Structures of Anabaena Calcium-binding Protein CcbP

Abstract

Ca²⁺-binding proteins play pivotal roles in both eukaryotic and prokaryotic cells. CcbP from cyanobacterium Anabaena sp. strain PCC 7120 is a major Ca²⁺-binding protein involved in heterocyst differentiation, a process that forms specialized nitrogen-fixing cells. The three-dimensional structures of both Ca²⁺-free and Ca²⁺-bound forms of CcbP are essential for elucidating the Ca²⁺-signaling mechanism. However, CcbP shares low sequence identity with proteins of known structures, and its Ca²⁺-binding sites remain unknown. Here, we report the solution structures of CcbP in both Ca²⁺-free and Ca²⁺-bound forms determined by nuclear magnetic resonance spectroscopy. CcbP adopts an overall new fold and contains two Ca²⁺-binding sites with distinct Ca²⁺-binding abilities. Mutation of Asp³⁸ at the stronger Ca²⁺-binding site of CcbP abolished its ability to regulate heterocyst formation in vivo. Surprisingly, the β-barrel subdomain of CcbP, which does not participate in Ca²⁺-binding, topologically resembles the Src homology 3 (SH3) domain and might act as a protein-protein interaction module. Our results provide the structural basis of the unique Ca²⁺ signaling mechanism during heterocyst differentiation.

Introduction

Whereas the significance of Ca²⁺ ions in eukaryotic cells has been well recognized for a long time (1), its importance in prokaryotic cells has only gained increasing interests recently (2–4). There is growing evidence that the intracellular Ca²⁺ concentration is tightly regulated in prokaryotes, and Ca²⁺ signaling is involved in cell structure maintenance, gene expression, cell cycle and cell differentiation processes, including the regulation of heterocyst formation in cyanobacteria (2–4).

Cyanobacteria are a group of ancient prokaryotes that appeared on earth at least 2∼3.5 billion years ago. Some cyanobacteria can simultaneously carry out oxygenic photosynthesis and nitrogen fixation, which are two biochemically incompatible processes. When combined nitrogen is scarce, some photosynthetic vegetative cells differentiate into specialized nitrogen-fixing cells called heterocysts (5–9). The signaling network during heterocyst differentiation is highly complex and recalls that of eukaryotic cells. One of the essential triggering signals for heterocyst formation is the increase of intracellular free Ca²⁺ concentration, and it could represent an earliest example of calcium required cellular differentiation in evolution (9–11).

Protein CcbP (cyanobacterial calcium binding protein) from cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena sp.) was identified as a major Ca²⁺-binding protein involved in Ca²⁺ sequestration and the regulation of heterocyst differentiation (10, 11). At the early stages of heterocyst differentiation, CcbP is degraded by a serine-type protease HetR, leading to a Ca²⁺ release and subsequent differentiation processes (11). Nevertheless, CcbP shares low sequence identity with proteins of known structure, and its Ca²⁺-binding sites remain unknown. Although CcbP shows certain biochemical and biophysical similarity to the sarcoplasmic Ca²⁺-binding protein calsequestrin in vertebrates, its Ca²⁺-binding capacity (∼2 Ca²⁺ per molecule) differs from calsequestrin (∼40–50 Ca²⁺ per molecule) (10–12). These results strongly suggest that CcbP may represent a novel class of Ca²⁺-binding proteins. Therefore, the structural information of CcbP is essential for elucidating the molecular mechanism of its role in Ca²⁺ signaling during heterocyst differentiation.

Here, we report the solution structures of CcbP in both Ca²⁺-free and Ca²⁺-bound forms determined by nuclear magnetic resonance (NMR)³ spectroscopy. The structures of CcbP in both forms reveal an overall new fold with an α-subdomain and a β-barrel subdomain. Ca²⁺ titration experiments by NMR and mutagenesis analysis identified two Ca²⁺-binding sites. The stronger Ca²⁺-binding site I locates at an α-turn-β region, whereas the weaker Ca²⁺-binding site II resembles a single EF-hand motif with defects. Furthermore, the β-barrel subdomain of CcbP unexpectedly reveals an SH3-like topology that might act as a protein-protein interaction module during the degradation of CcbP by HetR. Our study provides the structural basis for understanding the mechanism of Ca²⁺ signaling during heterocyst differentiation and further extends our knowledge of Ca²⁺-binding proteins.

