Mg²⁺-dependent Interactions of ATP with the Cystathionine-β-Synthase (CBS) Domains of a Magnesium Transporter

Background: CNNMs are evolutionarily conserved Mg²⁺ transporters that possess CBS domains.

Results: CBS domains are essential for molecular function, and they bind to ATP in a Mg²⁺-dependent manner.

Conclusion: The unique binding feature with ATP may contribute to Mg²⁺ transport by CNNMs.

Significance: This is the first report showing the Mg²⁺-dependent and specific interactions of ATP with CBS domains.

Abstract

Ancient conserved domain protein/cyclin M (CNNM) family proteins are evolutionarily conserved Mg²⁺ transporters. However, their biochemical mechanism of action remains unknown. Here, we show the functional importance of the commonly conserved cystathionine-β-synthase (CBS) domains and reveal their unique binding ability to ATP. Deletion mutants of CNNM2 and CNNM4, lacking the CBS domains, are unable to promote Mg²⁺ efflux. Furthermore, the substitution of one amino acid residue in the CBS domains of CNNM2, which is associated with human hereditary hypomagnesemia, abrogates Mg²⁺ efflux. Binding analyses reveal that the CBS domains of CNNM2 bind directly to ATP and not AMP in a manner dependent on the presence of Mg²⁺, which is inhibited in a similar pattern by the disease-associated amino acid substitution. The requirement of Mg²⁺ for these interactions is a unique feature among CBS domains, which can be explained by the presence of highly electronegative surface potentials around the ATP binding site on CNNM2. These results demonstrate that the CBS domains play essential roles in Mg²⁺ efflux, probably through interactions with ATP. Interactions with ATP, which mostly forms complexes with Mg²⁺ in cells, may account for the rapid Mg²⁺ transport by CNNM family proteins.

Introduction

Magnesium is an essential element involved in a wide variety of biological activities. The homeostasis of magnesium levels is strictly regulated by intestinal absorption and renal reabsorption, in which the epithelia function as a barrier that permits selective and regulated transport of Mg²⁺ from apical to basolateral surfaces. Genomic analyses of familial cases of hypomagnesemia have identified key molecules directly involved in these processes (1, 2). One of the least characterized members among these is the ancient conserved domain protein/cyclin M (CNNM)³ family.

CNNM family proteins commonly possess a domain that is highly conserved from bacteria to humans (3). In humans, the CNNM family consists of four members, CNNM1–4. These proteins show a multidomain structure, composed of a sequence motif present in the cyclin box, a cyclic nucleotide monophosphate binding domain, two tandem cystathionine-β-synthase (CBS) domains, and a DUF21 domain (3). Significant evidence has suggested the importance of CNNMs in Mg²⁺ transport (4–6). Our recent study revealed that CNNM4 localizes to the basolateral membrane of intestinal epithelial cells and extrudes intracellular Mg²⁺ to the outside of the cells in exchange for Na⁺, thereby mediating the transcellular transport of Mg²⁺ through the epithelia (7). However, the functional importance of each domain or motif present in the CNNM family is poorly understood. The DUF21 and CBS domains are supposed to be important for the biological function, as stated by the studies on human hereditary diseases caused by mutations in the CNNM family gene. Several point mutations that cause Jalili syndrome, which is characterized by recessive amelogenesis imperfecta, occur in the DUF21 domain-encoding region in CNNM4 (8, 9). In addition, a point mutation that causes familial dominant hypomagnesemia, a rare human disorder characterized by renal Mg²⁺ wasting, occurs in the CBS domain-encoding region in CNNM2 (6).

CBS domains are small protein modules (typically 60 amino acid residues in length) that usually exist as two tandem repeats, each of which is made up of highly conserved two α-helices and three β-strands arranged in a β-α-β-β-α order (10). CBS domains were originally identified in human cystathionine-β-synthase (11) and subsequently found in thousands of cytosolic and membrane-associated proteins from all kingdoms of life (10). In addition to CNNMs, there is an example of a CBS domain-possessing Mg²⁺ transporter, and in the case of bacterial MgtE, the CBS domains are proposed to be important for the regulation of Mg²⁺ transporting activity (12, 13). The physiological importance of CBS domains has been demonstrated by a number of observations that point mutations found in several hereditary diseases occur in the CBS domain-encoding region in the genes and disrupt their protein functions (14). Recent studies have shown that adenine nucleotides, such as ATP, ADP, or AMP, can bind to CBS domains of several proteins, such as AMP-activated protein kinase (AMPK), inosine-5′-monophosphate dehydrogenase (IMPDH), and chloride channel (ClC) family proteins (10, 15). The functional consequences and physiological importance of interactions with adenine nucleotides have been characterized most extensively in the case of AMPK. The CBS domains of the regulatory γ-subunit of AMPK have two discrete sites that can bind to AMP, ADP, and ATP (named as site 1 and site 3) (16, 17). Structural and biochemical studies have shown that AMP or ADP binding, but not ATP binding, to site 3 promotes phosphorylation and inhibits the dephosphorylation of Thr-172 on the catalytic α-subunit, thereby maintaining AMPK in its most active form (17, 18). Phosphorylated AMPK is further activated by AMP binding, but not by either ADP or ATP binding, to site 1 (17, 18). Such intricate regulations of the activity are impaired by disease-associated amino acid substitutions found in patients with Wolff-Parkinson-White syndrome, indicating the physiological importance of interactions with adenine nucleotides (15, 17).

