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J. Biol. Chem., Vol. 280, Issue 37, 32372-32378, September 16, 2005

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Structural Characterization of Calcineurin B Homologous Protein 1^*

Youichi Naoe, Kyouhei Arita, Hiroshi Hashimoto, Hiroshi Kanazawa, Mamoru Sato, and Toshiyuki Shimizu1

From the International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, and Department of Biological Science, Graduate School of Science, Osaka University, Machikaneyama-cho 1-16, Toyonaka, Osaka 560-0043, Japan

Received for publication, March 28, 2005 , and in revised form, June 23, 2005.

	ABSTRACT

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Calcineurin B homologous protein 1 (CHP1), also known as p22, is a calcium-binding EF-hand protein that plays a role in membrane trafficking. It binds to multiple effector proteins, including Na⁺/H⁺ exchangers, a serine/threonine kinase, and calcineurin, potentially modulating their function. The crystal structure of calcium-bound CHP1 from rat has been determined at 2.2 Å of resolution. The molecule has a compact -helical structure containing four EF-hands. The overall folding topology of the protein is similar to that of the regulatory B subunit of calcineurin and to that of calcium- and integrin-binding protein. The calcium ion is coordinated in typical fashion in the third and fourth EF-hands, but the first and second EF-hands contain no calcium ion. The first EF-hand is maintained by internal interactions, and the second EF-hand is stabilized by hydrophobic interactions. CHP1 contains a hydrophobic pocket on the opposite side of the protein to the EF-hands that has been implicated in ligand binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Calcineurin B homologous protein 1 (designated CHP1) is a calcium-binding EF-hand protein and shows substantial sequence similarity (39% identity) to the regulatory B subunit of the protein phosphatase calcineurin (CNB).² CHP1 was initially identified as a protein required for constitutive vesicular transport and was known as p22 (1). CHP1 was also identified as an accessory protein that associates tightly with Na⁺/H⁺ exchangers (NHEs) that catalyze the electroneutral influx of extracellular Na⁺ and efflux of intracellular H⁺ (2). Further biochemical studies revealed that CHP1 serves as an essential cofactor required for at least three NHE isoforms, NHE1–3 (3). Of these isoforms, NHE1 has been characterized in the most detail. NHE1 is ubiquitously expressed in the plasma membranes of essentially all tissues and plays a major role in intracellular pH homeostasis and cell volume regulation. The activity of NHE1 is controlled by various extrinsic factors, including growth factors, hormones, and mechanical stimuli, presumably through a regulatory site. Such regulation is thought to occur through the interaction of the C-terminal domain of NHE1 with a variety of signaling molecules, such as calmodulin, phosphatidylinositol 4, 5-bisphosphate, and actin-binding proteins of the ezrin, radixin, and moesin families (4). CHP1 has been shown to interact with the juxtamembrane region of the C-terminal domain of NHE1, and CHP1-binding-defective mutants of NHE1 show a marked acidic shift and have completely impaired ATP depletion-induced inhibition and cytoplasmic alkalinization in response to various stimuli (3). Furthermore, a mutation of CHP1, in which the displays lacked calcium-binding affinity, also has a significantly reduced Na⁺/H⁺ exchange activity (5).

Recently, another isoform of CHP1, referred to as CHP2, sharing a 61% amino acid identity with CHP1, has been reported (6, 7). CHP2 is expressed in extremely low amounts in most human tissues, except intestinal epithelia and tumor cells.

Recent biochemical studies (8) reveal that CHP1 possesses multiple cellular functions. CHP1 binds to the catalytic A subunit of calcineurin (CNA) directly and inhibits its phosphatase activity, suggesting that CHP1 is an endogenous inhibitor of calcineurin. CHP1 also binds to the death-associated protein kinase-related apoptosis-inducing protein kinase 2 (DRAK2) causing significantly reduced kinase activity. DRAK2 is a member of the death-associated protein kinase family and is involved in the induction of apoptosis in various cell types. The enzyme activity of some members of the death-associated protein kinase family is regulated via binding of calmodulin to a calmodulin-binding region. DRAK2, however, lacks the calmodulin-binding region and instead possesses a CHP1-binding region in the C-terminal region of the kinase domain (9). The inhibitory effect of CHP1 is dependent on the presence of calcium, whereas the interaction between CHP1 and DRAK2 is not calcium-dependent (9, 10). CHP1 also interacts with KIF1B2 and other members of the KIF1B family of microtubule-dependent motor proteins in a calcium-dependent manner (11). CHP1 is also reported to modulate the organization and dynamics of microtubule cytoskeleton and affects endoplasmic reticulum network assembly (12).