EXPERIMENTAL PROCEDURES

Sample preparations

The ccbP gene was cloned into a pET15b vector (Novagen) with an N-terminal cleavable His-tag and expressed in an Escherichia coli BL21(DE3) strain. The culture was grown in LB medium, centrifuged, and resuspended in M9 minimal medium with antibiotics and ¹⁵NH₄Cl in the presence or absence of ¹³C₆-glucose for preparations of ¹³C/¹⁵N-labeled or ¹⁵N-labeled samples, respectively (13). The CcbP protein was purified by nickel-nitrilotriacetic acid column (Qiagen). The sample was digested using thrombin to remove the N-terminal His-tag and further purified by passing through the nickel-nitrilotriacetic acid column and subsequently the gel filtration column (Superdex-75) using an ÄKTA FPLC system (Amersham Biosciences). The purity was determined to be >95% as judged by SDS-PAGE. NMR samples were prepared with 1 mm CcbP dissolved in 90% H₂O/10% D₂O buffer containing 20 mm Tris-HCl (pH 7.4) and 220 mm NaCl. The sample for Ca²⁺-free CcbP was pretreated with excess EGTA and subsequently buffer-exchanged to remove EGTA. Excess Ca²⁺ ions (40 mm CaCl₂) were added in the Ca²⁺-bound form. In addition, 2,2-dimethyl-2-silapentanesulfonic acid was added as the internal chemical shift reference. CcbP mutants E17A, D21A, E23A, D24A, E26A, E27A, D37A, D38A, T39A, and E41A were expressed in E. coli and purified following the same method as wild type CcbP.

NMR Spectroscopy

All NMR experiments for structural determination were performed on Bruker Avance 500-MHz (equipped with a cryoprobe) and 800-MHz spectrometers equipped with a triple-resonance probe with pulsed field gradients at 30 °C. The spectra were processed with the software package NMRPipe (14) and analyzed using the program NMRView (15). The resonance assignments of backbone and side chain atoms were obtained following the common procedures (16). The three-dimensional ¹⁵N- and ¹³C-edited NOESY-HSQC (mixing time of 100 ms) spectra were recorded to confirm the assignments and generate distance restraints for structure calculations. Hydrogen-deuterium exchange experiments were performed to obtain hydrogen bond information. The ¹H-¹⁵N residual dipolar coupling (RDC) constants of CcbP were measured. The measurements were performed by dissolving the CcbP protein in a dilute liquid crystal buffer containing a mixture of alkyl-poly (ethylene glycol) C12E5 and n-hexanol (17). The C12E5/water ratio was 5.5% (w/w), and the molar ratio of C12E5 to n-hexanol was 0.92. The RDC values were extracted from the difference in ¹H-¹⁵N splitting measured by ¹H-¹⁵N IPAP-HSQC spectra between the weakly aligned and the isotropic samples (18).

Structure Calculations

The structures of CcbP in both forms were calculated using interproton NOE-derived distance restraints in combination with dihedral angles and hydrogen bonds information. The program TALOS (19) was used to predict dihedral angles ψ and ϕ restraints. Hydrogen bond restraints were determined based on hydrogen-deuterium exchange experiments in conjunction with NOEs and secondary structural information. The initial structures were generated using the CANDID module of the CYANA program (20). The 20 conformers with the lowest target function were selected as the models for SANE (21) to extend the NOE assignments. Two hundred structures were iteratively calculated using CYANA (22), and the 100 structures with the lowest target function were selected for further refinement in the AMBER force field using the parm99 parameters (23). The RDC restraints were added during the AMBER refinement procedure. For the Ca²⁺-bound form, two Ca²⁺ ions and distance restraints between Ca²⁺ and CcbP also were added during the AMBER calculation steps. For each binding site, one Ca²⁺ ion was restrained by adding Ca²⁺-O restraints of 1.8–2.8 Å based on the experimental results from chemical shift perturbations and mutagenesis. Initially, ambiguous Ca²⁺-O restraints for the two oxygen atoms of side chain carboxyl groups were used. Based on the calculated structures with Ca²⁺ ions, the oxygen atoms with Ca²⁺-O distances <3 Å were identified to resolve the ambiguous Ca²⁺-O restraints and to remove those that were violated. Subsequent calculations were performed using unambiguous Ca²⁺-O restraints. Finally, 20 of 100 structures with the lowest AMBER energy were selected as the representative structures of CcbP in the Ca²⁺-free and Ca²⁺-bound forms, respectively. Programs PROCHECK-NMR (24) and MOLMOL (25) were used to analyze the quality of the structures.

