affinity for Ca²⁺ (Mardsen et al. 1988). On the other hand, the same study showed that there are two low-affinity sites for Ca²⁺, both of which are exothermic in nature and two high affinity sites for Ca²⁺, of which one is endothermic and one is exothermic. It also revealed that both the N-terminal Sites I and II in EhCaBP bind Ca²⁺ 100-fold less strongly compared to Site IV. In the present study, we rationalize the binding affinity of Yb³⁺ to EhCaBP. We restrict the discussion to the Gly markers present at the sixth position of each of the four Ca²⁺-binding loops of EhCaBP, although intensity profiles of other residues (shown in Figs. 3–6) have also been considered in the analyses. It is also assumed throughout the study that the two domains in EhCaBP do not show any interdomain cooperativity, and hence, bind metal independently.

As evident from Scheme 1, the third Ca²⁺-binding loop (Site III) in EhCaBP possesses four charged residues (D85, D87, D89, and E96) that coordinate to Ca²⁺ as opposed to only three in the other three loops (I, II, and IV). Hence, Site III is expected to be the most preferred site for tripositive Yb³⁺, in EhCaBP (Biekofsky et al. 1999). Also, out of the four Ca²⁺-binding loops present in EhCaBP, the third loop structurally diverges the most from the corresponding loops of its homologous proteins, CaM and TnC (Atreya et al. 2001; Fig. 8). Such a divergence of the third Ca²⁺-binding loop of EhCaBP could be attributed to the presence of a Tyr residue at position -4 with respect to the third loop, instead of a highly conserved Phe (Atreya et al. 2001). Previously, it has been shown that, in a given domain containing a pair of EF-hands, Phe at -4 position with respect to the first EF-hand loop (F[-4]) interacts with yet another conserved Phe at the 13^th position of the second EF-hand loop (F[13]), such that their respective aromatic rings are oriented perpendicular to each other (Rashidi et al. 1999; Fig. 9). Such an orientation results in their C-C distance of 7–8 Å (Rashidi et al. 1999). Further, proteins that lack such Phe-Phe interactions due to the absence of F(-4) or/and F(13), are found to have lower affinity for Ca²⁺ (Chandra et al. 1994; Ohya and Botstein 1994). In both CaM and TnC and in the N-terminal domain of EhCaBP, the respective Phe aromatic rings are oriented perpendicular to each other, with their C-C distances in the range of 7–8 Å (Fig. 9). However, in the C-terminal domain of EhCaBP, the aromatic ring of Tyr 81 faces that of Phe 129 almost in a parallel orientation, resulting in their C-C distance of 10.3 Å (Atreya et al. 2001). This causes the divergence of the third loop of EhCaBP compared to the corresponding loops of CaM and TnC (Fig. 8). Thus, the preference for Yb³⁺ to Site III can be attributed to both its higher negative charge compared to the other Ca²⁺-binding loops in the protein and to the presence of a Tyr at -4 position in the loop. Such a hypothesis is consistent with the NMR data, which show a rapid decrease in intensity of G90 amide cross peak during the initial stages of titration when the metal:protein ratio is increased from 0 to 1. Simultaneously, the pseudocontact shifted peak of G122 (¹_H^N = 9.3 ppm) increases in its integral volume and reaches maximum at a little over metal:protein ratio of 1 (Figs. 1, 4, 6). We can therefore conclude that Yb³⁺ first displaces Ca²⁺ from the Ca²⁺-binding Site III in EhCaBP, which results in a pseudocontact shift of residues belonging to Site IV.

View larger version (24K): [in this window] [in a new window]   Figure 8. Superposition of four Ca2+-binding loops of EhCaBP (shown in blue) with the corresponding loops of CaM (red) and TnC (dark green). Both X-ray derived and NMR-derived structures of CaM (PDB code: 1CMG and 1CLL) and TnC (PDB codes 1TNX and 1TN4) are used in the comparison. The loops are numbered in the order from N-terminus to the C-terminus of the individual proteins. Thus, loops 1 and 2 belong to the N-terminal domains, while loops 3 and 4 belong to the C-terminal domains.