In this study, we examined the functional importance of the CBS domains of CNNM family proteins. Binding analyses revealed that the CBS domains of CNNM2 bind to ATP, but not AMP, in a manner dependent on the presence of Mg²⁺. A mutation in the CBS domains of CNNM2 associated with hereditary hypomagnesemia and the equivalent mutation of CNNM4 completely abrogated their Mg²⁺ efflux functions. Furthermore, this mutation also abrogated the binding of the CBS domains of CNNM2 to ATP. These results demonstrate the crucial importance of the CBS domains for Mg²⁺ efflux function of CNNM family proteins probably via the interaction with ATP.

EXPERIMENTAL PROCEDURES

Materials

AMP sodium salt, ATP disodium salt hydrate, and the mouse and rabbit anti-FLAG antibodies were purchased from Sigma-Aldrich. [2,8-³H]AMP (15 Ci/mmol) was purchased from Movarek, and [α-³²P]ATP (3000 Ci/mmol) was purchased from Izotop. Magnesium Green-AM and rhodamine-phalloidin were purchased from Invitrogen.

Constructs

cDNAs for mouse CNNM2 and human CNNM3 were obtained by performing PCR and verified by DNA sequencing. Human CNNM1 cDNA was purchased from Invitrogen (IMAGE: 40006972), and human CNNM4 cDNA was generated in a previous study (7). Amino acid-substituted mutants of mouse CNNM2 (T568I) and human CNNM4 (T495I) were generated using the QuikChange Site-directed Mutagenesis kit (Agilent). ΔCBS mutants of CNNM2(Δ469–578) and CNNM4(Δ396–505) deletion constructs were generated by ligating two PCR fragments. These cDNA fragments were inserted into pCMV-4A (Stratagene) for expression in mammalian culture cells. cDNAs for the CBS domains of human CNNM1(425–568), mouse CNNM2(442–579), human CNNM3(310–447), human CNNM4(359–511), and human IMPDH2(112–232) were obtained by PCR and verified by DNA sequencing. cDNA for the CBS domains of human ClC-5(568–746) was kindly provided by Dr. R. Dutzler (19). These cDNA fragments were inserted into pGEX-6p1 (GE Healthcare) for bacterial expression.

Cell Culture and Transfection

HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Expression plasmids were transfected in each cell by using Lipofectamine 2000 (Invitrogen).

Mg²⁺ Imaging Analyses

Mg²⁺ imaging analyses with Magnesium Green were performed as described previously (7).

Preparation of Recombinant Proteins

Recombinant proteins of CBS domains of CNNM1–4, ClC-5, and IMPDH2 were expressed as GST fusion forms in Escherichia coli by using the expression constructs in pGEX-6p1 and were purified using glutathione-Sepharose beads (GE Healthcare). For surface plasmon resonance (SPR) analysis, the GST tag was cleaved off by digestion with PreScission protease (GE Healthcare).

SPR Analyses

The binding of Mg²⁺ and ATP to the CBS domains of CNNM2 was analyzed using Biacore T200 instrument with an S series CM5 sensor chip (GE Healthcare). GST-cleaved CBS-WT and CBS-T568I were immobilized onto the sensor chip by using a standard amine coupling protocol. Binding analyses were performed in running buffer (50 mm HEPES-KOH (pH 7.0), 140 mm KCl, 1 mm 2-mercaptoethanol, and 0.05% Tween 20) at 25 °C in the presence of 0, 0.5, or 10 mm MgCl₂. For analyses of Mg²⁺ binding, MgCl₂ was injected as an analyte in running buffer without MgCl₂. SPR data were analyzed using the Biacore T200 Evaluation software and fit to a 1:1 binding model using the following equation, R = (R_max × C)/(K_d + C), where R is the observed response, R_max is the maximal binding capacity, C is the analyte concentration, and K_d is the dissociation constant. For the injection of ATP with buffer switching, the dual injection mode was applied, according to the manufacturer's instructions. Observed responses were normalized to R_max for each experiment and are represented as normalized response unit (RU). For normalization of sensorgrams for ATP injection without significant responses, R_max of SPR responses to ATP injection in the presence of Mg²⁺ 10 mm from the CBS-WT surface was used.