Ca²⁺ Titration by NMR

For the Ca²⁺ titrations by NMR, ¹⁵N-labeled CcbP protein (final concentration of ∼0.5 or 0.05 mm) was dissolved in 20 mm Tris-HCl buffer (pH 7.4) in the absence of NaCl. CaCl₂ was dissolved in the same buffer and gradually added to the protein sample. A series of two-dimensional ¹⁵N-edited heteronuclear single-quantum coherence (HSQC) spectra were recorded, and the chemical shift changes of backbone ¹⁵N atoms of all residues were analyzed to identify the Ca²⁺-binding sites. Ca²⁺ titration by NMR was also performed in the presence of 220 mm NaCl, and the results were compared with those obtained in the absence of NaCl. In addition, Ca²⁺ titrations by NMR in the absence of NaCl were performed similarly for CcbP mutant proteins.

Backbone {¹H}-¹⁵N Heteronuclear NOE Measurements

The backbone steady-state heteronuclear {¹H}-¹⁵N NOE values of CcbP in the Ca²⁺-free and Ca²⁺-bound forms were measured on a Bruker Avance 600-MHz NMR spectrometer at 30 °C (26). The experiments were performed in the presence and absence of a 3-s proton presaturation period prior to the ¹⁵N excitation pulse.

Ca²⁺ Titration into CcbP by ITC

Binding of Ca²⁺ to CcbP was measured by isothermal titration calorimetric (ITC) using a MicroCal VP-ITC MicroCalorimeter (Northampton, MA). The protein samples used in the titration was extensively dialyzed against a buffer containing 20 mm Tris-HCl (pH 7.4), which is the same as used in NMR titration experiments. Stock solutions of CaCl₂ (2.9 and 5.8 mm), used as the titrant, were prepared the same buffer. Typical ITC experiments were performed at 25 °C according to the manufacturer's instructions. A total of 272 μl concentrated CaCl₂ (2.9 mm or 5.8 mm) was added into the protein solution (1.43 ml, 0.10 mm, or 0.22 mm) in 34 aliquots (8-μl each). The additions were 3-min apart to allow heat accompanying each increment to return to baseline prior to the next addition. The reference experiments by titrating CaCl₂ ligand into the buffer were subtracted before data analysis. All data were analyzed by fitting with different binding models using the program Origin (version 7.0; MicroCal, Northampton, MA), and best fits were obtained using two-site binding model.

RESULTS

NMR Structure of Ca²⁺-free CcbP

The solution structure of CcbP in the absence of Ca²⁺ was determined based on a total of 4,370 restraints derived from multidimensional NMR spectroscopy, including proton-proton distance restraints generated from NOE, hydrogen-bond restraints based on the hydrogen-deuterium exchange experiments, dihedral angle restraints based on chemical shifts, and ¹H-¹⁵N RDC restraints measured by weakly aligning the protein sample in a dilute liquid crystal buffer (Table 1). The ensemble of the 20 representative structures and a ribbon diagram are depicted in Fig. 1, A and C. The stereo images of the structures are shown in supplemental Fig. 1.

TABLE 1.