View larger version (32K): [in this window] [in a new window]   Figure 9. Orientation of the aromatic rings of F(-4)/Y(-4) and F(13) in N- and C-terminal domains of (A) Calmodulin (B) Troponin C, and (C) EhCaBP.

Further, the rate at which the pseudocontact shifted peaks of residues belonging to Site I increase in their integral volumes is relatively lower compared to the rate of decrease of the corresponding original peaks. This implies the coexistence of species (Yb³⁺)(I)-(Yb³⁺)(II)-(Yb³⁺) (III)-(Ca²⁺)(IV)-EhCaBP (species A) and species (Ca²⁺) (I)-(Yb³⁺)(II)-(Yb³⁺)(III)-(Ca²⁺)(IV)-EhCaBP (species B). Their relative populations can be determined from the knowledge of the ratio of integral volumes of cross peaks arising from the respective species.

Beyond a metal:protein ratio of 2.0, both the original and pseudocontact shifted cross peaks for residues in Site I show a decline in their integral volumes (Figs. 1, 5A). This implies the filling of Yb³⁺ in Site I only in the species wherein Site II is already filled with Yb³⁺. As observed, a net decrease in the pseudocontact shifted cross peak volumes implies that population of species B is relatively more compared to species A. Another important point to note is that, the reaction path that proceeds via the filling of Site I is completely ruled out in the present case, as we did not observe any pseudocontact shifted peaks for residues in Site II.

As far as the cooperativety is concerned, it is very well known that it exists between any two metal binding sites of adjacent EF-hands. Such cooperative binding of ions is the most important functional property of the calcium signaling pathways. So, it is not surprising that we also notice positive cooperativity during the process of our lanthanide titration discussed here. The most pertinent explanation for such cooperativity is that the binding of one metal facilitates the binding of the other metal ion, because the apo becomes half-holo, and causes the other site to loose its flexibility and become more structured. Such site–site communication in the EF-hand calcium binding proteins has been reported earlier (Maler et al. 2000). However, in the present case it does not go from apo to half-holo to holo. Instead, it is a displacement of Ca²⁺ ions by Yb³⁺. To start with, both sites are already well structured and remain so even after the metal displacement. This observation, however, raises questions such as what is the origin of the positive cooperativity seen in the present case? And why should the second Yb³⁺ care to go to a site only when another Yb³⁺, and not a Ca²⁺, is in its neighboring site of a given pair of EF-hands? The answers to these questions lie in the difference in charges present in individual sites. When both sites are negatively charged there is an intrinsic repulsion between them, which is decreased when a metal ion binds to one of the sites, resulting in the formation of a short stretch of ß-sheet, which in turn, facilitates the entry of the second metal ion. If a domain with a pair of EF-hands is already occupied by two Ca²⁺ ions, as is the case now, there could still be a slight excess of negative charge. Substitution of one of them with a tripositive lanthanide ion could decrease even more, or perhaps abolish, the residual electrostatic repulsion between the two loops, facilitating the entry of the second tripositive lanthanide ion. This argument is in line with the works reported earlier on the N-terminal domain of calbindin (Maler et al. 2000; Bertini et al. 2001c), where strong cooperativity was observed. When cerium ions occupy both the sites of the domain, the intermetal distance is reported to be sizably shorter than that observed between the two Ca²⁺ ions in the native protein.

As mentioned earlier, EF-hand proteins maintain specificity for Ca²⁺ against its strong competitor Mg²⁺ inside the cell. Hence, it could also be the same case with tripositive lanthanide ions against Mg²⁺.

Coming to the ITC data, it yields four macroscopic binding constants, K₁, K₂, K₃, and K₄, as shown in Table 1, which can be now assigned to the Ca²⁺ displacement by Yb³⁺ from the respective Ca²⁺-binding sites in EhCaBP.