Filter Binding Assays

Filter binding assays were performed in 10-μl reaction mixtures containing reaction buffer (50 mm HEPES-KOH (pH 7.0), 140 mm KCl, 1 mm 2-mercaptoethanol), 50 μm recombinant proteins, and various concentrations of [α-³²P]ATP (500 cpm/pmol) or [2,8-³H]AMP (60 cpm/pmol) in the presence or absence of 10 mm MgCl₂. The reaction systems were incubated for 20 min at 25 °C and immediately filtered through nitrocellulose membranes (25-mm diameter, 0.22-μm pore size; Millipore), which were prewashed with reaction buffer and washed with 1 ml of ice-cold reaction buffer. Filters were dried, and the radioactivity was determined by scintillation counting using a LS-6500 scintillation counter (Beckman). Nonspecific binding was determined by control assays with GST and was subtracted. The data were analyzed using the GraphPad Prism software (version 4.0) and fit to a 1:1 binding model using the same equation used in SPR analyses, where R was substituted with the amount of bound adenine nucleotides, C was substituted with the concentration of adenine nucleotides, and R_max was substituted with estimated maximum binding of adenine nucleotides. The data were normalized to the percentage of estimated maximum binding of ATP and are represented as the mean ± S.E. of three independent experiments. For competition assays, recombinant proteins were incubated with 5 μm [α-³²P]ATP (5000 cpm/pmol) and reaction buffer in the presence of increasing concentrations of ATP or AMP and were subsequently processed as described above.

Structural Analyses

Homology models for the CBS domains of CNNM1, CNNM2, and CNNM4 in complex with ATP were built using Spanner (20) based on the structures of the CBS domains of CorC (a bacterial homolog of CNNMs) from Bordetella parapertussis (Protein Data Bank (PDB) ID 3JTF), and that of IMPDH2 was built using Spanner based on the structure of and IMPDH from Pseudomonas aeruginosa (PDB ID 4DQW) (21). ATP was placed into the predicted binding sites, based on the similar structures in complex with ATP found from GIRAF (22). Electrostatic surface potentials were calculated for these modeled structures and the crystal structure of the CBS domains of human ClC-5 in complex with ATP (PDB ID 2J9L) (19) using the Adaptive Poisson-Boltzmann Solver software (23), mapped on the solvent-accessible surface, and visualized in PyMOL.

RESULTS

Indispensable Role of the CBS Domains in Mg²⁺ Efflux

In our previous study, we reported that CNNM4 can stimulate Mg²⁺ efflux when expressed in HEK293 cells (7). To examine whether the other CNNM family proteins also have the ability to stimulate Mg²⁺ efflux, we transfected HEK293 cells with CNNM1-FLAG, CNNM2-FLAG, CNNM3-FLAG, or CNNM4-FLAG (Fig. 1A, lower panel) and then, performed imaging analyses with Magnesium Green, a fluorescent indicator for Mg²⁺. Cells were first loaded with Mg²⁺ by bathing them in a solution containing 40 mm Mg²⁺ and then exposed to a Mg²⁺-free solution to promote Mg²⁺ efflux artificially. The intensity of fluorescent signals in the cells expressing CNNM4-FLAG rapidly decreased immediately after Mg²⁺ depletion, whereas only a very subtle decrease was observed in empty vector-transfected cells (Fig. 1A, top), thereby confirming our previous finding (7). A significant level of Mg²⁺ efflux was also observed in cells expressing CNNM2-FLAG. In contrast, only a very subtle level of Mg²⁺ efflux was observed in cells expressing CNNM1-FLAG, and no significant Mg²⁺ efflux was observed in the cells expressing CNNM3-FLAG. These data indicate that CNNM2 can also potently stimulate Mg²⁺ efflux, similar to the efflux induced by CNNM4, when expressed in HEK293 cells. To investigate the importance of the CBS domains of CNNM family proteins in their Mg²⁺ efflux functions, we constructed deletion mutants of CNNM2 and CNNM4 lacking CBS domains, called CNNM2-ΔCBS and CNNM4-ΔCBS, respectively. Similar Mg²⁺ imaging analyses showed that these ΔCBS mutants could not stimulate Mg²⁺ efflux (Fig. 1B). We performed confocal microscopy and confirmed that these ΔCBS mutants, like WT, were localized mostly to the plasma membrane (Fig. 1C). Collectively, these results indicate the importance of the CBS domains in Mg²⁺ efflux functions.

