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J. Biol. Chem., Vol. 280, Issue 18, 18008-18014, May 6, 2005

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Mg²⁺ and Ca²⁺ Differentially Regulate DNA Binding and Dimerization of DREAM^*

Masanori Osawa, Alexandra Dace, Kit I. Tong¶, Aswani Valiveti, Mitsuhiko Ikura¶, and James B. Ames||

From the Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850 and the ^¶Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Ontario M5G 2M9, Canada

Received for publication, January 11, 2005 , and in revised form, February 22, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca²⁺ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca²⁺ binding to the various mutants revealed that, in the absence of Mg²⁺, Ca²⁺ binds independently and sequentially to EF-3 (H = -2.4 kcal/mol), EF-4 (H = +5.2 kcal/mol), and EF-2 (H = +1 kcal/mol). By contrast, only two Ca²⁺ bind to DREAM in the presence of physiological levels of Mg²⁺ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg²⁺. ITC measurements demonstrate that one Mg²⁺ binds enthalpically with high affinity (K_d = 13 µM and H = -0.79 kcal/mol) and two or more Mg²⁺ bind entropically in the millimolar range. Size-exclusion chromatography studies revealed that Mg²⁺ stabilizes DREAM as a monomer, whereas Ca²⁺ induces protein dimerization. Electrophoretic mobility shift assays indicated that Mg²⁺ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca²⁺ levels, suggesting that binding of a single Ca²⁺ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca²⁺ and Mg²⁺ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg²⁺ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca²⁺-induced protein dimerization disrupts DNA binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
DREAM¹ (downstream regulatory element antagonist modulator also known as calsenilin (1) and KChIP3 (2)) is a 29-kDa EF-hand Ca²⁺-binding protein that serves as a transcriptional repressor for pain modulation (3–5). DREAM translocates into the nucleus during calcium signaling (6) and blocks transcription of prodynorphin and c-fos genes by binding to specific DNA sequences (DRE, downstream regulatory element (7, 8)) in a Ca²⁺-dependent fashion (9–11). The importance of DREAM as a calcium-sensitive transcriptional repressor has been demonstrated in DREAM-deficient mice that exhibit the very striking phenotype of ongoing analgesia due to up-regulated expression of prodynorphin (12, 13). The DREAM knock-out mice also exhibit mild effects on -amyloid production and long-term potentiation (13), but do not exhibit any motor or behavioral abnormalities. Hence, DREAM regulates pain transmission by controlling prodynorphin expression and represents an attractive therapeutic opportunity for managing pain.

DREAM contains four EF-hands similar to the Ca²⁺-binding motifs found in calmodulin and troponin C (14, 15). The four EF-hands of DREAM are >45% identical in sequence to recoverin and related Ca²⁺ sensors of the neuronal calcium sensor (NCS) subclass of the EF-hand superfamily (16, 17) (see Fig. 1). A characteristic feature of DREAM and the NCS family is the sequence CPXG (Cys-104 and Pro-105, see Fig. 1) that prevents the binding of Ca²⁺ to the first EF-hand (EF-1) as seen in the crystal structures of recoverin (18), KChIP1 (19, 20), neurocalcin (21), and frequenin (22). The second EF-hand (EF-2) of DREAM contains aspartate (Asp-150) instead of the usual glutamate at the 12-position of the EF-hand binding loop (Fig. 1). The presence of aspartate at the 12-position in other EF-hand proteins is known to diminish its binding selectivity of Ca²⁺ versus Mg²⁺ (23). Mg²⁺ binding to the second EF-hand of DREAM might be sufficient to affect DREAM structure and DNA binding. Indeed, a recent mass spectrometry study revealed that both Ca²⁺ and Mg²⁺ are bound by DREAM (24). Magnesium is not normally considered a regulator, but recent in vivo measurements have detected changes in free Mg²⁺ concentrations in cortical neurons after treatment with neurotransmitter (25). Other NCS proteins such as GCAPs, VILIP, and NCS-1 also bind Mg²⁺ and exhibit Mg²⁺-induced effects (26, 27). Therefore, it is of interest to investigate the functional role of Mg²⁺ binding to DREAM and determine whether Mg²⁺ might control the binding of DREAM to DNA targets.