CHP1, consisting of 195 amino acids, has an N-myristoylation site at its N terminus and is predicted to contain four EF-hand calcium-binding motifs (denoted EF-1–EF-4), two of which bind calcium (EF-3 and EF-4) with affinities of 90 nM (5). Myristoylation does not significantly affect the affinity for calcium (5) nor is it required for the interaction of CHP1 with NHE1 (3) or DRAK2 (10). However, CHP1 associates with microtubules in an N-myristoylation-dependent manner (13). CHP1 predominantly localizes to the cytoplasm because of two functional nuclear export signal sequences in its C terminus (14).

Despite the great importance of the multiple intracellular functions of CHP1, no structural information has been obtained for this protein. Here, we report the crystal structure of calcium-bound CHP1 and show that the structure of CHP1 is similar in overall folding topology to the structures of CNB and related proteins but differs in local conformation. The target recognition mechanism is also discussed.

	MATERIALS AND METHODS

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Expression and Purification—Details on the expression, purification, and crystallization of CHP1 are published (15). In brief, rat CHP cDNA, corresponding to amino acid residues 1–195 cloned into a pET21b vector (Novagen) as a fusion protein with a C-terminal His₆ tag, was expressed in the Escherichia coli BL21(DE3) codon plus RIL. Bacterial lysates treated with equilibrium buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM CaCl₂) were disrupted by sonication. The supernatant was applied to a nickel-nitrilotriacetic acid-agarose affinity column (Qiagen) and the adsorbed fraction eluted with equilibrium buffer containing 250 mM imidazole. CHP1 was further purified by a Hitrap Q HP column and HiLoad Superdex 75 gel filtration column (Amersham Biosciences). The purified protein was dissolved in 5 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl, 1 mM CaCl₂, and 1 mM dithiothreitol and was concentrated to 10 mg/ml at 277 K by an Amicon Ultra-15 10,000-cut filter (Millipore) for crystallization. Homogeneity of the purified protein was confirmed by SDS-PAGE. The purified CHP1 possesses 13 amino acid residues derived from the pET21b vector at the C terminus. For ultracentrifugation experiments, rat CHP encoding 1–195 was expressed as a fusion protein with glutathione S-transferase. After cleaving the glutathione S-transferase tag with thrombin, CHP1 was purified in a similar manner to the His-tagged CHP1. This CHP1 has no extra sequence at the C terminus, although there is a Gly-Ser sequence from the vector at the N terminus.

Analytical Ultracentrifugation—Sedimentation velocity experiments were carried out using an Optima XL-I analytical ultracentrifuge (Beckman Coulter, Fullerton, CA) using a Beckman An-50 Ti rotor. For sedimentation velocity experiments, cells with a standard Epon two-channel centerpiece and sapphire windows were used. Sample (400 µl) and reference buffer (420 µl) were loaded into cells. The rotor temperature was equilibrated at 20 °C in the vacuum chamber for 1–2 h prior to start-up. A₂₈₀ scans were collected at 5-min intervals during sedimentation at 40,000 revolutions/min. The sedimentation velocity experiments for CHP1 were conducted at concentrations of between 3.2 and 4.7 mg/ml. The resulting scans were analyzed using the continuous distribution (c(s)) (described below under the heading "Monomeric State of CHP1") analysis module in the program Sedfit, version 8.9 (16, 17). Partial specific volume of the protein, solvent density, and solvent viscosity was calculated from standard tables using the program SEDNTERP, version 1.08 (18), which was obtained from the Reversible Associations in Structural and Molecular Biology program depository.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Sequence alignment of rat CHP1, rat CHP2 and rat CNB with the secondary structure elements of CHP1. The secondary structure elements are shown at the top, with gray tubes for the -helices (A–I) and shaded rectangles for the 310 helices. The dotted lines show disordered regions. The secondary structure elements of CNB (PDB code 1AUI [PDB] ) (20), as determined from the tertiary structure, are boxed. The residues corresponding to the EF-hand are marked at the bottom of the alignment (X,Y,Z, -Y, -X, and -Z), according to the typical EF-hand motif.