FIGURE 1.

Indispensable role of the CBS domains in Mg²⁺ efflux. A and B, HEK293 cells transfected with the indicated constructs were subjected to Mg²⁺ efflux assays. The means of the relative fluorescence intensities of 10 cells are indicated. The arrowheads indicate the start point of Mg²⁺ depletion. The results of Western blotting analyses with the anti-FLAG antibody are also shown to indicate the similar level of expression for each CNNM isoform (A, lower panel). C, HEK293 cells transfected with the indicated constructs were subjected to immunostaining with anti-FLAG (green) and rhodamine-phalloidin (red) and were observed with a confocal microscope. Scale bar, 10 μm.

Disease-associated Amino Acid Substitution in the CBS Domains Completely Abrogates Mg²⁺ Efflux

Mutations in the CNNM2 gene have recently been reported to be associated with human familial dominant hypomagnesemia (6). Two different mutations have been shown to occur in patients, and one of the mutations is an amino acid substitution of the threonine 568 residue with isoleucine, which is located in the CBS domains of CNNM2 (6). As shown in Fig. 2A, this threonine residue is completely conserved among CNNM family members across various species, ranging from bacteria to humans (indicated with the asterisk). To assess the importance of this mutation in the Mg²⁺ efflux function, we constructed the amino acid substitution mutants CNNM2-T568I and CNNM4-T495I, which is an equivalent mutant of CNNM4, and performed Mg²⁺-imaging analyses with Magnesium Green, as described previously. Almost no Mg²⁺ efflux was observed in the cells expressing CNNM2-T568I or CNNM4-T495I (Fig. 2B). These mutant proteins, like WT proteins, were also localized mostly to the plasma membrane (Fig. 2C). These results confirm the importance of the highly conserved and disease-associated threonine residue in Mg²⁺ efflux functions, further supporting the role of the CBS domains.

FIGURE 2.

Disease-associated amino acid substitution in the CBS domains abrogates Mg²⁺ efflux. A, sequence alignment of two tandem CBS domains of CNNM family proteins from the following various species (NCBI protein database accession numbers are given in parentheses): Homo sapiens CNNM2 (NP_060119.3), Mus musculus CNNM2 (NP_291047.2), Gallus gallus CNNM2 (XP_426532.3), Danio rerio CNNM2 (NP_001138257.1), Drosophila melanogaster Unextended (ELP57407.1), Caenorhabditis elegans C52D10.12 (CAM36329.1), Saccharomyces cerevisiae Mam3p (EIW07873.1), and Bordetella parapertussis CorC (NP_883455.1). Identical residues in all of the species are boxed in black, and residues with one exception are boxed in gray. The asterisk indicates the conserved threonine residue, whose amino acid substitution in human CNNM2 is associated with familial dominant hypomagnesemia. B, HEK293 cells transfected with the indicated constructs were subjected to Mg²⁺ efflux assays. The means of relative fluorescence intensities of 10 cells are indicated. The arrowhead indicates the start point of Mg²⁺ depletion. C, HEK293 cells transfected with the indicated constructs were subjected to immunostaining with anti-FLAG (green) and rhodamine-phalloidin (red) and were observed using a confocal microscope. Scale bar, 10 μm.

ATP Binds to the CBS Domains of CNNM2 in a Mg²⁺-dependent Manner

Adenine nucleotides have been shown to interact with the CBS domains of several proteins and to regulate their activities (10, 15). To investigate the possibility that adenine nucleotides can also interact with the CBS domains of CNNM family proteins, we performed binding analyses using SPR. Recombinant proteins of the CBS domains of wild-type CNNM2 (CBS-WT) were immobilized on the surface of a sensor chip, and various concentrations of ATP were injected over the SPR sensor surface as an analyte by using the running buffer that does not contain any divalent cation. However, no significant response was observed (data not shown). Because there are examples of proteins for which binding to ATP depends on the presence of Mg²⁺ (24–27), we determined to examine the interaction in the presence of Mg²⁺. Before ATP binding analyses, we checked SPR responses using Mg²⁺ itself as an analyte. The results in Fig. 3A show a gradual and dose-dependent increase of responses, indicating the occurrence of direct interaction between Mg²⁺ and the CNNM2 CBS domains. However, this increase of the SPR response never reached plateau even at 10 mm, and thus, it was impossible to determine the K_d value for the interaction within the physiological range (0.5–1 mm). We then performed SPR analyses to examine the interaction of CBS-WT with ATP by setting the concentrations of Mg²⁺ to 0.5 mm and 10 mm. As shown in Fig. 3B, significant and dose-dependent SPR responses were observed, and the levels of the responses were similar, indicating that ATP can bind to the CNNM2 CBS domains under both conditions. Thus, we determined to set the concentration of Mg²⁺ in the running buffer as 10 mm in the following experiments. First, we quantitatively compared the ATP binding ability of CBS-WT and CBS-T568I. In the absence of Mg²⁺, we observed no SPR response (Fig. 4A). In the presence of Mg²⁺, significant and concentration-dependent SPR responses were observed from the CBS-WT surface but not from the CBS-T568I surface (Fig. 4B), indicating the occurrence of the Mg²⁺-dependent interaction between the CNNM2 CBS domains and ATP, which is abolished by the disease-associated mutation. As one adenine nucleotide typically binds to two tandem CBS domains (10), we fitted the steady-state plot of the CBS-WT surface response in the presence of Mg²⁺ to a 1:1 binding model (Fig. 4C) and estimated K_d of 141 ± 12 μm (represented as the mean ± S.E. from three independent experiments), which is within the range of those reported for other CBS domains (15).

