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Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin IIb cytoplasmic domain

Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4

Reprint requests to: Hans J. Vogel, Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4; e-mail: vogel{at}ucalgary.ca; fax: (403) 289-9311.

(RECEIVED December 23, 2004; FINAL REVISION February 22, 2005; ACCEPTED March 14, 2005)

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Calcium- and integrin-binding protein (CIB) is a small EF-hand calcium-binding protein that is involved in hemostasis through its interaction with the IIb cytoplasmic domain of integrinIIb₃. We have previously demonstrated that CIB lacks structural stability in the absence of divalent metal ions but that it acquires a well-folded conformation upon addition of Ca²⁺ or Mg²⁺. Here, we have used fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry to demonstrate that both Ca²⁺-bound CIB (Ca²⁺-CIB) and the Mg²⁺-bound protein (Mg²⁺-CIB) bind with high affinity and through a similar mechanism to IIb cytoplasmic domain peptides, but that metal-free CIB (apo-CIB) binds in a different manner. The interactions are thermodynamically distinct for Ca²⁺-CIB and Mg²⁺-CIB, but involve hydrophobic interactions in each case. Since the Mg²⁺ concentration inside the cell is sufficient to saturate CIB at all times, our results imply that CIB would be capable of binding to the IIb cytoplasmic domain independent of an intracellular Ca²⁺ stimulus in vivo. This raises the question of whether CIB can act as a Ca²⁺ sensor in IIb₃ signaling or if other regulatory mechanisms such as fibrinogen-induced conformational changes in IIb₃, post-translational modifications, or the binding of other accessory proteins mediate the interactions between CIB and IIb₃. Differences in NMR spectra do suggest, however, that Ca²⁺-binding to the Mg²⁺- CIB-IIb complex induces subtle structural changes that could further modulate the activity of IIb₃.

Keywords: calcium- and integrin-binding protein; calcium; magnesium; integrin; peptide; isothermal titration calorimetry; nuclear magnetic resonance spectroscopy; fluorescence spectroscopy

Abbreviations: CIB, calcium- and integrin-binding protein • Ca²⁺-CIB, calcium-bound CIB • Mg²⁺-CIB, magnesium-bound CIB • apo-CIB, metal-free CIB • CaM, calmodulin • CnB, calcineurin B • can, calcineurin A • NCS, neuronal calcium sensor • TnC, troponin C • TnI, troponin I

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041312805.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Integrins are a large family of transmembrane glycoproteins that are important in mediating adhesion between cells as well as the attachment of cells to the extracellular matrix. The highly abundant platelet integrin IIb₃ links the cytoplasmic and extracellular environments, acting as a bidirectional signaling molecule in the platelet aggregation process leading to blood clot formation (Calvete 2004). Platelet agonists trigger inside-out signaling, where ligand binding to the cytoplasmic domains causes conformational changes that increase the affinity of the extracellular domains for fibrinogen and the von Willebrand factor, initiating platelet aggregation. The subsequent ligand binding by the extracellular domains also triggers outside-in signaling events that increase the avidity of binding and enhance the aggregation process (Calvete 2004; Mould and Humphries 2004; Xiao et al. 2004). Despite their relatively small sizes, the cytoplasmic domains of IIb and ₃ interact with each other and many intracellular ligands and play an important role in mediating these signaling events (Vinogradova et al. 2002; Weljie et al. 2002).