NMR statistics for Anabaena CcbP

	Ca2+-free	Ca2+-bound
NMR restraints
Distance restraints
Total NOE	4131	4430
Total unambiguous NOE	3246	3538
Intra-residue	1397	1506
Inter-residue
Sequential (|i − j| = 1)	849	875
Medium range (|i − j| < 4)	352	404
Long range (|i − j| > 5)	648	753
Total ambiguous NOE	885	892
Hydrogen bonds	37	42
Total dihedral angle restraints
Phi (ϕ)	67	73
Psi (ψ)	67	73
Total RDCs	68	64
Total Ca2+-O restraints		12
Energy (kcal/mol)
Mean AMBER energy	−7398.06 ± 10.50	−8022.30 ± 12.24
Distance restraints violation energy	7.84 ± 1.41	20.24 ± 1.42
Torsion angle restraints violation energy	0.66 ± 0.10	0.97 ± 0.15
Structure statistics
Violationsa
Maximum distance restraint violation (Å)	0.33	0.35
Maximum dihedral angle violation	0.00°	0.00°
r.m.s.d.b from mean structure (Å)
Heavy (for all residues)	1.45 ± 0.15	1.44 ± 0.17
Backbone (for all residues)	0.90 ± 0.13	0.93 ± 0.20
Secondary structure heavy	0.99 ± 0.10	0.96 ± 0.13
Secondary structure backbone	0.49 ± 0.14	0.48 ± 0.15
Ramachandran statisticsc (%)
Most favored regions	82.1 (86.2)	81.9 (88.3)
Additional allowed regions	16.5 (13.7)	16.6 (11.5)
Generously allowed regions	1.2 ( 0.1)	0.9 ( 0.2)
Disallowed regions	0.2 ( 0.0)	0.6 ( 0.0)

^a None of the structures have distance violation >0.4 Å or dihedral angle violations >5°. For Ca²⁺-O restraints, the largest violations were <0.2 Å.

^b The root mean square deviation (r.m.s.d.) values were calculated for 20 refined structures.

^c The values of Ramachandran statistics given outside of the parentheses were calculated for all residues in the 20 refined structures. The values given inside of the parentheses were calculated for residues 9–50, 58–85, and 92–122 in the 20 refined structures; the excluded residues were the N and C termini and loop regions with high flexibility and few NOE restraints.

FIGURE 1.

Solution structures of Anabaena CcbP in the Ca²⁺-free and Ca²⁺-bound forms. A and B, superimpositions of the 20 representative structures of Anabaena CcbP in the Ca²⁺-free (A) and Ca²⁺-bound forms (B). C and D, ribbon diagram representations of CcbP in Ca²⁺-free (C) and Ca²⁺-bound (D) forms, with the secondary structures labeled. The α-helical subdomain is shown in red, the β-barrel subdomain is shown in green, and the two short 3₁₀ helices located in the β-barrel subdomain are shown in yellow. The Ca²⁺ ions in the Ca²⁺-bound form are not shown. The figures were prepared by MOLMOL (25).

The Ca²⁺-free CcbP shows a mixed α-β fold, which contains an α-helical and a β-barrel subdomains. The three α-helices (residues 9–17 (α1), 25–39 (α2), and 107–122 (α3)) are formed by the N- and C-terminal parts of CcbP, with the short helix α1 packed almost perpendicularly onto the two long helices α2 and α3, forming a triangular shaped conformation. The five anti-parallel β-strands (residues 44–50 (β1), 59–68 (β2), 79–83 (β3), 92–96 (β4), and 100–103 (β5)) form a β-barrel. Two short 3₁₀ helices H1 and H2 link the β-strands β2–β3 and β4–β5, respectively. The α-helical and β-barrel subdomains are packed tightly with exclusive orientation and form a single globular architecture. The contact between the two subdomains mainly involves hydrophobic interactions. The N terminus of the protein, the extended loops connecting β2–β3 and β3–β4, and the loop linking helix α1-α2 are relatively flexible (supplemental Fig. 2).

A search using DALI (27) or CATH (28) did not find structural homologues with significant overall similarity, suggesting a new protein fold. The best hit by DALI is a partial region of a human cell cycle protein splindin-1 (Protein Data Bank code 2NS2, chain A) with a Z score of 4.8, and an root mean square deviation of 4.1 Å over 73 aligned C^α atoms. However, these aligned residues are strictly limited to the β-barrel subdomain of CcbP. Interestingly, a closer inspection reveals that this β-barrel subdomain is topologically reminiscent of the eukaryotic SH3 domain (Fig. 2A). A structural alignment by DaliLite (29) between the SH3-like subdomain of CcbP and the structure of c-Src SH3 domain (Protein Data Bank code 1QWE) showed that the overall structures are similar, with a 2.0 Å root mean square deviation for 44 aligned backbone C^α atoms. The loops consisting of residues 51–58, 69–78, and 84–91 in CcbP correspond to the RT loop, the n-Src loop and the distal loop in the c-Src structure, respectively (Fig. 2B). The structure-based sequence alignment (Fig. 2C) shows no identity (0%) between the two protein sequences. However, several residues located on the β-strands are relatively similar in amino acid types and biochemical properties. Most of these residues have hydrophobic side chains and form the hydrophobic core of the β-barrel, indicating their importance in maintaining the overall structure.