FIGURE 3.

ATP binds to the CBS domains of CNNM2 in a Mg²⁺-dependent manner. A, sensorgrams showing the binding of Mg²⁺ to immobilized CBS-WT (left panel). Mg²⁺ concentration varied from 0.1 to 10 mm (0.1, 0.25, 0.5, 1, 2.5, 5, or 10 mm). Representative data of three independent experiments are shown. RU, response unit. Steady-state analyses for binding of Mg²⁺ to CBS-WT surface are also shown (right panel). Equilibrium responses (R_eq) were plotted as a function of Mg²⁺ concentration and fitted with a 1:1 binding model. B, sensorgrams showing the binding of ATP to immobilized CBS-WT in the presence of 0.5 mm Mg²⁺ (left panel) or 10 mm Mg²⁺ (right panel). ATP concentration varied from 10 to 500 μm (10, 25, 50, 75, 100, 250, or 500 μm). Representative data of three independent experiments are shown.

FIGURE 4.

Disease-associated amino acid substitution abrogates the interaction with ATP. A and B, sensorgrams showing the binding of ATP to immobilized CBS-WT (left panel) or CBS-T568I (right panel) in the absence of Mg²⁺ (A) or in the presence of 10 mm Mg²⁺ (B). ATP concentration varied from 10 to 500 μm for each experiment. Representative data of three independent experiments are shown. RU, response unit. C, steady-state analysis for binding of ATP to CBS-WT and CBS-T568I surfaces in the presence or absence of 10 mm Mg²⁺. Equilibrium responses (R_eq) extracted from A and B were plotted as a function of ATP concentration and fitted with a 1:1 binding model. D, sensorgrams showing the binding of ATP to immobilized CBS-WT (left panel) or CBS-T568I (right panel) under the sequential changes of Mg²⁺ concentration. ATP (100, 250, or 500 μm) was injected with a buffer containing no Mg²⁺ for 2 min, and then the buffer was switched to one containing 10 mm Mg²⁺. The arrowhead indicates the time point of the buffer switch. Representative data of three independent experiments are shown.

To further confirm the Mg²⁺ dependence of ATP binding to the CBS domains of CNNM2, we performed SPR analysis with sequential changes of Mg²⁺ concentration in the running buffer. ATP was first injected in the running buffer without Mg²⁺ and then in a buffer containing Mg²⁺, and changes in SPR responses were observed. As shown in Fig. 4D, significant SPR responses were observed with CBS-WT just after the injection of Mg²⁺, and the amplitude of the responses increased in a manner dependent on the ATP concentration. In contrast, there was no significant SPR response observed from CBS-T568I even after the Mg²⁺ injection. Thus, ATP binding to the CBS domains of CNNM2 requires the presence of Mg²⁺, indicating that the CBS domains of CNNM2 bind to ATP in a Mg²⁺-dependent manner.