Here, we present a structural analysis of Mg²⁺ and Ca²⁺ binding to the individual EF-hands in DREAM. The results reveal that Mg²⁺ is required for sequence-specific DNA binding; Mg²⁺ binds constitutively at EF-2; Mg²⁺ stabilizes a monomeric form of DREAM; Ca²⁺ induces protein dimerization; and Ca²⁺ binding at either EF-3 or EF-4 is sufficient to abolish DNA binding at high Ca²⁺. On the basis of these results, a structural model for Ca²⁺-sensitive DNA binding by DREAM will be presented.

View larger version (60K): [in this window] [in a new window]   FIG. 1. Alignment of amino acid sequence of mouse DREAM with various members of the recoverin branch of the EF-hand superfamily. The Ca2+ binding loops of the four EF-hands are underlined. A negatively charged residue at the 12-position of the functional EF-hand loops (Glu or Asp in bold) were individually mutated to the corresponding neutral residue (Gln or Asn) to disable functional Ca2+ binding to the respective EF-hands.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Size Exclusion Chromatography—Determination of the molecular weight of the purified DREAM-C in solution was carried out on a Superdex 200 HR 10/30 column (Amersham Biosciences) at 4 °C in the buffers containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, and 5 mM CaCl₂ for Ca²⁺-DREAM-C, or 1 mM EGTA for Mg²⁺-bound DREAM-C. 0.1 ml of DREAM-C (protein concentration ranged from 10 to 460 µM) was loaded onto the column and eluted at a flow rate of 0.5 ml/min. Apparent molecular weights were calculated using a standard curve of V_e/V_o versus the log of the molecular masses of standard proteins: -amylase (200 kDa), alcohol dehydrogenase (150 kDa), transferrin (81 kDa), carbonic anhydrase (29 kDa), and myoglobin (17 kDa). V_o is a void volume obtained using blue dextran (2000 kDa), and V_e is a volume of elution.

Preparation of Decalcified Protein Solutions—Decalcified ITC buffer (150 mM NaCl and 10 mM Tris-HCl, pH 7.6) was prepared by treatment with Chelex 100 resin (Bio-Rad). 1 mM dithiothreitol and 5 mM MgCl₂ were added after treatment with Chelex 100 and immediately before use. Containers and pipette tips were rinsed with 1 M HCl twice followed by a rinse with decalcified buffer. EDTA was removed from protein solution using Econo-Pac 10 DG gel filtration column (Bio-Rad Laboratories, Inc.) pre-equilibrated in the decalcified buffer. Before the ITC experiment, the sample cell and injection syringe of ITC machine (Microcal Inc.) was cleaned by the decalcified buffer extensively.

Mg²⁺ and Ca²⁺ Titration into DREAM-C by ITC—Binding of Mg²⁺ and Ca²⁺ to DREAM-C was measured by ITC (28) using a MicroCal VP-ITC MicroCalorimeter (MicroCal Inc.). The protein solution used in the titration was exchanged with decalcified buffer using Econo-Pac 10 desalting gel-filtration column as described above. Stock solutions of MgCl₂ and CaCl₂ (0.5–2.0 mM), used as the titrant, were prepared using decalcified buffer as a diluent. A stock solution of CaCl₂ with 5 mM MgCl₂ was the titrant for Ca²⁺ titration of Mg²⁺-bound DREAM-C. A typical ITC experiment was performed at 25 °C on a protein solution (1.38 ml, 35–100 µM) by adding a total of 290 µl of concentrated MgCl₂ or CaCl₂ (0.50–2.0 mM) in 29 aliquots (10 µl each). The additions were 3 min apart to allow heat accompanying each increment to return to baseline prior to the next addition. For each addition, the molar heat (Q_t) was measured as a function of total ligand concentration (X_t),

(Eq. 1)

where n is the number of sites, M_t is the total protein monomer concentration, K_a is the metal-binding association constant (1/K_d), and V is the cell volume. The differential molar heat (dQ_t) measured in the ITC experiment was fit to various kinetic models (sequential versus independent sites) using a nonlinear least squares minimization method (28, 29). Thermodynamic parameters in the analysis are defined by,

(Eq. 2)

and

(Eq. 3)

where n is the number of moles, T is the absolute temperature, and R = 8.3151 J mol^-1 K^-1.