Up to a metal:protein ratio of 1.0, Site III is predominantly filled with Yb³⁺, leaving Sites I, II, and IV with Ca²⁺. Thereafter, Sites I and II are filled completely at a metal: protein ratio slightly above 3.0. Further, it is interesting to note that, Site IV is left with 30% Ca²⁺ even when we go beyond a metal:protein ratio of 4.0. This implies that binding constants for Yb³⁺ in Sites III and IV of EhCaBP differ by a large factor, resulting in the sequential displacement of Ca²⁺ in the C-terminal domain. In the light of such observations, we can ascribe the highest binding constant (K₁) obtained in ITC to the Ca²⁺ displacement by Yb³⁺ in Site III (k_III), while the lowest (K₄) to the similar process in Site IV. It is important to note that K₄ is the binding constant of Yb³⁺ for Site IV when Yb³⁺ is already bound to Site III (k_IV,III). The assignment K₄ to Site IV is in line with the thermodynamics study of Ca²⁺-binding in EhCaBP carried out earlier, which showed that Site IV has the highest affinity for Ca²⁺ in EhCaBP (Gopal et al. 1997). In the present study, the remaining two macroscopic binding constants, K₂ and K₃ obtained using ITC (Table 1) can be assigned to the Ca²⁺ displacement by Yb³⁺ in Sites I and II in the N-terminal domain. As discussed above, during the course of titration wherein the metal:protein ratio is changed from 1 to 3, NMR studies indicate that Yb³⁺ preferentially displaces Ca²⁺ in Site II. Further, at a metal:protein ratio of 2.0, the population of species B is about 45% and that of species A is about 25% of the total species present. The presence of species B in excess to that of species A implies that k_I,II > k_II. Thus, out of K₂ and K₃, the smaller one (K₃) can be assigned to the Ca²⁺ displacement by Yb³⁺ in Site II (k_II); K₂ then represents the equilibrium constant of Ca²⁺ displacement by Yb³⁺ in Site I, when Yb²⁺ is already bound in its neighboring Site II (k_I,II).

To verify whether the above assignments of thermodynamic constants to the various reactions are correct, plots of normalized volumes of cross peaks (Figs. 2, 5, 6) were back calculated using the equilibrium constants obtained from ITC (Table 1, last column). The reactions considered in the simulations are given in the Electronic Supplemental Material.

The experimental and simulated plots of intensity profiles are shown in Figure 10 for the original and pseudocontact shifted cross peaks of G15 and G122. As evident from the figure, simulated plots (Fig. 10B) obtained using the thermodynamic constants given in Table 1 (last column) are almost identical to the intensity profiles obtained for various species based on NMR titration data (Fig. 10A). The values of the thermodynamic constants used in the simulation are same as the experimentally observed ones, except for K₁, which is found to be three times higher). To verify the assignments of K₂ and K₃, simulated plots of intensity profiles were generated by interchanging the values of K₂ and K₃. This is shown in Figure 10C. A lower value for K₂ compared to K₃ results in 40% population of the pseudocontact shifted cross peak of G15 at a metal:protein ratio 2. However, NMR data indicates about 25% population of this species at this metal:protein ratio (Fig. 5). Thus, a higher value of K₂ compared to K₃ (as given in Table 1) more appropriately describes the system.

View larger version (27K): [in this window] [in a new window]   Figure 10. (A) Experimental and (B) simulated plots of intensity profiles of the original and pseudocontact shifted cross peaks of G15 and G122. The thermodynamic constants used in simulations are listed in Table 1 (last column; K2 > K3). (C) Simulated plots of the intensity profiles of original and pseudocontact shifted cross peaks of G15 and G122 obtained by interchanging the values of K2 and K3 values as 1.55 * 104 and 2.55 * 104 M-1, respectively.

