1Department of Chemistry, Center of Drug Design, 2Department of Biology and 3Department of Computer Science, Georgia State University, Atlanta, GA 30303, USA
4 To whom correspondence should be addressed. e-mail: chejjy{at}panther.gsu.edu
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Abstract |
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Keywords: calcium binding/calmodulin/CD2.D1/EF-hand protein/FRET
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Introduction |
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Using the grafting approach, three criteria must be met to obtain an intrinsic metal-binding affinity of each EF-loop in EF-hand proteins. First, the host protein must retain its native structure in the presence and absence of calcium ions so that the large conformational entropy that arises from a conformational change in the protein is avoided. Second, the calcium-binding loop should be able to maintain its native calcium binding and structural properties in a foreign host protein. Third, the influence of the host protein environment on the calcium binding properties of the grafted EF-loop needs to be minimized if not completely decoupled. It is known that charged side chains that are present on the surface of the protein exhibit strong influences on metal-binding affinity even if they are not directly involved as ligands (George et al., 1993; Bonagura et al., 1999
; Pidcock and Moore, 2001
). Linse and Forsén and their co-workers have shown that removal of three negative surface charges Glu, Asp and Glu at positions 17, 19 and 26 in the vicinity of the EF-hand calcium-binding sites of calbindinD9k leads to up to a 45-fold decrease in average affinity (per site) (Linse et al., 1988
; Linse et al., 1991b
). In addition, Ababou and Desjarlais have recently demonstrated that the replacement of polar side chains glutamine and lysine (positions 41 and 75) outside of EF-loop I and II with non-polar side chains leads to dramatic decreases in the calcium-binding affinity of N-terminal domains of calmodulin (Ababou and Desjarlais, 2001
). Their studies further support the contention that the protein environment plays an important role in calcium binding.
Three different locations in CD2.D1 were chosen for insertion of EF-loop III of calmodulin based on several considerations (Figure 1). First, CD2.D1, a non-calcium binding cell adhesion molecule, has been shown to be an excellent host protein with a strong ability to maintain its native structure upon large changes of electrostatic interactions (Davis and McCammon, 1990; Driscoll et al., 1991
; Jones et al., 1992
; Yang et al., 2000a
, 2001). These three insertion positions, 2223, 5253 and 8384, are largely solvated and tolerant to mutations. In addition, these loops in the host protein have significantly different relative electrostatic potentials predicted using AMMP (Another Molecular Mechanics Program) (Table I) (Harrison, 1999
). While position 5253 of loop C''D has a positive potential of 47, position 2223 of loop strands BC and position 8384 in loop FG have negative potentials of about 42 and 21 kcal/mol, respectively. Furthermore, these loops connect with different ß-strands and at opposite sides/ends of the protein structure representing different protein environments with different hydrogen bonding and hydrophobic interactions. To provide the flexibility required for calcium binding and native geometry, three Gly residues (a total of 6G or 5G) were used as attachments at either side of the EF-loop (Ye et al., 2001
). Three chimeric CD2 variants with the isolated EF-loop III of calmodulin inserted after positions 22, 52 and 83 are named CaM-CD2-III-6G-22, CaM-CD2-III-5G-52 and CaM-CD2-III-6G-83, respectively.
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Materials and methods |
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Visual analysis of the electrostatic map of the native CD2.D1 domain was calculated to allow for the finding of approximate regions of the molecule where negative charge is concentrated. After selection of these approximate locations, a positively charged probe (sphere of 5 Å radius) was placed at each location. Then the locations of the probe spheres were refined using the energy minimization with AMMP (Harrison, 1999). Finally, electrostatic potential (Eq) was calculated in the center of each sphere.
Protein engineering, expression and purification
The loop III of calmodulin was inserted into different locations of domain 1 of rat CD2 (residues 199) using the same approach as with CaM-CD2-III-5G-52 (Ye et al., 2001). Recombinant CaM-CD2-III-5G-52, CaM-CD2-III-5G-22 and CaM-CD2-III-5G-83 were expressed in LB medium as a fusion construct with the enzyme glutathione S-transferase (GST) of Schistosoma japonicum in a pGEX plasmid vector transformed into Escherichia coli BL21 (DE3) (Driscoll et al., 1991
; Yang et al., 2000a
; Ye et al., 2001
). CD2 variants were first purified as a GST fusion protein using glutathione Sepharose 4B beads (Pharmacia). After being cleaved by thrombin, CD2 variants were further purified using a Superdex 75 column (Pharmacia) in 10 mM TrisHCl, pH 7.4 buffer. The identity of CD2 variants was confirmed by MALDI-TOF and electrospray mass spectrometry at the Georgia Institute of Technology. The concentration of CD2 variants was measured by its absorption at 280 nm with the extinction coefficient of CD2
280 = 11 700 M1 cm1.