Calcium- and integrin-binding protein (CIB) is a 22-kDa calcium (Ca²⁺)-binding protein of the helix-loop-helix EF-hand superfamily. It was originally identified as a specific binding partner for the IIb cytoplasmic domain of platelet integrin (Naik et al. 1997), but has since been suggested to bind to several other proteins(Wu and Lieber 1997; Kauselmann et al. 1999; Stabler et al. 1999; Ito et al. 2000; Fang et al. 2001; Haataja et al. 2002; Henderson et al. 2002; Hollenbach et al. 2002; Whitehouse et al. 2002). Both in vivo and in vitro binding studies using intact IIb₃ as well as IIb cytoplasmic domain peptides have shown that CIB specifically binds to the cytoplasmic domain of II band that its affinity is enhanced in the presence of Ca²⁺ (Shock et al. 1999; Barry et al. 2002; Tsuboi 2002). CIB is also myristoylated on its N terminus (Stabler et al. 1999), although it binds to IIb independent of myristoylation (Shock et al. 1999; Tsuboi 2002). The sequence homology of CIB to calmodulin (CaM), calcineurin B (CnB), and members of the neuronal calcium sensor (NCS) family suggests that CIB might function as a Ca²⁺ sensor in IIb₃-mediated inside-out signaling (Hwang and Vogel 2000). Indeed CIB binds two Ca²⁺ ions with its C-domain EF-hands EF-III and EF-IV with dissociation constants (K_d) near 1.9 µM and 0.5 µM, respectively (Yamniuk et al. 2004). However, there have also been reports of apo-CIB binding to IIb (Shock et al. 1999; Vallar et al. 1999; Tsuboi 2002). Moreover, unlike many other Ca²⁺-regulatory proteins, a single Mg²⁺ binds to EF-III of CIB (K_d=120 µM) and causes conformational changes similar to those induced by Ca²⁺ (Yamniuk et al. 2004). The binding of either Ca²⁺ or Mg²⁺ to CIB significantly increases the structural stability of the protein in comparison to apo-CIB, which is a molten globule (Yamniuk et al. 2004). Here we have studied the binding of IIb cytoplasmic domain peptides to all three forms of CIB, metal-free CIB (apo-CIB), Ca²⁺-bound CIB (Ca²⁺-CIB), and Mg²⁺-bound CIB (Mg²⁺-CIB). Our results demonstrate that both Ca²⁺-CIB and Mg²⁺-CIB bind strongly to the IIb cytoplasmic domain in a similar manner, whereas apo-CIB binds differently and with lower affinity. Because the free intracellular Mg²⁺ concentration in platelets is maintained between 0.5 and 1.3mM (Matsuno et al. 1993), our results suggest that CIB would be capable of binding IIb independent of a Ca²⁺ stimulus in vivo. Therefore, other regulatory mechanisms are likely involved in vivo, in mediating the interactions of CIB with IIb.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Fluorescence spectroscopy
We first studied the interactions between CIB and the cytoplasmic domain of IIb by fluorescence spectroscopy, taking advantage of the single Trp residue in the IIb cytoplasmic domain (W988). Studying peptide binding directly in this fashion is possible here because CIB does not contain any Trp residues in its sequence. Titrations were performed to establish the stoichiometry and affinity of binding, while steady state fluorescence emission spectra and quenching studies were used to determine the environment of W988 in each complex. Studies were performed using a "long" peptide (IIb-L) encompassing the entire cytoplasmic domain or a "short" peptide (IIb-S) encompassing only the membrane proximal region. To test the role of ionic interactions all experiments were performed in the presence and absence of 100 mM KCl. NMR studies indicate that the conformation of CIB is unchanged between 0 and 100 mM KCl and therefore any salt-dependent binding effects are unlikely to be caused by structural modifications (data not shown).

The steady state fluorescence emission spectrum of free IIb-L is characteristic of a solvent-exposed Trp having an emission wavelength maximum near 355 nm (Fig. 1A). Addition of Ca²⁺-CIB increased the steady state emission intensity up to a stoichiometry of 1:1 with a K_d near 0.2 µM, consistent with a previous study (Table 1; Barry et al. 2002). Interestingly, we found that peptide binding to Mg²⁺-CIB was also stoichiometric with a comparable affinity, suggesting that a similar interaction can occur in the presence of Mg²⁺. In each complex the emission wavelength maximum shifted only 2–4 nm to shorter wavelengths, indicating that although the Trp is held rigidly, it is not buried within a hydrophobic binding pocket. Fluorescence quenching studies also indicated that W988 is on the surface of each complex since the fluorescence emission was quenched ~70% as efficiently when bound to either Ca²⁺-CIB or Mg²⁺-CIB in comparison to the free peptide (Table 1). Experiments performed in the absence of salt gave very similar results to those in the presence of 100 mM KCl, suggesting that ionic interactions play a minor role in the binding of Ca²⁺-CIB or Mg²⁺-CIB to IIb-L. All studies with IIb-S also gave similar results to those with IIb-L, although the emission wavelength maxima were shifted an additional ~2–4 nm toward shorter wavelengths, and binding to Mg²⁺-CIB was about fivefold weaker than with the full-length peptide (Table 1). The similar interactions with each peptide and lack of salt dependency suggest that both Ca²⁺-CIB and Mg²⁺-CIB bind to the membrane proximal region of IIb predominantly through hydrophobic interactions.