View larger version (17K): [in this window] [in a new window]   Scheme 2

Finally, studies such as this will help in unraveling the number of Ca²⁺-binding sites in a protein. It assumes importance for EF-hand proteins such as neuronal Ca²⁺ sensor 1 (NCS-1), wherein an exact number of Ca²⁺-binding sites in the protein have not yet been resolved unambiguously (Cox et al. 1994; Ames et al. 2000). Moreover, in multidomain proteins, if we can observe pseudocontact shifts in one domain due to the filling of Ln³⁺ in the other, then such shifts help in fixing the interdomain orientations. Efforts are on in this direction to define the relative orientation of the two domains in EhCaBP.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
Protein sample preparation
The plasmid encoding EhCaBP was transformed into Escherichia coli BL21(DE3) cells. The protocol described earlier (Sahu et al. 1990a; Atreya et al. 2001) was used for overexpression of EhCaBP in a minimal (M9) medium. In the present study, two samples were prepared: (1) [u-¹⁵N] and (2) [ALK]-¹⁵N-labeled (Ca²⁺)₄-EhCaBP (holo) samples. The details of growth condition and purification procedure have been described earlier (Sahu et al. 1999a; Atreya et al. 2001). The final step in the purification of EhCaBP involves the elution of protein from the anion exchange column using 10 mM CaCl₂. Excess of Ca²⁺ in the protein sample was removed using a 3-kD cutoff Amicon centricon unit, by washing the protein solution six times with deionized water followed by three times with 0.1 M KCl solution containing 10% ²H₂O and pH 6.0, and finally concentrating it to 0.6 mL for NMR studies. The protein concentration was estimated as 1.2 mM (12.5 mg of protein in 0.6 mL) by measuring the absorbance at 280 nm (₂₈₀ = 5120 M^-1 cm^-1) on a Varian Inc. Cary 50 scan spectrophotometer. NMR experiments were performed at 35°C on 0.6 mL of 1 mM protein samples (pH 6.0), in a mixed solvent of 90% H₂O and 10% ²H₂O.

Yb³⁺ titration of [u-¹⁵N] and [ALK]-¹⁵N-labeled (Ca²⁺)₄-EhCaBP
NMR experiments on EhCaBP were carried out in the form of a titration. The displacement of Ca²⁺ in the protein by Yb³⁺ was achieved by adding small aliquots from a 20 mM stock solution of YbCl₃ to the protein solution. To start with, an aliquot of YbCl₃ from a 0.02 M stock solution corresponding to a metal:protein ratio of 0.1 was taken each time and added directly to the protein solution in the NMR sample tube. The sample was mixed thoroughly before transferring it to the 600/700 MHz magnet. As the metal: protein ratio reached 0.5, large changes were observed in the HSQC spectrum and, hence, further titration was carried out in smaller steps of 0.05 metal:protein ratios. From a metal:protein ratio of 2.0 to 4.6, titrations were carried out in larger step sizes (at a variable metal:protein ratio of 0.1, 0.2, and 0.4). This resulted in a total number of 47 titration points. The change in pH on addition of the lanthanide salt was found to be negligible.

NMR experiments
NMR experiments were recorded either on a Bruker Avance 700 MHz NMR spectrometer (CERM) or Varian Unity⁺ 600 MHZ NMR spectrometer (NFMR), both equipped with pulsed field gradient units and triple resonance probes with actively shielded Z-gradients. Sensitivity enhanced 2D [¹⁵N-¹H] HSQC spectrum using pulsed field gradients for coherence selection (Kay et al. 1992) was acquired during each step of the titration. During the entire course of titration, the sample was found to be stable and soluble. ¹H chemical shift calibrations were carried out with respect to the methyl signal (at 0.0 ppm) of 3-(trimethylsilyl)[3,3,2,2-²H] propionated₄ (TSP), which has been used as an external reference. ¹⁵N chemical shifts were referenced with respect to an external standard of ¹⁵N-labeled ammonium chloride (2.9 M in 1 M HCl). The ¹H and ¹⁵N chemical shifts published earlier (Sahu et al. 1999a, 1999b) were used to identify individual cross peaks in the 2D [¹⁵N-¹H] HSQC spectrum of EhCaBP.