Mass spectrometry
Metal-binding uptake by the proteins was analyzed by mass spectrometry using the electrospray technique. A Micromass Quattro LC instrument was used to acquire the data in the positive ion mode by syringe pump infusion of the protein solutions at a flow rate of 10 µl/min. All samples were run in either 100% water or 1.0 mM Tris buffer at pH 7.4 to maintain non-denaturing conditions. Calcium (CaCl2) or lanthanum (LaCl3) was added in 0200 molar excess over the protein concentration to observe specific binding. Both ions bind with 1:1 stoichiometry such that the metal-bound mass peak was observed at either M + 37 for calcium or M + 137 for lanthanum.
Circular dichroism (CD)
CD spectra were measured using a Jasco-710 spectropolarimeter equipped with a temperature-controlled water-bath (Neslab 110). A CD cell with a 10 mm lightpath was used for far-UV CD spectra. All spectra were the average of four or eight scans with a scan rate of 50 nm/min. The protein concentrations were 26 µM in the far-UV CD measurements. All solutions were prepared using 10 mM TrisHCl10 mM KCl, pH 6.9 buffer that was pre-purified on a Chelex-100 (Bio-Rad) chelating column.
Intrinsic Trp fluorescence
Fluorescence experiments were performed using a PTI lifetime fluorimeter equipped with a temperature-controlled water-bath (Neslab 110) at 25°C. A fluorescence cell with a 1 cm pathlength was used. The protein concentration used for fluorescence was about 2 µM. The scan wavelength for the emission spectrum was from 300 to 400 nm with the excitation wavelength at 283 nm. The slit widths for excitation and emission spectra were 4 and 8 nm, respectively. Raman scattering from water was not corrected owing to its negligible contribution (<2%).
The effect of metal ions on the Trp fluorescence was investigated by gradually adding stock solutions of La3+ (1.0 or 10 mM) with 2 µM protein to a protein sample of 2 µM in 10 mM Tris10 mM KCl, pH 6.9. An incubation time of 2030 min was used to reach equilibrium between each measurement. Light bleaching was minimized by closing the excitation shutter during incubation. CD2.D1 was used as a negative control. The net change of Trp fluorescence of CD2 variants due to La3+ binding was calculated by subtracting the contribution of quenching using the same concentration of CD2.D1 at each metal concentration.
Tyrosine-sensitized Tb3+ energy transfer
Tyrosine-sensitized Tb3+ energy transfer was carried out using a 1 cm pathlength cell with a protein concentration of 5 µM in 10 mM Tris-HCl, 10 mM KCl at pH 6.9 at room temperature. Tb3+ emission fluorescence spectra were acquired from 500 to 600 nm with excitation at 283 or 292 nm. The slit widths for excitation and emission were 8 and 16 nm, respectively. A glass filter was used to avoid secondary Raleigh scattering. Protein samples (5 µM) were titrated with Tb3+ by gradually adding different volumes of Tb3+ stock solutions (0.1 mM, 1.0 mM with 5 µM protein) in 10 mM Tris10 mM KCl at pH 6.9. An equilibrium time of 1020 min was used between each point. Tb3+ emission signals in the presence and absence of protein (intrinsic Tb3+ signal) at 545 nm were calculated by integrating areas of Tb3+ emission spectra between 527 and 568 nm and subtracting the areas of its baseline between points 527 and 568 nm. Tb3+ fluorescence enhancement signals at 545 nm by the CD2 variants are the difference of the net area of emission signals between 527 and 568 nm in the presence of protein and in the absence of protein.
The competition of La3+ or Ca2+ for the Tb3+ binding pocket using Tb3+ fluorescence enhancement was carried out using 5 µM protein with 30 µM Tb3+ equilibrated with 0.10 mM La3+ or with 10 mM Ca2+ in 10 mM Tris10 mM KCl, pH 6.9 for 1 h. Under this condition, no precipitation was observed at Tb3+ concentrations <0.5 mM. The Tb3+ fluorescence enhancements with CD2 variants in the presence of Ca2+ or La3+ were calculated by subtracting the Tb3+ fluorescence in the presence of Ca2+ or La3+ in the absence of the protein.