	RESULTS

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Overall Structure—Two CHP1 molecules (designated molecules A and B) in the asymmetric unit of the crystal have essentially the same structure with a root mean square deviation of 0.7 Å for C- atoms (amino acids 15–93 and 107–195). The significant difference appears within the N-terminal region. Although this region forms a continuous -helix in molecule B, a part of the -helix is collapsed and disordered in molecule A. The linker between E and F also shows differences as stated below.

The polypeptide chain of CHP1 is folded into two globular domains (N-lobe and C-lobe) composed of an -helical structure with 10 -helices (A–J) and two 3₁₀ helices. The two lobes are superimposed with a root mean square deviation of 2.2 Å for C- atoms corresponding to four -helices (B–E, F–I). It should be noted that the continuous tenth -helix (J) is composed of eight residues from the C terminus of the native protein together with part of the tag sequence (KLAAALEH) derived from the pET21 vector (Figs. 1 and 2a).

The structure of CHP1 is similar in overall folding topology to the structures of other EF-hand-containing proteins. In fact, the MSD-fold server (30) showed that CHP1 is similar to CNB (PDB code 1TCO [PDB] , Z-score 6.5) (31), calcium- and integrin-binding protein (CIB1) (PDB code 1XO5 [PDB] , Z-score 5.2) (32), and Kv channel-interacting protein (KChIP1) (PDB code 1S6C [PDB] , Z-score 4.7) (33). A common structural feature of these proteins is that they all have a four-EF-hand scaffold with EF-hands working in pairs, such that EF-1 and EF-2 form the N-lobe and EF-3 and EF-4 form the C-lobe. In addition to the eight -helices forming the four EF-hands, these proteins have an N-terminal -helix and C-terminal helix that flank the EF-hand domains. These structural features are also observed in recoverin (34), frequenin (neuronal calcium sensor 1; NCS-1) (35), Arabidopsis thaliana calcineurin B-like protein (AtCBL2) (36), and neurocalcin (37). Indeed, CHP1 possesses these structural features, but significant differences are observed in local conformation at the domain-domain interface in CHP1 and the related proteins. For example, the superposition of the C- atoms, corresponding to eight -helices (B–I) of CHP1 on CNB, yields a large root mean square deviation of 2.2 Å, whereas the N- and C-lobes can both be separately superimposed, with root mean square deviations of 1.5 and 0.9 Å, respectively. This indicates that different domain-domain hinge motions occur between CHP1 and CNB. In fact, when the N-lobe of CHP1 and CNB are superimposed, a swiveling of the N-lobe of 20° is observed (Fig. 2b). It should be noted that no domain motion is observed in the two independent molecules in the asymmetric unit.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Overall structure of CHP1. a, ribbon representation of secondary structure elements. -Helices are colored blue, and calcium ions are depicted by yellow balls. The -helix derived from the vector is colored green. b, ribbon representation of the structure of CHP1 superimposed on that of CNB. CHP1 and CNB are represented as blue and red ribbons, respectively. The figure was generated by superimposing the -helices of the C-lobes. These are shown in pale blue and red. N- and C-terminal helices (A and J) are omitted for clarity. The angle of rotation between the N-terminal domains of CHP1 and CNB is 20°.

A nuclear export signal sequence has been assigned to amino acids 138–147 and 176–185 of CHP1 (14). These regions correspond to the middle of G to the following loop and from the middle of I to the following loop, respectively. The structure of these regions is consistent with that of other nuclear export signals, which tend to be -helical in the N-terminal part of the signal (38).