ITC measurements and analyses
ITC measurements were performed with a Microcal Omega titration calorimeter. Samples were centrifuged and degassed prior to the titration and examined for precipitation, if any, after the titration. A typical titration consisted of injecting 1.2 µL aliquots of 20 mM YbCl₃ solution into 0.24 mM (1 mL) of the protein solution after every 3.5 min to ensure that the titration peak returned to the baseline prior to the next injection. A total of 31 injections were carried out. No precipitation was observed during the titration. Aliquots of more concentrated ligand solution were injected into the buffer solution (without the protein) in a separate ITC run, to subtract the heat of dilution. All experiments were repeated at least twice with different protein and ligand concentrations to establish reproducibility of the data. The ITC data were analyzed using the software ORIGIN (supplied with Omega Microcalorimeter). The amount of heat released per addition of the titrant was fitted to two/four or sequential/independent binding sites on the protein as given by Wisemen et al. (1989).

Considering a two-metal binding site system depicted in Scheme 3 (e.g., N- or C-terminal domain of EhCaBP), a situation can arise in which the values of the microscopic binding constants k_I and k_II for the two sites under consideration are widely different (one of them might be 100–1000-fold larger than the other). In such a situation, the protein will exhibit a step-wise or sequential binding/displacement of Ca²⁺.

View larger version (16K): [in this window] [in a new window]   Scheme 3

ITC experiments yield only the values of the macroscopic binding constant K₁ and K₂. However, if k_II >> k_I, the reaction path shown as dashed lines in Scheme 3 is completely ruled out and the macroscopic binding constants K₁ and K₂ essentially coincide with the microscopic binding constants k_II and k_I,II. Further, the values of k_II and k_I,II are reflected in the relative populations of species A and B shown in Scheme 3. If k_II is much larger than k_I,II (say 100-fold), then the population of species A will be largely predominant until all of the Site II is filled with the metal. On the other hand, if k_II and k_I,II are comparable, both species A and B coexist and their relative population can then be determined by the ratio: k_II/k_I,II.

	Electronic supplemental material

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
The reactions considered for simulating the intensity profiles of cross peak volumes in EhCaBP. Based on the reactions, the concentration of various experimentally observed species are derived using thermodynamic constants obtained from ITC data.

	Acknowledgments

 
We gratefully acknowledge the facilities provided by the National Facility for High Field NMR, supported by the Department of Science and Technology (DST), Department of Biotechnology (DBT), Council of Scientific and Industrial Research (CSIR), and Tata Institute of Fundamental Research, Mumbai, India. This work was supported under an Indo-Italian Joint Research Project, supported by the DST, New Delhi, India. Support by the EC, Contract No. HPRI-CT-19999-50006 (Transient NMR), and by CNR, Progetto Finalizzato Biotecnologie, 99.XX, is also acknowledged. We thank Prof. Alok Bhattacharya (JNU, New Delhi) for providing the EhCaBP clone. ITC measurements carried out in Prof. R. Varadarajan’s laboratory (MBU, IISc, Bangalore) are gratefully acknowledged.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
Akerfeldt, K.S., Coyne, A.N., Wilk, R.R., Thulin, E., and Linse, S. 1996. Ca2+-binding stoichiometry of calbindin D28k as assessed by spectroscopic analyses of synthetic peptide fragments. Biochemistry 35: 3662–3669.[CrossRef][Medline]

Allegrozzi, M., Bertini, I., Janik, M.B.L., Lee, Y.M., Liu, G., and Luchinat, C. 2000. Lanthanide induced pseudocontact shifts for solution structure refinements of macromolecules in shells up to 40 A from the metal ion. J. Am. Chem. Soc. 122: 4154–4161.[CrossRef]

Ames, J.B., Hendricks, K.B., Strahl, T., Huttner, I.G., Hamasaki, N., and Thorner, J. 2000. Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae. Biochemistry 39: 12149–12161.[CrossRef][Medline]

Atreya, H.S., Sahu, S.C., Bhattacharya, A., Chary, K.V.R., and Govil, G. 2001. NMR derived solution structure of an EF-hand calcium-binding protein from Entamoeba histolytica. Biochemistry 40: 14392–14403.