View larger version (24K): [in this window] [in a new window]   Figure 1. Interactions between CIB and IIb peptides studied by fluorescence spectroscopy. (A) Steady state fluorescence emission spectra of free IIb-L (——), and IIb-L in the presence of 1.2 molar equivalents of Ca2+-CIB (- - - -), Mg2+-CIB (• • •), or apo-CIB (–• •–). Titrations of Ca2+-CIB (B), Mg2+-CIB (C), or apo-CIB (D) into IIb-L in the absence (•) or presence () of 100 mM KCl or similar titrations into IIb-S in the absence () or presence () of 100 mM KCl. The Kd was calculated from the best fit to the titration data (——), except for the titrations of apo-CIB into IIb-S, which were not fit due to nonspecific interactions and are instead connected by dotted lines (• • • • • •) and (• • • • • •).

View this table: [in this window] [in a new window]   Table 1. Fluorescence parameters for IIb-peptides binding to CIB

Isothermal titration calorimetry
Thermodynamic analysis of IIb-L binding to CIB was performed by isothermal titration calorimetry (ITC). Under all conditions the binding was exothermic, and the data were best fit to a single site binding model (Fig. 2). The calorimetrically derived K_d values for IIb-L binding to Ca²⁺-CIB or Mg²⁺-CIB were similar to each other and in the high nanomolar range, but slightly larger than the values calculated by fluorescence spectroscopy (Table 2). All thermodynamic parameters were similar in the absence of salt, confirming again that electrostatic interactions are not prevalent in either complex. The interactions were also thermodynamically distinct, with IIb-L binding to Ca²⁺-CIB proceeding with small but favorable changes in both enthalpy and entropy, whereas binding to Mg²⁺-CIB was considerably more enthalpically favorable and entropically unfavorable (Table 2). Both Hand TS displayed strong linear but opposite temperature dependence known as enthalpy–entropy compensation (Fig. 3; Cornish-Bowden 2002). Therefore, the changes in H and TS offset to give similar binding free energy changes over the entire temperature range. The slope of the temperature dependence of H was used to calculate the change in heat capacity (Cp) for binding to each form of the protein. This in turn can be correlated to the change in solvent accessible nonpolar surface area accompanying peptide binding (Loladze et al. 2001). Importantly, the Cp for IIb-L binding to Ca²⁺-CIB (–1.1 kJ/mol K) and Mg²⁺-CIB (–1.3 kJ/mol K) were each negative and of similar magnitude, indicating that the amount of nonpolar surface buried upon formation of each complex is comparable. In the presence of saturating concentrations of both Ca²⁺ and Mg²⁺, the thermodynamics of binding were similar to Ca²⁺ alone, consistent with the higher affinity of CIB for Ca²⁺ over Mg²⁺ (Yamniuk et al. 2004).

View larger version (19K): [in this window] [in a new window]   Figure 2. Representative isothermal titration calorimetry experiments for IIb-L binding to Ca2+-CIB (A), Mg2+-CIB (B), and apo-CIB (C) in 20 mM HEPES, 100 mM KCl (pH 7.3) at 37°C. The top panels display the baseline corrected calorimetric titration data while the bottom panels display the derived binding isotherms for each experiment.

View this table: [in this window] [in a new window]   Table 2. Thermodynamics of IIb-L binding to CIB as determined by isothermal titration calorimetry in 20 mM HEPES, 100 mM KCl (pH 7.3)

View larger version (15K): [in this window] [in a new window]   Figure 3. Temperature dependence of the enthalpy (solid symbols) or entropy (open symbols) of IIb-L binding to CIB in the presence of 2 mM Ca2+ (circles), 4 mM Mg2+ (squares), or 2 mM Ca2+ and 4 mM Mg2+ (triangles). The linear regression is shown as solid, dotted, and dashed lines, respectively.