NMR Spectroscopy—Samples for NMR analysis were prepared by dissolving ¹⁵N-labeled DREAM-C (0.2–0.5 mM) in 0.3 ml of a 95% H₂O/5% [²H]H₂O solution containing 10 mM [²H₁₁]Tris (pH 7.4), 5 mM [²H₁₀]dithiothreitol, 50 mM KCl, and either 5 mM EDTA (apo), 5 mM MgCl₂ (Mg²⁺-bound), or 5 mM CaCl₂ plus 5 mM MgCl₂ (Mg²⁺/Ca²⁺-bound). All NMR experiments were performed at 32 °C on a Bruker Avance 600-MHz spectrometer equipped with a four-channel interface and triple-resonance probe with triple-axis pulsed field gradients. The ¹⁵N-¹H HSQC spectra (see Fig. 6) were recorded on samples of ¹⁵N-labeled DREAM-C (in 95% H₂O, 5% ²H₂O). The number of complex points and acquisition times were: 256, 180 ms (¹⁵N (F₁)), and 512, 64 ms (¹H (F₂)).

Fluorescent EMSA—Synthetic oligonucleotides (purchased from ACGT Corp., Toronto, Canada) representing the DRE of human prodynorphin (9, 10) were 5'-labeled using the fluorescent dye, Cy5 (Amersham Biosciences). The precise nucleotide sequence was 5'-GAAGCCGGAGTCAAGGAGGCCCCTG-3'. Complementary strands were denatured at 95 °C for 10 min and annealed by slowly cooling to room temperature in the presence of 20 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, and 0.1 mM EDTA. Formation of double-stranded DNA was checked on a 20% non-denaturing polyacrylamide/0.25 x TBE/1%glycerol gel prior to binding.

DREAM-C proteins (wild-type, D150N, E186Q, and E234Q) were exchanged firstly to 20 mM Tris-HCl, pH 7.4, 10% glycerol, 100 mM NaCl, 5 mM dithiothreitol, 0.5 mM EDTA using PD-10 columns and concentrated by Centricon 10 (Millipore) at 4 °C. Protein (0–70 µM, Fig. 7) was then incubated with 5 nM Cy5-labeled duplex DNA for 5 h at room temperature in 10 mM HEPES, pH 7.9, 8 mM MgCl₂, 0.1 mM EDTA, 2 mM dithiothreitol, 1 nM (0.05 unit/ml) of poly(dI-dC) (Amersham Biosciences), and with or without 10 mM CaCl₂. Protein·DNA complexes were resolved in 5% non-denaturing polyacrylamide/0.25 x TBE/1% glycerol gels at 200 V for 30 min in 0.25 x TBE and 0.5 mM MgCl₂ running buffer. Wet fluorescent gels were scanned using the red fluorescence mode of a Storm 860 system (Amersham Biosciences) with a voltage setting of 1000 V and a scanning resolution of 200 µM.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
ApoDREAM-C Binds Three Ca²⁺ Ions—Isothermal titration calorimetry (ITC, see Equations 1, 2, 3) was used in this study to analyze the energetics of Ca²⁺ and Mg²⁺ binding to the individual EF-hands of DREAM-C. Intrinsic metal binding to proteins is usually entropically driven (H > 0) due to very high dehydration energies of divalent cations. However, the enthalpy of binding can become exothermic (H < 0) if metal binding is coupled to a protein conformational change (30).