Bertini, I., Janik, M.B., Lee, Y.M., Luchinat, C., and Rosato, A. 2001a. Magnetic susceptibility tensor anisotropies for a lanthanide ion series in a fixed protein matrix. J. Am. Chem. Soc. 123: 4181–4188.[CrossRef][Medline]

Bertini, I., Janik, M.B.L., Liu, G., Luchinat, C., and Rosato, A. 2001b. Crystal structures of the helix-loop-helix calcium-binding proteins. J. Magn. Reson. 148: 23–30.[CrossRef][Medline]

Bertini, I., Lee, Y.-M., Luchinat, C., Piccioli, M., and Poggi, L. 2001c. Locating the metal ion in calcium-binding proteins by using cerium(III) as a probe. ChemBioChem 2: 550–558.[CrossRef][Medline]

Bertini, I., Luchinat, C., and Parigi, G. 2001d. Solution NMR of paramagnetic molecules. Elsevier, Amsterdam.

Biekofsky, R.R., Muskett, F.W., Schmidt, J.M., Martin, S.R., Browne, J.P., Bayley, P.M., and Feeney, J. 1999. NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca²⁺ by Tb³⁺. FEBS Lett. 460: 519–526.[CrossRef][Medline]

Chandra, M., McCubbin, W.D., Oikawa, K., Kay, C.M., and Smillie, L.B. 1994. Ca2+, Mg2+, and troponin I inhibitory peptide binding to a Phe-154 to Trp mutant of chicken skeletal muscle troponin C. Biochemistry 33: 2961–2969.[CrossRef][Medline]

Cox, J.A., Durussel, I., Comte, M., Nef, S., Nef, P., Lenz, S.E., and Gundelfinger, E.D. 1994. Cation binding and conformational changes in VILIP and NCS-1, two neuron-specific calcium-binding proteins. J. Biol. Chem. 269: 32807–32813.[Abstract/Free Full Text]

Falke, J.J., Drake, S.K., Hazard, A.L., and Peersen, O.B. 1994. Molecular tuning of ion binding to calcium signaling proteins. Quart. Rev. Biophys. 27: 219–290.[Medline]

Gilli, R., Lafitte, D., Lopez, C., Kilhoffer, M., Makarov, A., Briand, C., and Haiech, J. 1998. Thermodynamic analysis of calcium and magnesium binding to calmodulin. Biochemistry 37: 5450–5456.[CrossRef][Medline]

Gopal, B., Swaminathan, C.P., Bhattacharya, S., Bhattacharya, A., Murthy, M.R.N., and Surolia, A. 1997. Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica. Biochemistry 36: 10910–10916.

Hohenester, E., Maurer, P., and Timpl, R. 1997. Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40. EMBO J. 16: 3778–3886.[CrossRef][Medline]

Horrocks, Jr., W.D. and Sudnick, D.R. 1981. Lanthanide ion luminescence probes of the structure of biological macromolecules. Acc. Chem. Res. 14: 384.[CrossRef]

Imaizumi, M., Tanokura, M., and Yamada, K. 1990. Calorimetric studies on calcium and magnesium binding by troponin C from bullfrog skeletal muscle. J. Biochem. (Tokyo) 107: 127–132.[Abstract/Free Full Text]

Kawasaki, H., Nakayama, S., and Kretsinger, R.H. 1998. Classification and evolution of EF-hand proteins. BioMetals 11: 277–295.[CrossRef][Medline]

Kay, L.E., Keifer, P., and Saarinen, T. 1992. Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J. Am. Chem. Soc. 114: 10663–10665.[CrossRef]

Kretsinger, R.H. and Nockolds, C.E. 1973. Carp muscle calcium-binding protein. II. Structure determination and general description. J. Biol. Chem. 248: 3313–3326.[Abstract/Free Full Text]

Lewit-Bentley, A. and Réty, S. 2000. EF-hand calcium-binding proteins. Curr. Opin. Struct. Biol. 10: 637–643.[CrossRef][Medline]

Linse, S. and Forsén, S. 1995. Determinants that govern high-affinity calcium binding. Adv. Sec. Mesg. Phospho. Res. 30: 89–151.