NMR spectroscopy
To investigate the extent of peptide-induced conformational changes in CIB, we recorded heteronuclear ¹H, ¹⁵N-HSQC NMR spectra of uniformly ¹⁵N-labeled Ca²⁺-CIB, Mg²⁺-CIB, and apo-CIB, in the presence and absence of unlabeled IIb-L. These spectra are essentially the fingerprint for the protein where each peak represents a backbone or side chain amide group. As has been discussed in more detail previously (Yamniuk et al. 2004), the spectra of Ca²⁺-CIB and Mg²⁺-CIB are characteristic of folded proteins, where the downfield Gly121 and Gly166 peaks represent Ca²⁺ orMg²⁺ coordination at EF-III or EF-IV, respectively (Fig. 4A,B). Less than the expected number of peaks are observed in each spectrum, presumably due to chemical exchange broadening from weak self-association or flexible internal regions. There are also more distinct backbone amide peaks detected for Ca²⁺-CIB (~160) than Mg²⁺-CIB (~140) in the absence of peptide, consistent with greater structural stability in the presence of Ca²⁺ than Mg²⁺. The binding of IIb-L induces chemical shift changes for most of the peaks in both the Ca²⁺-CIB and Mg²⁺-CIB spectra, indicative of large peptide-induced conformational changes in each form of the protein. Interestingly ~160 distinct backbone amide peaks are now observed in the spectra of each complex, indicating that peptide binding stabilizes the structure of some previously exchange-broadened regions of Mg²⁺-CIB. This includes a peak for G166 of Mg²⁺-CIB that was not observed in the absence of IIb-L, suggesting that the structure of EF-IV of Mg²⁺-CIB is stabilized in the complex, possibly by Mg²⁺ binding directly to this site. Also noteworthy is that many of the peaks in each complex have similar chemical shifts, indicating that regions of the protein adopt similar structures when bound to IIb-L in the presence of Ca²⁺ or Mg²⁺ (Fig. 4D). However these spectra are not identical, indicating that Ca²⁺ binding to the Mg²⁺-CIB-IIb-L complex does induce some small conformational changes.

View larger version (31K): [in this window] [in a new window]   Figure 4. 1H, 15N HSQC NMR spectra of uniformly 15N-labeled Ca2+-CIB (A), Mg2+-CIB (B), and apo-CIB (C) in the presence and absence of unlabeled IIb-L at 37°C. (D) Overlaid spectra of the Ca2+-CIB-IIb-L and Mg2+-CIB-IIb-L complexes. G121 and G166 represent Gly6 of the Ca2+-binding loop from EF-III and EF-IV, respectively, as determined previously (Yamniuk et al. 2004). Note that the G166 peaks for Ca2+-CIB in the presence and absence of IIb-L overlap in A, whereas G166 of Mg2+-CIB is only observed in the presence of IIb-L in B.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

 
Previous studies have shown that CIB is capable of binding to the IIb cytoplasmic domain in the absence of divalent metal ions, and that its affinity increases in the presence of Ca²⁺ (Shock et al. 1999;Vallar et al. 1999;Barry et al. 2002; Tsuboi 2002). Our structural studies have demonstrated that apo-CIB is a molten globule, but adopts a folded structure in the presence of Ca²⁺ or Mg²⁺ (Yamniuk et al. 2004). Here we demonstrate that both Ca²⁺-CIB and Mg²⁺-CIB bind to IIb peptides with high affinity and by a similar binding mode, but that binding to apo-CIB is of lower affinity and involves a different mechanism. The apo-CIB binding mechanism might involve some nonspecific interactions between the exposed hydrophobic surfaces of apo-CIB and the nonpolar residues of IIb, including W988, as evidenced by the slow ITC baseline recovery and binding of several IIb-S peptides observed by fluorescence spectroscopy. However, the interaction with apo-CIB is unlikely to be of biological importance since CIB would never exist in vivo devoid of divalent metal ions due to the high intracellular Mg²⁺ concentration. Instead, Mg²⁺-CIB would be the major intracellular form of the protein, with Ca²⁺ potentially displacing Mg²⁺ upon intracellular Ca²⁺ influx in a stimulated cell.