The Ca²⁺ binding properties of metal-free DREAM-C (apo-DREAM-C) were monitored by ITC (Fig. 2). Titration of CaCl₂ into apoDREAM-C (in the absence of Mg²⁺) resulted in a binding isotherm that is multiphasic and could be fit by the binding of three or more Ca²⁺ ions, consistent with previous binding studies (10, 24). The isotherm exhibited stoichiometric binding of two Ca²⁺ (K_d 1–10 µM) followed by endothermic binding of one or more Ca²⁺ ion(s) with much lower affinity. The binding isotherm is too complex to accurately quantitate the thermodynamic properties of each of the binding sites. To simplify the analysis, we have constructed various single-site mutants that disable functional Ca²⁺ binding to the individual EF-hands (Fig. 1): D150N (EF-2), E186Q (EF-3), and E234Q (EF-4). In each mutant, a negatively charged Glu or Asp at the 12-position in the EF-hand loop has been substituted with a corresponding neutral residue (Gln or Asn) that dramatically lowers the Ca²⁺ binding affinity of the respective EF-hand outside of the physiological range of Ca²⁺ (31, 32).

View larger version (19K): [in this window] [in a new window]   FIG. 2. Isothermal titration microcalorimetric analysis of Ca2+ binding to apoDREAM-C. Trace of the calorimetric titration of 29 x 10-µl aliquots of 1.3 mM CaCl2 into the cell containing 46 µM apo-DREAM-C (A), and integrated binding isotherm (B). No correction was needed for the heat of dilution, because negligible heat was observed when adding aliquots of CaCl2 (or MgCl2) into a decalcified buffer blank.

In summary, EF-2 is an endothermic site with low affinity that gives rise to the non-stoichiometric binding in the wild-type isotherm. EF-3 and EF-4 are relatively high affinity sites (stoichiometric binding) whose enthalpies nearly cancel one another in the wild-type isotherm. A recent mass spectrometry analysis of DREAM suggested that a fourth Ca²⁺ might bind to DREAM at EF-1 (24), although the affinity of this site was not reported, and the binding of a fourth Ca²⁺ might be artificially enhanced in gas phase species analyzed in mass spectrometry experiments. The primary sequence of EF-1 contains unfavorable substitutions (e.g. Cys-104, Pro-105, and Thr-113) that would be expected to abolish physiological Ca²⁺ binding at this site as has been demonstrated in other NCS proteins (16, 18, 19). Accordingly, our ITC Ca²⁺ binding measurements performed on samples of DREAM-C in solution do not detect any Ca²⁺ binding at EF-1, suggesting that either EF-1 does not bind Ca²⁺ under physiological conditions or the enthalpy of binding is zero.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Comparison of Ca2+ binding isotherms for DREAM-C and EF-hand mutants (D150N, E186Q, and E234Q). Ca2+ binding isotherms of apoDREAM-C (without MgCl2) and Mg2+-saturated protein (5 mM MgCl2) are depicted in A and B, respectively. Wild-type DREAM-C, D150N, E186Q, and E234Q are shown in black, red, green, and blue, respectively. A dotted line in panel B shows the normalized sum of isotherms for E186Q and E234Q mutants.

Constitutive Mg²⁺ Binding at EF-2—The presence of physiological levels of Mg²⁺ (5 mM Mg²⁺) had a profound affect on the ITC Ca²⁺ binding isotherms of wild-type and EF-hand mutants (Fig. 3B). The Ca²⁺ binding isotherm of wild-type in the presence of physiological Mg²⁺ was saturated with a Ca²⁺ binding stoichiometry of two, in contrast to a stoichiometry of three in the absence of Mg²⁺ (Fig. 3A). The Ca²⁺ binding isotherm of D150N in the presence of physiological Mg²⁺ looked remarkably similar to that of wild-type and was saturated with a Ca²⁺ binding stoichiometry of two. The Ca²⁺ binding isotherms of E186Q and E234Q in the presence of physiological Mg²⁺ both saturated with a Ca²⁺ binding stoichiometry of one. These results imply that Ca²⁺ does not bind to EF-2 in the presence of Mg²⁺ (Fig. 3B), suggesting that EF-2 binds constitutively to Mg²⁺ under physiological conditions (i.e. 5 mM Mg²⁺ and 0.1–100 µM Ca²⁺). The presence of Asp-150 (instead of Glu) at the 12-position of the Ca²⁺ binding loop of EF-2 might explain the loss of Ca²⁺ selectivity by EF-2. A shorter side chain at the 12-position would energetically favor binding of Mg²⁺ over Ca²⁺ as has been seen in other EF-hand proteins (23). Constitutive Mg²⁺ binding at EF-2 might somehow mediate the binding of DREAM to DNA targets.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Isothermal titration microcalorimetric analysis of the Mg2+ binding to DREAM-C. Trace of the calorimetric titration of 29 x 10-µl aliquots of 2.0 mM MgCl2 into the cell containing 69 µM apoDREAM-C (A) and integrated binding isotherm (B). The binding isotherm in B was fit using a two-site model where n1 = 0.91 ± 0.01, K1 = (7.5 ± 0.7) x 104 [M-1], H1 = -791 ± 90 [cal·mol-1]; n2 = 2 (fixed), K2 = (4.3 ± 0.2) x 103 [M-1], H2 = (3.97 ± 0.06) x 103 [cal·mol-1].