Monomeric State of CHP1—Because two molecules are packed in the asymmetric unit of the crystal, we investigated whether CHP1 forms a dimer in solution. Gel filtration experiments using His-tagged CHP1 and CHP1 lacking a tag show that CHP1 exists in the monomeric state.³ We also performed an ultracentrifugation experiment. The sedimentation velocity experimental data were analyzed using the program Sedfit to determine the distribution of protein species with different sedimentation coefficients. The resultant continuous distribution (c(s)) contains a single predominant peak with a sedimentation coefficient (s) of 2.4 (±0.1) S (data not shown), which means the protein is very homogeneous. The weight:average frictional ratio (f:f_o) was optimized by least-squares regression and converged to a best-fit value of 1.2 (±0.1), suggesting the component has a globular shape. When the distribution (c(s)) contains a single major peak, it can be transformed to a molar mass distribution c(M) that allows estimation of the molar mass of the main species (39). The component corresponds to a molecular mass of 24 kDa, which agrees very well with the theoretically calculated monomeric CHP1 mass based on the amino acid sequence. In the 3.2–4.7 mg/ml protein concentration range, CHP1 did not show significant protein concentration dependence. Pang et al. (5) also reported that CHP1 is in the monomeric state and that CHP1 in complex with the NHE1 fragment also exists as a monomer in a 1:1 molar ratio. We therefore concluded that CHP1 is a monomer in solution.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Representation of the four EF-hand motifs (EF-1, EF-2, EF-3, EF-4). a, calcium-bound EF-hand motifs (EF-3 and EF-4). Calcium ions and water molecules are represented as yellow and red balls, respectively. An omit map, contoured at the 7 level around each calcium ion, is superimposed. Amino acid residues involved in the calcium coordination are shown. b, calcium-unbound EF-hand motifs (EF-1 and EF-2). Non-covalent interactions within EF-hands are represented as dotted lines. In EF-2, residues forming the hydrophobic core are also shown.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Hydrophobic pocket of CHP1 and related proteins. a, positive (blue) and negative (red) potentials are mapped on the van der Waals surfaces, excluding the C-terminal region, in the range -20 kBT (red) to +20 kBT (blue), where kB is the Boltzmann constant and T is the absolute temperature. The C-terminal region and residues from the vector of CHP1 are represented as a tube model colored yellow and green, respectively. A tube model colored yellow and green shows the C-terminal region of KChIP1 and the Kv4.2 peptide (33). A tube model colored green represents an -helix protruding from CNA in the CNB·CNA complex (20). Tube models colored yellow show the C-terminal region of recoverin (34), neurocalcin (37), and NCS-1 (35), respectively. b, conserved region between CHP1 and CHP2 mapped on the molecular surface. Orange indicates conserved identical residues and white non-conserved residues, whereas lighter shades of orange indicate semi-invariant residues.

The calcium ion is coordinated in the typical EF-hand fashion of pentagonal bipyramidal geometry. In EF-3, the calcium coordination is through the side chain carboxylate of Asp-123 (X), Asp-125 (Y), Asp-127 (Z), Glu-134 (-Z), the main chain carbonyl of Lys-129 (-Y), and the water molecule (-X) in EF-3. In EF-4, the calcium coordination is through the side chain carboxylate of Asp-164 (X), Asp-166 (Y), Asp-168 (Z), Glu-175 (-Z), the main chain carbonyl of Ala-170 (-Y), and the water molecule (-X) (Fig. 3a). In contrast, EF-1 and EF-2 significantly deviate from the canonical sequence, which explains the absence of calcium ions in these EF-hands. EF-1 contains several internal interactions, whereas EF-2 adopts a -turn conformation (Fig. 3b). In EF-2, Phe-71 (X) stacked against Arg-48 is located at the inner side of the EF-hand so as to stabilize the EF-hand. Phe-71 also forms a hydrophobic core with Phe-51, Phe-70, and Val-78 (Fig. 3b).

The helices of EF-hands reorient in relation to one another when the calcium ion is bound. Structural analyses of several calcium-binding proteins have shown that the helices of each EF-hand exhibit open conformation with an interhelical angle of 90° when the calcium ion is present. In the apostructures, the helices become anti-parallel with interhelical angles of 130–140° (40). The angles of EF-1–EF-4 are 96, 130, 95, and 96°, respectively, indicating that at least three of the EF-hands adopt the open conformation. The open conformation of EF-1, which does not contain calcium ion, is maintained by several hydrogen bonds and water-mediated interactions.