Both fluorescence spectroscopy and ITC demonstrate that the similar mode of IIb-binding to Ca²⁺-CIB and Mg²⁺-CIB involves predominantly hydrophobic interactions with little contribution from electrostatic interactions. Studies using mutant IIb peptides have indicated that L983, W988, F992, and F993 of IIb are particularly important in binding, and that this region of IIb likely interacts with the C domain of CIB (Barry et al. 2002). We also found that W988 of IIb-L is on the surface of the complex with either Ca²⁺-CIB or Mg²⁺-CIB, similar to W352 of CnA bound to CnB, but unlike many CaM-binding peptides where the Trp is completely shielded from solvent by the protein (Crivici and Ikura 1995; Ke and Huai 2003; Yamniuk and Vogel 2004). This supports the previous proposal that the CIB-IIb interaction is similar to the CnB–CnA interaction (Barry et al. 2002). Peptide binding to Ca²⁺-CIB or Mg²⁺-CIB is also accompanied by comparable changes in heat capacity, indicating that the nonpolar binding interface is similar in the complexes with Ca²⁺-CIB or Mg²⁺-CIB. However, the Cp of IIb-L binding to CIB was considerably less than that associated with peptides binding to other EF-hand Ca²⁺-regulatory proteins such as CaM (Wintrode and Privalov 1997; Brokx et al. 2001) and S100P (Gribenko et al. 2002), suggesting that the hydrophobic binding surface of CIB is somewhat smaller than these proteins. In fact the Cp associated with the CIB-IIb-L interaction (~–1.2 kJ/mol K) is quite similar to that associated with peptides binding to a single domain of CaM (~–1.6 kJ/mol K) (Brokx et al. 2001), suggesting that IIb-L binds to only one domain of CIB, possibly the C domain as suggested by Parise and coworkers (Barry et al. 2002).

The high affinity interaction between CIB and IIb in the presence of Mg²⁺ indicates that CIB should be capable of binding to the integrin in vivo independent of a Ca²⁺ stimulus. This feature distinguishes CIB from many related Ca²⁺-regulatory proteins that show strict Ca²⁺-dependent target protein binding (Ikura 1996; Bhattacharya et al. 2004). However, the majority of evidence suggests that CIB binds only to activated IIb₃, indicating that it does not remain constitutively bound in vivo (Vallar et al. 1999; Naik and Naik 2003). Therefore, other mechanisms must regulate the interaction between CIB and IIb in vivo. For example, the membrane proximal CIB-binding region of IIb also interacts with the ₃ cytoplasmic domain, and it is this interaction that is believed to maintain IIb₃ in an inactive conformation (Hughes et al. 1995, 1996; Vinogradova et al. 2002; Weljie et al. 2002). This association between the IIb and ₃ cytoplasmic domains could prevent the binding of CIB in vivo. Alternatively, CIB binding could be regulated by other accessory proteins that associate with either the IIb or ₃ domains. The protein phosphatase PP1 was recently shown to interact with the ⁹⁸⁹KVGF⁹⁹² region of IIb, but to dissociate upon IIb₃ binding to fibrinogen (Vijayan et al. 2004). Likewise the ion channel-forming protein ICln has been shown to influence the activity of IIb₃ through a high affinity interaction with the ⁹⁸⁹KVGFFKR⁹⁹⁵ region of IIb (Larkin et al. 2004). Since these binding regions also overlap with the CIB-binding domain, dissociation of PP1 or ICln might be a prerequisite to CIB binding. Other proteins such as the ₃ binding protein Talin might also be required to disrupt the IIb–₃ interaction (Vinogradova et al. 2002; Garcia-Alvarez et al. 2003; Tadokoro et al. 2003), which could free IIb for a subsequent binding to CIB. Moreover, CIB contains several consensus phosphorylation sequences (Naik et al. 1997) that might require dephosphorylation prior to IIb-binding, while phosphorylation of Tyr and Thr residues of the ₃ integrin tail has also been demonstrated (Jenkins et al. 1998; Lerea et al. 1999; Kirk et al. 2000) and this in turn could also fine-tune its interactions with the IIb domain.