DREAM-C Has a High Affinity Mg²⁺ Site—The affect of Mg²⁺ on the Ca²⁺ binding isotherm of DREAM-C (Fig. 3) prompted us to quantitate the Mg²⁺-binding properties of DREAM using isothermal titration calorimetry (ITC). Titration of MgCl₂ into DREAM-C resulted in a two-phase binding isotherm for wild-type (Fig. 4) and each of the EF-hand mutants (data not shown). An exothermic phase at the beginning of the isotherm represents binding of a single Mg²⁺ followed by endothermic binding of non-stoichiometric Mg²⁺ (i.e. 1 or more Mg²⁺ with K_d > protein concentration used for ITC). Analysis of the binding isotherm using a "two sites" model (Microcal Origin software) revealed that DREAM-C has one Mg²⁺ site with relatively high affinity (dissociation constant of 13 µM and H = -0.79 kcal/mol), and two or more sites with much lower affinity (K_d in the millimolar range and H = +3.97 kcal/mol). Intrinsic Mg²⁺ binding to most proteins is usually entropically driven (30). The enthalpic binding of Mg²⁺ to DREAM-C suggests a possible Mg²⁺-induced conformational change. NMR spectra of DREAM-C change quite dramatically upon the addition of saturating Mg²⁺ (see below), suggesting that Mg²⁺ binding stabilizes tertiary structure of DREAM-C.

Also noteworthy was our observation that each EF-hand mutant exhibited Mg²⁺ binding similar to that of wild-type. Apparently, the EF-hand mutation at the 12-position in our study does not impair Mg²⁺ binding like it does for Ca²⁺. Different coordination numbers for Mg²⁺ and Ca²⁺ might explain their different binding behavior in this regard (33).

Mg²⁺/Ca²⁺ Effects on Oligomerization of DREAM-C—Previous dynamic light scattering and size-exclusion chromatography (SEC) analysis of metal-free DREAM-C (apoDREAM-C) originally indicated a mixture of aggregated protein species in solution (10). The presence of 10 mM lauryldimethylamine oxide detergent increased the solubility of apoDREAM-C and stabilized the metal-free protein in a tetrameric state. The DREAM tetramer is believed to bind functionally to DNA response elements in the prodynorphin and c-fos genes (9). In our current SEC study (Fig. 5), we now determine that DREAM-C in the presence of physiological Mg²⁺ levels (5 mM MgCl₂ and absence of detergent or DNA) eluted with an apparent molecular mass of 30 kDa in solution, which is slightly higher than the mass calculated for a protein monomer (25 kDa). The slightly higher apparent molecular mass in solution (30 versus 25 kDa) might be attributed to a non-spherical shape factor or perhaps a small percentage of dimer formation and was independent of protein concentration below 0.4 mM. Dynamic light scattering analysis similarly revealed a monomeric hydrodynamic radius for Mg²⁺-bound DREAM-C. These results suggest that Mg²⁺ binding to one or more EF-hands (Fig. 4) stabilizes the protein mainly in a monomeric state at protein concentrations below 0.4 mM. By contrast, Ca²⁺-saturated DREAM-C in the presence of Mg²⁺ eluted with an apparent molecular mass of 50 kDa in solution (Fig. 5), indicating that the Ca²⁺-bound protein forms a stable dimer even at very dilute protein concentrations (10). Similar calcium-sensitive hydrodynamic properties were observed for each of the EF-hand mutants (data not shown), suggesting that each mutant is structurally intact. In summary, DREAM-C appears to be a monomer under basal physiological conditions (5 mM Mg²⁺ and 100 nM Ca²⁺) and forms a stable dimer at saturating Ca²⁺ levels. A similar Ca²⁺-induced dimerization has been reported previously for other NCS proteins such as neurocalcin (21).