The Specificity and Uniqueness of ATP Binding to the CBS Domains of CNNM2

To further ascertain the Mg²⁺ dependence of ATP binding to the CBS domains of CNNM2, we performed filter binding assays using [α-³²P]ATP as a ligand to detect the amount of ATP bound to proteins trapped on the filter. The results of assays in the presence of 10 mm Mg²⁺ showed a clear saturated binding curve with apparent K_d of 159 ± 28 μm, whereas no binding was observed in the absence of Mg²⁺ (Fig. 5A and Table 1). 10 mm Mg²⁺ is much more than the normal cytosolic concentration of Mg²⁺, and thus, we also performed filter binding assays using the same concentrations of Mg²⁺ as those of ATP. As shown in Fig. 5A, CBS-WT showed similar levels of ATP binding signals compared with the experiments using constant 10 mm Mg²⁺, when the concentrations of ATP and Mg²⁺ were set to 200, 400, and 800 μm, which are far above the reported K_d value between ATP and Mg²⁺ (∼78 μm) (28). As most cytosolic ATP forms complexes with Mg²⁺ and the free cytosolic Mg²⁺ level is ∼0.5–1 mm, we concluded that the interaction between ATP and CBS-WT is physiological. In contrast, the affinity of CBS-T568I to ATP was significantly lower than that of CBS-WT (Fig. 5B), and the estimated K_d value was >1000 μm (Table 1). These data are consistent with the results shown in Fig. 4 and confirm the notion that the CBS domains of CNNM2 bind specifically to ATP in a Mg²⁺-dependent manner.

FIGURE 5.

Comparative analyses of the various CBS domains for ATP binding. A, filter binding assays were performed by incubating increasing concentrations of [α-³²P]ATP with 50 μm recombinant protein (GST or GST-CBS-WT) in a reaction buffer in the presence of Mg²⁺ at concentrations of 0, 10 mm, or equal to that of ATP for 20 min at 25 °C. Samples were filtered through a nitrocellulose membrane, and the amount of ATP bound to each filter was quantitated by performing liquid scintillation counting. Nonspecific binding was determined by performing control assays with GST and was subtracted. The data were normalized to the percentage of ATP bound at saturation and are represented as the means ± S.E. (error bars) of three independent experiments. B, filter binding assays using GST-CBS-WT and GST-CBS-T568I in the presence of 10 mm Mg²⁺ were performed as in A. C and D, filter binding assays using the CBS domains of ClC-5 (C) and IMPDH2 (D) in the presence or absence of 10 mm Mg²⁺ were performed as in A. E, filter binding assays using the CBS domains of CNNM1–4 in the presence or absence of 10 mm Mg²⁺ were performed as in A. F, competition assays were performed to examine the binding of GST-CBS-WT to ATP or AMP. ATP or AMP (0.4, 0.8, 1.6, or 3.2 mm) was added to reactions containing 5 μm [α-³²P]ATP, 10 mm Mg²⁺, and GST-CBS-WT, and the assays were performed as in A. The data are represented as a percentage of specific binding in the absence of any competitor, and the means ± S.E. (error bars) of three independent experiments are shown.

TABLE 1.

K_d values obtained from filter binding assays

The K_d values are indicated as the means ± S.E. of three independent measurements.

Protein	ATP		AMP with Mg2+
	With Mg2+	Without Mg2+
	μm	μm	μm
CNNM2 WT	159 ± 28	No binding	>3200a
CNNM2 T568I	>1000	NDb	ND
CNNM1	915 ± 389	No binding	ND
CNNM3	No binding	No binding	ND
CNNM4	43.4 ± 8.9	No binding	ND
ClC-5	177 ± 28	37.5 ± 11.3	241 ± 56
IMPDH2	242 ± 78	56.4 ± 7.9	141 ± 58

^a Estimated from the IC₅₀ value obtained from competition assays.

^b ND, not determined.

The CBS domain is evolutionarily conserved and present in the proteome of archaebacteria, prokaryotes, and eukaryotes, and there are a number of reports stating the binding of CBS domains to ATP (10). However, there have been no reports, to date, showing such clear Mg²⁺ dependence of this interaction as in the case of CNNM2. To examine whether the Mg²⁺-dependent interaction is a unique feature of the CBS domains of CNNM2, we also performed ATP binding assays using two other typical CBS domains derived from ClC-5 and IMPDH2, both of which are reported to bind to ATP (15, 19), in the presence or absence of Mg²⁺. As shown in Fig. 5, C and D, both ClC-5 and IMPDH2 showed saturable binding with ATP, regardless of the presence or absence of Mg²⁺. In the presence of Mg²⁺, K_d values were 177 ± 28 μm for ClC-5 and 242 ± 78 μm for IMPDH2, and in the absence of Mg²⁺, the K_d values decreased to 37.5 ± 11.3 μm and 56.4 ± 7.9 μm, respectively (Table 1). Therefore, the affinities of the CBS domains of these proteins for ATP were stronger in the absence of Mg²⁺ than in the presence of Mg²⁺. These results support the notion that Mg²⁺ dependence of the CBS domains of CNNM2 for binding to ATP is a unique feature among the CBS domains. To investigate whether this unique feature is conserved among CNNM family proteins, we also performed filter binding assays using CBS domains derived from CNNM1, CNNM3, and CNNM4. In the presence of Mg²⁺, all of these proteins, except for CNNM3, showed saturable binding with ATP (Fig. 5E). K_d values were 43.4 ± 8.9 μm for CNNM4 and 915 ± 389 μm for CNNM1 (Table 1). Interestingly, these K_d values inversely correlate with the capacities for Mg²⁺ efflux shown in Fig. 1A. In the absence of Mg²⁺, no binding was observed from all of the proteins (Fig. 5E). These results confirm the Mg²⁺ dependence of the interaction of the CNNM CBS domains with ATP and suggest its importance in Mg²⁺ efflux.