Although CIB is capable of binding to IIb in the absence of Ca²⁺, subtle Ca²⁺-induced conformational changes in CIB could be important for regulating the activity of IIb₃. Indeed although the NMR spectra of Ca²⁺-CIB or Mg²⁺-CIB bound to IIb-L were similar, they were not identical. This type of mechanism takes place in the troponin complex in vertebrate skeletal muscle where the Mg²⁺-bound C domain of troponin C (TnC) constitutively tethers the protein to troponin I (TnI), awaiting subtle Ca²⁺-induced conformational changes that activate the complex (Zot and Potter 1982; Finley et al. 2000). Notably, Mg²⁺ binds to EF-III but not EF-IV of TnC in the absence of TnI, but to both EF-III and EF-IV in the presence of TnI (Finley et al. 2000). Moreover the Cp associated with Ca²⁺-TnC or Mg²⁺-TnC binding to TnI are nearly identical (Calvert et al. 2000). These characteristics were also observed with CIB. Although N-terminal myristoylation of CIB is not required for binding to IIb, a Ca²⁺-induced myristoyl switch mechanism similar to the NCS proteins (Ames and Ikura 2002; O’Callaghan and Burgoyne 2003) could also target CIB toward the cytoplasmic membrane, which would increase the probability of an interaction with IIb. Therefore, although the Mg²⁺-dependent binding of CIB to IIb canoccur in the absence of Ca²⁺, Ca²⁺-binding to CIB could potentially alter its interactions with IIb₃ and contribute to the regulation of integrin-mediated signaling events.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Protein expression, purification, and peptide synthesis
Unlabeled and uniformly ¹⁵N-labeled CIB were expressed and purified as previously described (Yamniuk et al. 2004). Both peptides used in this study were synthesized commercially and were determined to be more than 95% pure by high pressure liquid chromatography and matrix-assisted laser desorption/ionization mass spectroscopy. Peptide IIb-L (Ac-LVLAMWKVGFFKRNRPPLEEDDEEGQ-OH) corresponds to amino acids 983–1008 of the IIb subunit of platelet integrin IIb₃, and comprises the entire cytoplasmic domain. This peptide contains the E1008Q post-translational modification that was suggested by mass spectrometry (Calvete et al. 1990). Peptide IIb-S (Ac-LVLAMWKVGFFKRNR-NH₂) is a shortened version of IIb-L containing only the membrane proximal region of IIb.

Fluorescence spectroscopy
All fluorescence spectra were recorded on a Varian Cary Eclipse spectrofluorimeter. In each experiment the Trp residue of IIb-L or IIb-S was selectively excited at 295 nm using an excitation slit width of 5 nm, and emission spectra were recorded from 300 to 450 nm using an emission slit width of 10 nm. All samples consisted of 8–10 µM peptide in 20 mM HEPES, 1 mM DTT (pH 7.3) with or without 100 mM KCl. Studies with Ca²⁺-CIB were performed in 2 mM CaCl₂, while Mg²⁺-CIB samples contained 2 mM MgCl₂ and 0.5 mM EGTA, and apo-CIB samples contained 2 mM EDTA and 2 mM EGTA. This strategy for producing Ca²⁺-, Mg²⁺-, and apo-CIB has been described previously (Yamniuk et al. 2004). Titration experiments involved sequential addition of microliter volumes of 100–200 µM CIB in the respective buffer into 1-ml samples of IIb-L or IIb-S. The changes in fluorescence intensity at the emission wavelength maxima of each complex were used to calculate the K_d for the interaction. In the case of IIb-L binding to apo-CIB, a second estimate for the K_d was obtained using the wavelength change of this emission maximum (_max). For fluorescence quenching experiments, 1-ml samples of 10 µM peptide saturated with 12 µM CIB were titrated with aliquots from a 5 M solution of CsCl to a final concentration of 1.4 M, and the fluorescence emission intensity was recorded at 351 nm for Ca²⁺-CIB and Mg²⁺-CIB, 333 nm for apo-CIB, and 355 nm for the unbound peptides. A total of six titration points were used in each case to generate Stern-Volmer plots that follow Equation 1:

(1)

where Q is the quencher CsCl, I_o is the fluorescence intensity at 0 M CsCl, I is the intensity at each titration point, and K_sv is the quenching constant. All Stern-Volmer plots were linear and there were no changes in the emission wavelength maxima during the CsCl titrations, indicating that no significant structural changes occurred in the presence of large amounts of CsCl.