Mg²⁺ and Ca²⁺ Stabilizes Tertiary Structure of DREAM-C— NMR spectroscopy was used to probe protein conformational changes in DREAM-C induced by Mg²⁺ and/or Ca²⁺ binding (Fig. 6). The peaks in the ¹H-¹⁵N HSQC NMR spectra represent main-chain and side-chain amide groups and provide a fingerprint of the overall protein conformation. The two-dimensional ¹H-¹⁵H HSQC spectrum of apoDREAM-C exhibited poorly resolved and overlapping peaks with narrow chemical shift dispersion in the amide proton dimension (Fig. 6A). The number of observed peaks was far less than the expected number of amide groups, and the intensities of many peaks were quite weak perhaps due to exchange broadening caused by conformational heterogeneity. The poor chemical shift dispersion demonstrates that apoDREAM-C adopts an unstructured molten-globule state similar to that described for apo states of many other EF-hand proteins (30, 34, 35). Consistent with this interpretation is a lack of long range NOEs (particularly between aromatic and aliphatic resonances) in the homonuclear NOESY spectrum of apoDREAM-C (data not shown). However, circular dichroism analysis suggested that apoDREAM-C adopts a high degree of helical content, consistent with the formation of the four EF-hands, and the helical content did not change much upon binding Mg²⁺ and/or Ca²⁺. Taken together, our structural studies indicate that apoDREAM-C adopts native secondary structure but is in a flexible molten-globule state.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Molecular weight estimation of DREAM-C by size exclusion chromatography (SEC). A, size exclusion chromatograms of Mg2+-(solid line) and Mg2+/Ca2+-DREAM-C (dotted line). 100 µl of 200 µM protein solution were applied onto Superdex 200 HR (10/30) column (Amersham Biosciences) pre-equilibrated with the buffers containing 10 mM Hepes·NaOH (pH 7.6), 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and 1 mM EGTA or 5 mM CaCl2 for Mg2+-DREAM-C and Ca2+-DREAM-C, respectively. B, the standard curve for the molecular mass determination was created using -amylase (200 kDa), alcohol dehydrogenase (150 kDa), transferrin (81 kDa), carbonic anhydrase (29 kDa), and myoglobin (17 kDa). Void volume of the column (Vo) was obtained as 7.81 ml using blue dextran (2000 kDa), and Ve is a volume of elution of each sample. Arrows indicate Ve/Vo values for Mg2+- or Ca2+-bound DREAM-C (50 µM). The molecular mass based on amino acid sequence of DREAM-C is 25 kDa.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Two-dimensional 15N-1H HSQC NMR spectra of apo (A), Mg2+-bound (B), and Ca2+-bound (C) DREAM-C. Each protein sample was uniformly labeled with nitrogen 15, and spectra were recorded at 600-MHz 1H frequency.