Hydrophobic Pocket—EF-hand-containing proteins, such as CNB, recoverin, and related proteins have a hydrophobic pocket on the opposite side of the molecule from the EF-hands. The conformation of the tenth -helix (J in CHP1) varies and affects the degree to which the hydrophobic pocket is exposed. For example, in NCS-1, it is fully exposed (35); in recoverin, it is partially shielded (34); and in AtCBL2, it is almost shielded by the C-terminal region (36). The hydrophobic pocket is often involved in target recognition. For example, the pocket of CNB accommodates CNA (Fig. 4a) (20, 31), and Kv4.2, which is a target protein of KChIP1, binds to the pocket of KChIP1 (Fig. 4a) (33). In CHP1, as stated above, J, comprising eight residues of the C terminus together with the residues from the vector, is plunged into the hydrophobic pocket (Fig. 4a, upper left). Phe-193 and Leu-194 of J interact with the residues forming the pocket. If the protein lacked the extra residues arising from the vector, the pocket would be partially exposed as observed in recoverin and neurocalcin.

	DISCUSSION

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In this study, we have characterized rat CHP1 structurally. Pang et al. (3) reports that amino acids 515–530 of NHE1 (found in the cytoplasmic domain) are required for CHP1 binding and are predicted to form an amphipathic -helix. Mutants carrying amino acid substitutions of hydrophobic (Phe, Leu) to hydrophilic (Gln, Arg) residues failed to bind to CHP1 and dramatically reduced the exchange activity, suggesting a cluster of hydrophobic residues are important for the interaction. Therefore, the hydrophobic pocket of CHP1 is a potential ligand-binding site. In this regard, the structure of the KChIP1·Kv4.2 complex (33) gives a good indication of the ligand recognition mechanism of CHP1. The binding region of Kv4.2 forms an 19-residue-long helix, and its two hydrophobic residues (Phe and Trp) of Kv4.2 provide the side chains at the interface that bind into the deep hydrophobic pocket formed by side chains from the N-lobe groove of the binding pocket of KChIP1 (Fig. 4a). It should be noted that the -helix of Kv4.2 binds as a continuation of the C-terminal -helix of KChIP1 in a head-to-tail fashion. In the case of CHP1, the sequence derived from the vector is not related to that of NHE1, but interestingly, two leucine residues (KLAAALEH) are involved at the interface. The amino acids forming the N-lobe groove of KChIP1 and the recoverin family are conserved. The corresponding residues in CHP1 are mostly hydrophobic, and thus ligand recognition by CHP1 could be carried out through a similar mechanism.

Binding studies have shown that CHP1 binds to a minimal 15-amino-acid region of NHE1, which corresponds to four-helical turns. The hydrophobic pocket (19 Å across) would be slightly too small to bind four turns of the -helix (>20 Å). It is possible that J undergoes a conformational change, such that a larger ligand-binding pocket is formed. Zhou et al. (33) proposed that the C-terminal -helix (H10) of KChIP1, corresponding to J of CHP1, is dynamic, such that it is stably locked in the C-lobe groove only when the N-lobe groove of the pocket is filled with 1 of Kv4.2. This mechanism may be applicable to CHP1.

The region of DRAK2 that binds to CHP1 is assumed to be located in the C-terminal region (amino acids 227–293) of the kinase domain. The sequence identity between this region and that of NHE1 is very low (3%). It therefore remains unclear whether CHP1 binds to DRAK2 in a similar way.

CHP1 is known to undergo calcium-mediated conformational changes as ascertained by electrophoretic mobility shift experiments (1). Structural studies of the recoverin family have shown that a hinge motion between the N- and C-lobes is dependent on the calcium-binding state (41). The binding of calcium ions to the EF-hands in recoverin leads to local structural changes that alter the domain interface and cause a swiveling of the N- and C-lobes. Likewise, it is supposed that calcium binding to the EF-hands of CHP1 induces a rearrangement of the domain interface. In the structure of CHP1, EF-1 and EF-2 forming the N-lobe lack bound calcium. Thus, it raises an obvious question as to how calcium binding to the C-lobe could affect the conformation. Because the N- and C-lobes are closely contacted, conformational change or domain reorganization triggered by calcium binding into EF-3 and EF-4 will occur. We compared the swiveling angles of some CHP1-related proteins. CIB1 shows a similar angle, but NCS-1 and recoverin show different angles. Therefore, the swiveling angle itself is an intrinsic value of the protein, but the angle would be changed according to the existence of calcium.