Isothermal titration calorimetry
All ITC experiments were performed on a MicroCal VP-ITC microcalorimeter. CIB was dissolved in 20 mM HEPES, 100 mM KCl (pH 7.3) and 2–10 mM DTT, and incubated at room temperature for several hours to reduce all sulfhydryl groups. Since the presence of DTT causes undesirable noise in ITC baselines, it was removed prior to ITC analysis by passing the samples through an Econo-Pac 10DG column (BioRad) equilibrated with 20 mM HEPES, 100 mM KCl (pH 7.3). Then concentrated metal ion or chelator solutions were added to give final concentrations of 2 mM CaCl₂ for Ca²⁺- CIB, 4 mM MgCl₂ and 1 mM EGTA for Mg²⁺-CIB, 2 mM CaCl₂ and 4 mM MgCl₂ for Ca²⁺(Mg²⁺)-CIB, or 2 mM EDTA and 2 mM EGTA for apo-CIB. The IIb-L peptide was simply dissolved in the respective ITC buffer. Titrations consisted of 4–11-µL injections of 280–500 µM CIB into a 1.43-mL sample cell containing 15–25 µM IIb-L at various temperatures. The heat of dilution was estimated from the heat of injection after saturation and subtracted before performing the curve fitting (Pierce et al. 1999). The stoichiometry (N), association constant (K_a), and enthalpy change (H) were each obtained directly from the data, while the entropy change (S) and Gibbs free energy change (G) were calculated using Equations 2 and 3:

(2)

(3)

By performing the titrations at multiple temperatures, the change in heat capacity associated with binding was also calculated using Equation 4:

(4)

All data were fit to a one-site model using MicroCal Origin software.

NMR spectroscopy
All ¹H, ¹⁵N HSQC NMR spectra were recorded at 37°C on a Bruker Avance 500 NMR spectrometer equipped with a triple resonance inverse cryoprobe with a single axis Z-gradient. Samples of 570 µM CIB in 20 mM HEPES, 100 mM KCl, 10 mM d₁₀-DTT, 10% D₂O (pH 7.5±0.1) with an additional 3 mM CaCl₂ for Ca²⁺-CIB, 5 mM MgCl₂+1 mM EGTA for Mg²⁺-CIB, or 2.5 mM EDTA+2.5 mM EGTA for apo-CIB, were titrated with 1.1–1.2 mM IIb-L in the respective buffer. Each spectrum was recorded with sweep widths of 8012.82 Hz and 1600.0 Hz and carrier frequencies of 500.13235 MHz and 50.68365 MHz for the ¹H and ¹⁵N dimensions, respectively. Quadrature detection in the F1 dimension was obtained using the echo/anti-echo time-proportional phase increment method. A total of 2048x256 real data points were recorded in ¹H and ¹⁵N, respectively, with 16 scans for peptide-free CIB and 48 scans for peptide-bound CIB in order to obtain a similar signal-to-noise ratio with the peptide-diluted samples. Each spectrum was zero filled once in each dimension and referenced in ¹H to 0 ppm using the DSS signal in the one-dimensional ¹H spectrum of each sample and in ¹⁵N using the conversion factor of 0.101329118 as suggested by Sykes and coworkers (Wishart et al. 1995). Data analysis was performed using NMRpipe and NMRDraw software (Delaglio et al. 1995).

	Acknowledgments

 
This research is supported by the Canadian Institutes of Health Research (CIHR). H.J.V. holds a senior scientist award from the Alberta Heritage Foundation for Medical Research (AHFMR), while A.P.Y. is a recipient of AHFMR and Natural Sciences and Engineering Research Council of Canada (NSERC) studentship awards. The isothermal titration microcalorimeter was purchased through a grant from the Alberta Science and Research Authority (ASRA) to the Alberta Network of Proteomics Innovation. The Bio-NMR center at the University of Calgary is maintained through funds provided by the CIHR and the University of Calgary. We wish to thank Dr. D.D. McIntyre for the continuous upkeep of the NMR instrumentation and Dr. Tomohiko Murase for his expertise in fluorescence data analysis.

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