DNA Binding by EF-hand Mutants versus Ca²⁺—Electrophoresis mobility shift assays (EMSA) were performed on the EF-hand mutants to monitor their Ca²⁺-sensitive binding to duplex oligonucleotide derived from the downstream response element (DRE) of the prodynorphin gene (Fig. 7). The EMSA binding data for wild-type and single-site EF-hand mutants look very similar, suggesting that each of the mutants bind to the DRE and are functionally intact. A typical EMSA for DREAM is shown in Fig. 7 and exhibits two shifted bands that represent different oligomeric species of DREAM bound to DRE. The lower shifted band has variable intensity, is not sequence-specific, is less responsive to Ca²⁺, and may represent DNA binding by the DREAM dimer or monomer. The uppermost band (believed to represent a DREAM tetramer) has been characterized previously to represent sequence-specific binding to DRE (9, 10), and we found in this study that this band requires saturating levels of Mg²⁺. The Mg²⁺-dependent DRE binding to D150N mutant suggests that this mutation does not impair high affinity Mg²⁺ binding at EF-2 (Fig. 4) perhaps needed for DRE binding. The EMSA shifted bands for wild-type and each of the EF-hand mutants (D150N, E186Q, and E234Q) disappear upon the addition of saturating Ca²⁺, suggesting that the Ca²⁺-bound forms of the EF-hand mutants (like wild-type) do not bind to DRE. In summary, Mg²⁺ is required for sequence-specific binding to DRE and Ca²⁺ binding at either EF-3 or EF-4 is sufficient to abolish DNA binding.

View larger version (80K): [in this window] [in a new window]   FIG. 7. Electrophoretic mobility shift assay of DREAM-C/DRE interaction. 5 nM Cy5 labeled DRE oligonucleotide in 10 mM HEPES, pH 7.9, 2 mM dithiothreitol, and 8 mM Mg2+ was incubated with 0–70 µM wild-type (A), D150N (B), E186Q (C), and E234Q (D) DREAM-C in the presence and absence of 10 mM Ca2+.

A second implication of this study is that Ca²⁺ binding at EF-3 and EF-4 promotes protein dimerization that somehow disrupts DNA binding by DREAM. In the Ca²⁺-bound protein, residues at the dimer interface (perhaps in EF-3 or EF-4) might sterically block residues of the Ca²⁺-free protein that contact DNA. The recent x-ray structure of a closely related DREAM-like protein, KChIP1, shows a dimeric structure for the Ca²⁺-bound protein with intermolecular contacts between a hydrophobic groove (formed by EF-1 and EF-2) and a C-terminal helix adjacent to EF-4 (19). The C-terminal helix of KChIP1 (residues 205–215) forms extensive intramolecular contacts with both EF-3 and EF-4, whereas the opposite face of the C-terminal helix forms intermolecular contacts with EF-1 and EF-2. It is plausible that Ca²⁺-induced conformational changes in either EF-3 or EF-4 would alter the disposition of the C-terminal helix and promote intermolecular contact with EF-1 and EF-2. An intriguing hypothesis follows that Ca²⁺-induced dimerization might sterically alter the structure of EF-2 (due to an interaction with the C-terminal helix) and thereby disrupt an Mg²⁺ ion bridge with DNA. In the future, we plan to determine the atomic-level structure of DREAM-DNA complex and Ca²⁺-induced conformational changes to more rigorously understand the structural basis of Ca²⁺-sensitive DNA binding by DREAM.

	FOOTNOTES

 
^* This work was supported in part by a Beckman Foundation Young Investigator Award (to J. B. A.) and Grants NS45909 and EY12347 from the National Institutes of Health (to J. B. A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by postdoctoral fellowships from the Human Frontier Science Program and Uehara Memorial Foundation.

^|| To whom correspondence should be addressed: The Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Dr., Rockville, MD 20850. Tel.: 301-738-6120; Fax: 301-738-6255; E-mail: james{at}carb.nist.gov.

¹ The abbreviations used are: DREAM, downstream regulatory element antagonist modulator; DREAM-C, deletion mutant of mouse DREAM consisting of residues 65–256; apoDREAM-C, metal-free DREAM; Mg²⁺-DREAM-C, Mg²⁺-bound DREAM; Ca²⁺-DREAM-C, Ca²⁺-bound DREAM; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; KChIP, potassium channel-interacting protein; SEC, size exclusion chromatography; EMSA, electrophoretic mobility-shift analysis; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy.

	ACKNOWLEDGMENTS

 
We thank Fred Schwarz for assistance with the ITC experiments, Fenhong Song for oligonucleotide DNA synthesis, and Nese Sari for help with NMR experiments.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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