FIGURE 2.

The β-barrel subdomain of Anabaena CcbP resembles the SH3 topology. A, a schematic representation of the topology of Anabaena CcbP with secondary structural elements labeled. The dashed box indicates the β-barrel subdomain that shows similarity to the SH3 topology. B, structural comparison between the SH3-like subdomain of Anabaena CcbP and the c-Src SH3 domain (Protein Data Bank code 1QWE). The topologically equivalent β-strands in the two structures are labeled and shown in the same color, as depicted in A. The RT loop, n-Src loop, and the distal loop of c-Src SH3 domain are labeled. The corresponding loops in CcbP SH3-like subdomain are also shown and labeled with the starting and ending residue numbers. The figures were prepared by MOLMOL (25). C, structure-based sequence alignment between the SH3-like subdomain of Anabaena CcbP and the c-Src SH3 domain. Among the 44 aligned residues (shown in uppercase letters), the substitutions by the same class of amino acids according to the side chain properties are shown in red boxes and those by amino acids that share certain biochemical similarity are shown in green.

NMR Structure of Ca²⁺-bound CcbP

Similar to the Ca²⁺-free CcbP, the structure of Ca²⁺-bound CcbP was determined with a total of 4,694 restraints, including 12 Ca²⁺-O restraints (Table 1). The addition of excess CaCl₂ appears to slightly stabilize the whole structure of CcbP, as evidenced by the increased number of interproton NOE restraints. This also was supported by the fact that under a Ca²⁺-free condition, residues Ser⁵³–Ser⁵⁶ were missing in the two-dimensional ¹⁵N-edited HSQC spectrum, which appeared in the presence of excess CaCl₂. Nevertheless, comparison of the overall architecture of CcbP between the Ca²⁺-bound and Ca²⁺-free forms reveals considerable similarity (Fig. 1, B and D). All secondary structural elements as well as the relative orientations between the two subdomains are retained essentially upon Ca²⁺ binding. The backbone root mean square deviation between the mean structures of the two forms is 1.0 Å for all residues and is only 0.5 Å for residues in regular secondary structures. The results indicate that Ca²⁺ binding does not induce notable conformational changes in CcbP. In addition, we compared the motional flexibility on picosecond-to-nanosecond time scales of both forms of CcbP by measuring the backbone {¹H}-¹⁵N heteronuclear NOE values (for more details see supplemental “Results and Discussion”). The results demonstrated that Ca²⁺ binding does not induce significant changes in the motional flexibility of CcbP as well (supplemental Fig. 2).

Because CcbP exists as oligomer under low ionic strength, which forbids its structure determination by NMR, the structures of CcbP in both forms were determined in the presence of 220 mm NaCl. Taking into consideration that the intracellular environments generally contain high K⁺ concentration (∼ 100–200 mm) (30), we compared the two-dimensional ¹⁵N-edited HSQC spectra of both forms of CcbP in excess K⁺ with those in excess Na⁺. The HSQC spectra with Na⁺ or K⁺ are similar, demonstrating essentially identical conformations of CcbP (supplemental Fig. 3). Therefore, under conditions near physiological environment, the binding of Ca²⁺ ions does not induce considerable conformational changes of CcbP.

Ca²⁺ Titration by NMR

Ca²⁺ titration experiments by NMR were performed to identify the Ca²⁺-binding sites of CcbP, which were monitored by HSQC spectroscopy (additional discussions are available in supplemental “Results and Discussion” and Figs. 3–10). During the gradual increase of Ca²⁺ concentrations, two regions in CcbP showed responses (supplemental Fig. 10). When the molar ratio of Ca²⁺:CcbP changed from 0:1 to 1:1, the backbone nitrogen chemical shifts of several residues located at the C terminus of helix α2 were largely perturbed, suggesting that these residues constitutes the stronger Ca²⁺-binding site (site I). When the molar ratio Ca²⁺:CcbP continued to increase, another region (residues Ile¹⁹–Glu²⁷) located at the loop linking helix α1 and α2 showed moderate sensitivity to Ca²⁺ ions, suggesting a weaker Ca²⁺-binding site (site II).