In general, CBS domains that bind to ATP can also bind to other adenine nucleotides, such as AMP (15). To examine whether the CBS domains of CNNM2 bind to AMP, we performed filter binding assays using [³H]AMP as a ligand in the presence of Mg²⁺. Saturable binding curves were obtained from ClC-5 and IMPDH2 with K_d values of 241 ± 56 μm and 141 ± 58 μm, respectively (Table 1), demonstrating their interactions with AMP as reported previously (15, 19). In contrast, AMP binding to CBS-WT of CNNM2 was not saturable, and we could not determine the K_d value (data not shown). Therefore, we performed competition assays using nonradiolabeled ATP or AMP to serve as a competitor against [α-³²P]ATP (5 μm). Although nonradiolabeled ATP effectively competed with [α-³²P]ATP, even 3.2 mm AMP (the maximum concentration we tested) could only outcompete 24% of [α-³²P]ATP, indicating that the IC₅₀ value of AMP binding to the CBS domains of CNNM2 is significantly >3.2 mm (Fig. 5F). Therefore, AMP does not virtually bind to the CBS domains of CNNM2. Collectively, the interactions between ATP and the CNNM2 CBS domains require the terminal phosphate groups of ATP, which are a unique feature of CNNM2. Because Mg²⁺ is known to form complexes with ATP through interactions with the phosphate groups of ATP, these results suggest that Mg-ATP is the true ligand for the CNNM2 CBS domains.

Electronegative Potentials around the ATP Binding Site of the CBS Domains of CNNMs

There are several examples of proteins that require Mg²⁺ to bind to ATP. In the cases of sarcoplasmic reticulum Ca²⁺-ATPase and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (26, 27), Mg²⁺ neutralizes the electrostatic repulsion force between the electronegative surface potentials around the ATP binding site and negatively charged phosphate groups of ATP. Therefore, we compared the electrostatic surface potentials of the ATP binding sites between CNNM2, ClC-5, and IMPDH2. The electrostatic surface potentials were calculated for homology models of the CBS domains of CNNM2 and IMPDH2 based on the crystal structures of their bacterial homologs (21) and the previously solved crystal structure of the CBS domains of ClC-5 in complex with ATP (19). As shown in Fig. 6A, CNNM2 showed highly electronegative surface potentials around the ATP binding site, whereas both ClC-5 and IMPDH2 showed highly electropositive surface potentials, which would be suitable for the electrostatic interactions with the phosphate groups of ATP. This clear difference in the surface potentials presumably explains the unique requirement of Mg²⁺ for the interactions between ATP and the CNNM2 CBS domains. We also calculated the electrostatic surface potentials for homology models of the CBS domains of CNNM1 and CNNM4. As shown in Fig. 6B, both CNNM1 and CNNM4 showed similar electronegative potentials around the ATP binding site as did CNNM2, which is consistent with their unique requirement of Mg²⁺ for the interactions with ATP.

FIGURE 6.

Comparison of electrostatic surface potential maps of the ATP binding sites. A, the electrostatic surface potential maps of the entire CBS domains (upper) and magnified images around the ATP binding site (lower) are visualized with colors ranging from blue to red (from +2 kT/e to −2 kT/e). ATP is represented as sticks in atomic coloring (blue, nitrogen; green, carbon; orange, phosphorus; and red, oxygen). To calculate electrostatic surface potentials, the following structures of CBS domains in complex with ATP were used: a homology model based on the crystal structure of CorC from B. parapertussis (PDB ID 3JTF) for CNNM2, the crystal structure of human ClC-5 (PDB ID 2J9L) for ClC-5, and a homology model based on the crystal structure of IMPDH from P. aeruginosa (PDB ID 4DQW) for IMPDH2. B, the electrostatic surface potential maps of the entire CBS domains (upper) and magnified images around the ATP binding site (lower) of CNNM1 and CNNM4 were created and visualized as CNNM2 in A.