1D 1H NMR
NMR samples were prepared by diluting proteins in 10 mM TrisHCl10 mM KCl, 10% D2O at pH 6.9 and 7.4. Protein concentrations varied between 110 and 170 µM. Spectral widths of 6600 and 8000 Hz were used for 1D NMR at 500 and 600 MHz, respectively. A modified WATERGATE pulse sequence was used for 1D NMR with 16K complex data points at 25°C. Different amounts of Ca2+ and La3+ (1040 µl of 1 mM, 10 mM and 1 M metal ion stock solutions at pH 6.9 and 7.4) were gradually added to the NMR sample tube. The 1D NMR spectra at different metal ion concentrations were the average of 1024 scans. Samples were incubated with the addition of metal ions about 30 min before the next acquisition. The data were processed with the program FELIX98 (MSI) with a squared sine-bell window function shifted over 75°. Post-acquisition suppression of the water signal was achieved by a Gaussian deconvolution function with a width of 20.
Data analysis
La3+ and Tb3+ binding affinities of proteins were calculated using data obtained from La3+ and Tb3+ titrations by Trp fluorescence change and Tb3+ fluorescence enhancement, respectively. The fluorescence intensity at 327 nm and the area of Tb3+ fluorescence enhancement at 545 nm in the absence of metal ions were used as S0 with 0% metal binding and the Trp fluorescence intensity at 327 nm and the area of Tb3+ fluorescence enhancement in saturated metal concentrations (2 mM LaCl3 or 0.3 mM TbCl3) were used as S100 with 100% metal binding. The fractional change values, f, at different metal concentrations were calculated using the equation f = (S S0)/(S100 S0).
Kd values of proteins that form a 1:1 proteinmetal complex were calculated by fitting the titration curves from fractional changes of Trp fluorescence at 327 nm or the area of Tb3+ fluorescence enhancement using the following equation:
where f is the fractional change of the signal and [M]T and [P]T are the total concentrations of metal ions and protein, respectively (Yang et al., 2000b).
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Results and discussion |
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Since the grafted EF-hand loop attached to Gly linkers inserted at three different protein environments has similar metal binding affinity, the insertion EF-loop is most likely to be largely solvated and functions independently from the host protein. Our previous work has shown that the calcium-binding affinity (Kd) of CaM-CD2-III-5G-52 measured by monitoring chemical shift changes as a function of Ca2+ concentration by NMR is 0.186 mM in 10 mM Tris and 10 mM KCl at pH 7.4 (Ye et al., 2001). This Kd is about 1030-fold greater than the published calcium-binding affinity of the isolated peptide/fragments of the same EF-loop under similar conditions (Malik et al., 1987
; Borin et al., 1989b
; Dadlez et al., 1991
; Linse and Forsén, 1995
; Kuhlman et al., 1997
; Yang et al., 2000b
). Our work has also shown that the attachment of three Gly residues at each end of the EF-loop is essential for metal binding since the calcium-binding affinity of EF-loop III in CD2 without Gly residues is reduced by more than 10-fold (Ye et al., 2001
). A similar effect was observed for other EF-hand proteins; for example, the EF-loop III of troponin C directly inserted into myoglobin without the glycine linker has a very weak calcium-binding affinity with a Kd of >10 mM (M.Nakamura, personal communication). Our modeling study demonstrated that the connection of three Gly residues at each end of the calcium-binding loop allows the EF-loop to have native-like calcium-binding geometry.
The field of calcium signaling has long wrestled with a difficult problem, how to evaluate the contributions of various amino acid chains to the coordination of the Ca2+ ion, when the free energy of binding includes an interaction with the other member of a pair of EF-hands as usually found in nature. In addition, an isolated EF-hand as peptide fragments from EF-hand proteins, such as calmodulin, troponin C and calbindin D9k, is not stable (Reid, 1990; Shaw et al., 1990
; Julenius et al., 2002
). Our measured metal-binding affinity of the EF-loop in the host protein should reflect the intrinsic metal-binding affinity of the grafted EF-loop, since neither native structure nor folding behavior of the host protein is changed by the insertion of the EF-loop. Our work opens up a new avenue to obtain the site-specific calcium-binding affinity of EF-hand proteins without the complexities encountered in cooperative, multi-site systems of natural EF-hand proteins, the conformational change of coupled EF-hand motifs or the limitations associated with peptide models in order to understand the calcium-signaling process.
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Acknowledgements |
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Received May 25, 2002; revised January 23, 2003; accepted April 25, 2003.