Maler, L., Blankenship, J., Rance, M., and Chazin, W.J. 2000. Site-site communication in the EF-hand Ca²⁺-binding protein calbindin D_9k. Nat. Struct. Biol. 7: 245–250.[CrossRef][Medline]

Marcus, Y. 1985. Ion solvation. John & Wiley Sons Ltd, Chichester, UK.

Mardsen, B.J., Hodges, R.S., and Sykes, B.D. 1988. 1H-NMR studies of synthetic peptide analogues of calcium-binding site III of rabbit skeletal troponin C: Effect on the lanthanum affinity of the interchange of aspartic acid and asparagine residues at the metal ion coordinating positions. Biochemistry 27: 4198–4206.[CrossRef][Medline]

Moeschler, H.J., Schaer, J., and Cox, J.A. 1980. A thermodynamic analysis of the binding of calcium and magnesium ions to parvalbumin. Eur. J. Biochem. 111: 73–78.[CrossRef][Medline]

Nelson, M.R. and Chazin, W.J. 1998. Structures of EF-hand Ca²⁺-binding proteins: Diversity in the organization, packing and response to Ca²⁺ binding. BioMetals 11: 297–318.[CrossRef][Medline]

Ohya, Y. and Botstein, D. 1994. Structure-based systematic isolation of conditional-lethal mutations in the single yeast calmodulin gene. Genetics 138: 1041–1154.[Abstract]

Rashidi, H.H., Bauer, M., Patterson, J., and Smith, D.W. 1999. Sequence motifs determine structure and Ca++-binding by EF-hand proteins. J. Mol. Microbiol. Biotechnol. 1: 175–182.[Medline]

Sahu, S.C., Atreya, H.S., Chauhan, S., Bhattacharya, A., Chary, K.V.R., and Govil, G. 1999a. Sequence-specific 1H, 13C and 15N assignments of a calcium binding protein from Entamoeba histolytica. J. Biomol. NMR 14: 93–94.

Sahu, S.C., Bhattacharya, A., Chary, K.V.R., and Govil, G. 1999b. Secondary structure of a calcium binding protein (CaBP) from Entamoeba histolytica. FEBS Lett. 459: 51–56.

Shannon, R.D. 1976. Revised effective ionic radii in halides and chalcogenides. Acta. Crystallogr. A32: 751–767.

Sharma, Y., Chandani, S., Sukhaswami, M.B., Uma, L., Balasubramanium, D., and Fairwell, T. 1997. Modified helix-loop-helix motifs of calmodulin—The influence of the exchange of helical regions on calcium-binding affinity. Eur. J. Biochem. 243: 42–48.[Medline]

Strynadka, N.C. and James, M.N. 1989. Crystal structures of the helix-loop-helix calcium-binding proteins. Annu. Rev. Biochem. 58: 951–998.[CrossRef][Medline]

Waltersson, Y., Linse, S., Brodin, P., and Grundstrom, T. 1993. Mutational effects on the cooperativity of Ca2+ binding in calmodulin. Biochemistry 31: 7866–7871.

Wiseman, T., Williston, S., Brandts, J.F., and Lin, L.N. 1989. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 79: 131–137.

Wu, S.L., Franklin, S.J., Raymond, K.N., and Horrocks, Jr., W.D. 1996. Kinetics of the formation of macrocyclic polyaminocarboxylate ligand complexes: A laser-excited luminescence study of the Eu³⁺-DTPA-dien system. Inorg. Chem. 35: 162–167.[CrossRef][Medline]

Wu, X. and Reid, R.E. 1997. Structure/calcium affinity relationships of site III of calmodulin: Testing the acid pair hypothesis using calmodulin mutants. Biochemistry 36: 8649–8656.[CrossRef][Medline]

CiteULike    Connotea    Del.icio.us