DISCUSSION

In this study, we have shown that the CBS domains of CNNM2 bind to ATP in a Mg²⁺-dependent manner, which presumably contributes to the Mg²⁺ efflux function of CNNM2. The possibility of ATP binding to the CBS domains of CNNM2 was theoretically predicted in a previous report by using a homology model, which was based on the structural data of B. parapertussis CorC (29). In this model, the evolutionary conserved threonine (Thr-568) is located at the region predicted to form the ATP binding pocket and also forms a hydrogen bond that stabilizes the position of the Glu-570 and Asp-571 residues. The substitution of Thr-568 into the bigger isoleucine residue was predicted to cause steric bumps with the ATP molecule and also alter the position of the Glu-570 and Asp-571 residues. Therefore, the substitution was proposed to severely affect ATP binding. In this study, we confirmed these predictions experimentally by performing binding analyses; ATP was determined to bind to the CBS domains of CNNM2, and this binding was abrogated by T568I mutation. Moreover, we further demonstrated the Mg²⁺ dependence of this binding, which was not accounted for in the predicted model. To the best of our knowledge, Mg²⁺ dependence for binding to ATP has never been reported to occur in other CBS domains. Comparative analyses of electrostatic surface potentials of the CBS domains revealed the presence of highly electronegative potentials around the ATP binding site of the CBS domains of CNNM2, which may explain the unique requirement of Mg²⁺ in the case of CNNM2.

So far, the biochemical importance of the interaction between CNNM2 and ATP remains unknown. Notably, AMPK binds to AMP or ADP with higher affinity than that of ATP (in the presence of Mg²⁺, similar to the physiological condition) and is regulated in response to changes in the intracellular AMP/ATP or ADP/ATP ratio, enabling AMPK to respond dynamically to the energetic state of cells (17, 18). Thus, the CBS domains of AMPK are believed to function as a cellular energy sensor (30, 31). However, in the case of CNNM2, the CBS domains have a unique feature such that they selectively bind to ATP with a relatively low K_d of ∼150 μm, which is considerably lower than the ATP concentrations typically found in cells (1–10 mm) (32), and virtually does not bind to AMP. Therefore, the CBS domains of CNNM2 are considered to constitutively bind to ATP irrespective of the energetic state of cells. Another unique feature of the CBS domains of CNNM2 is the strict Mg²⁺ dependence of its binding ability to ATP. In mammalian cells, the majority of intracellular Mg²⁺ is present in the form of complexes with various intracellular molecules, such as phospholipids, proteins, and nucleic acids (28, 33). Among these Mg²⁺-binding biomolecules, ATP represents a major component because of its abundance (1–10 mm) and relatively high Mg²⁺ binding affinity (K_d of ∼78 μm); hence, most ATP is considered to exist as Mg-ATP within the cells (28). Taking this into account, we hypothesize that the CBS domains of CNNM2 may utilize their ATP binding ability for efficiently recruiting Mg²⁺. Indeed, intracellular Mg-ATP (1–10 mm) is much more abundant than free Mg²⁺ (0.5–1 mm) (28), and the size of Mg-ATP is much larger than that of Mg²⁺, which would be beneficial for molecular recognition. Once Mg²⁺ is dissociated from Mg-ATP, it is supplied as a substrate for efflux, and Mg²⁺-free ATP dissociates from the CBS domains due to the loss of affinity. Then, another Mg-ATP can immediately occupy the binding site. Repeating this sequential process, Mg²⁺ can be efficiently supplied to facilitate Mg²⁺ efflux via CNNM2. This hypothesis should be tested in the future by much more detailed studies, such as structural analyses.

The T568I mutation of CNNM2 associated with human hereditary hypomagnesemia abrogated not only its ATP binding ability but also its Mg²⁺ efflux function, suggesting the physiological importance of Mg²⁺ efflux by CNNM2 in the regulation of magnesium homeostasis at the organismal level. In a previous study, we showed that CNNM4 localizes to the basolateral membrane of intestinal epithelial cells and mediates transcellular Mg²⁺ transport across epithelia by extruding intracellular Mg²⁺ to the outside of cells (7). In addition, CNNM4-knock-out mice exhibited mild hypomagnesemia due to the intestinal malabsorption of magnesium. Considering these similarities between CNNM2 and CNNM4, we speculate that CNNM2 also mediates transcellular Mg²⁺ transport across epithelia, like CNNM4 does. In support of this possibility, two previous papers have reported a strong expression and localization of CNNM2 at the basolateral membrane of distal convoluted tubule cells in the kidney (6, 29), where renal reabsorption of magnesium by transcellular Mg²⁺ transport is known to occur. CNNM2 presumably plays an important role in the renal reabsorption of magnesium by promoting Mg²⁺ efflux from the distal convoluted tubule cells to the inner parts of the body, which should be the focus of future studies on CNNM2.