The relative rotation between the N- and C-lobes around the conserved Gly (Gly-96 in recoverin and Gly-121 in KChIP1) is essential to the alignment of the N- and C-lobe pockets into one continuous pocket (41). CHP1, however, lacks this Gly. Instead, a long linker (amino acids 90–109) performs the role of the Gly. The orientation of the long linker of the two independent molecules in the asymmetric unit is almost similar, although the linker is largely disordered in molecule A.

As described, CHP1 binds to DRAK2 in a calcium-independent manner. Such a calcium-independent binding to the target protein is observed in several proteins. For example, the association of KChIP1 with Kv4 channels is calcium-independent, but the modulatory functions of KChIP1 are dependent on calcium (42). Guanylyl cyclase activator protein 2 (GCAP-2) can form a complex with guanylyl cyclase in a calcium-free form, and calcium-loaded GCAP-2 inhibits its activity (43). The interaction between CIB1 and integrin IIb also occurs in the absence of calcium, although with reduced affinity (44). Of interest, KChIP1 and CIB1 have functional EF-hands only in the C-lobe in a similar manner to CHP1. Probably, the target recognition pocket is retained even in a calcium-free form, and calcium binding induces a domain rearrangement or conformational change, causing modulatory effects to the target protein. Further structural studies are required to elucidate the mechanism. To our knowledge, no CHP1-related proteins have the same EF-1 and EF-2 amino acids of CHP1. But, some proteins, similar to CIB1 and KChIP1, have functional EF-hands in the C-lobe (EF-3 and EF-4) and non-functional EF-hands in the N-lobe (EF-1 and EF-2). As described above, KChIP1 has a similar feature in the target recognition. Therefore, proteins with two functional EF-hands in the C-lobe can work in a similar manner to other EF-hand-containing proteins.

As mentioned above, CHP2 exhibits a high homology to CHP1. The Leu-99 insertion in CHP2 is located in the disordered region, which would suggest that the insertion region induces no conformational change. Several hydrophobic residues form the possible ligand-binding pocket in CHP1, and these residues are mostly conserved in CHP2. We mapped conserved residues on the molecular surface of CHP1. The possible binding site is covered with conserved identical and semi-invariant residues (Fig. 4b). This indicates that CHP2 is likely to have a similar ligand-binding pocket and recognize the target protein in a similar manner to CHP1. In fact, CHP2 is reported to have the regulatory ability of NHE1 in a calcium-dependent manner (6).

Further structural studies of CHP1 with peptides from target proteins or directly with target proteins, in combination with further biochemical and cellular assays, will extend our understanding of the role of this protein in cellular functions.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2CT9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Grant-in-aid (14780515) for Young Scientists (B) from the Japan Society of the Promotion of Science (JSPS) (to H. H.), Grant-in-aid (15570101) for Scientific Research (C) from the JSPS (to T. S.), Grants-in-aid for Scientific Research on Priority Areas (13142207) (to H. K.) and (16048226) (to H. H.), and by the Protein 3000 Project of The Ministry of Education, Culture, Sports, Science, and Technology. 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.

¹ To whom correspondence should be addressed: International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Tel.: 81-45-508-7226; Fax: 81-45-508-7365; E-mail: shimizu{at}tsurumi.yokohama-cu.ac.jp.

² The abbreviations used are: CNB, catalytic B subunit of calcineurin; CNA, catalytic A subunit of calcineurin; NHE, Na⁺/H⁺ exchanger.

³ Y. Naoe, K. Arita, H. Hashimoto, H. Kanazawa, M. Sato, and T. Shimizu, unpublished data.

	ACKNOWLEDGMENTS

 
We thank the staff at beamline BL-5 Photon Factory, Tsukuba, Japan, for data collection support. Dr. Unzai is acknowledged for the measurement and useful comments on the analytical ultracentrifugation.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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