Characterization of Ca²⁺-binding Site I

Site I locates at the segment Asp³⁷–Glu⁴¹, which is an α-turn-β motif connecting helix α2 and the first β-strand of the β-barrel subdomain (Fig. 3, A and B). Although CcbP has a large negatively charged surface, this Ca²⁺-binding site is highly specific and shows the highest sensitivity to the presence of Ca²⁺ ions (Fig. 3D, supplemental Figs. 7–10). In NMR spectroscopy, metal coordination to a backbone carbonyl can cause deshielding of the backbone nitrogen atom of the succeeding residue (31). The backbone ¹⁵N atoms of residues Asp³⁸, Thr³⁹, and Glu⁴¹ at this region showed significant downfield chemical shift changes, suggesting the main chain carbonyls of the preceding residues Asp³⁷, Asp³⁸, and Leu⁴⁰ might be potential Ca²⁺ ligands.

FIGURE 3.

Ca²⁺ binding to Anabaena CcbP. A, ribbon diagram representation of CcbP structure bound with two Ca²⁺ ions (purple balls). B and C, local structures of the Ca²⁺-binding site I (B) and II (C) in CcbP. The figures were prepared by MOLMOL (25). D and E, chemical shift changes at the binding sites. The downfield shifts of backbone nitrogen atoms of Asp³⁸, Thr³⁹, and Glu⁴¹ (D) and Val²⁰, Asp²¹, and Ala²² (E) are shown as a function of the Ca²⁺:CcbP molar ratio. F and G, in vivo assay of wild type CcbP and a CcbP mutant protein (D38A) in regulating heterocyst differentiation. Anabaena sp. PCC 7120 cells containing a wild type ccbP gene (F) or the mutant (D38A) gene (G) under a copper-inducible promoter were subjected to a nitrogen step-down in the presence of 1 μm copper for 36 h. Overexpression of CcbP suppressed heterocyst formation in Anabaena sp. PCC 7120 (F), whereas a normal heterocyst pattern was observed when CcbP-D38A was overexpressed (G). Arrows indicate heterocysts, and scale bars indicate 10 μm.

To further characterize this binding site, plasmids carrying genes encoding CcbP mutants D37A, D38A, T39A, and E41A were constructed. The Ca²⁺ titrations by NMR were performed similarly using these mutant proteins, and the Ca²⁺-binding abilities of this site were examined by comparing the backbone nitrogen chemical shift changes with the wild type CcbP. Results showed that the Ca²⁺ sensitivity of this site was impaired largely by D37A, D38A, and E41A mutants, but not T39A mutant (data not shown). Thus, it is highly possible that the side chain oxygen atoms of Asp³⁷, Asp³⁸, and Glu⁴¹ also are involved in Ca²⁺ coordination. The local structure of site I was further calculated based on the above results using the AMBER force field (see “Experimental Procedures”). The bound Ca²⁺ ion is in proximity (Ca²⁺-O distances < 3 Å) with six oxygen atoms, three from the carboxyl group of Asp³⁷, Asp³⁸, and Glu⁴¹ and three backbone carbonyls of Asp³⁷, Asp³⁸, and Leu⁴⁰ (Fig. 3B).

Characterization of Ca²⁺-binding Site II

Site II locates at the loop linking helix α1 and α2 (Fig. 3, C and E, and supplemental Fig. 10B). Similarly, we investigated the possible Ca²⁺ ligands of this site. The backbone ¹⁵N chemical shifts of residues Val²⁰, Asp²¹, and Ala²² were most perturbed during Ca²⁺ titration experiments by NMR, although the chemical shift changes were small (Fig. 3E and supplemental Figs. 7F, 9F, and 10B). CcbP mutants E17A, D21A, E23A, D24A, E26A, and E27A were purified, and Ca²⁺ titrations by NMR were performed. Results showed that the E17A and E26A mutations did not affect Ca²⁺ binding, D21A mutation only had minor effects, whereas E23A, D24A, and E27A mutations significantly decreased Ca²⁺ binding (data not shown). The local structure of site II (Fig. 3C) shows that the second Ca²⁺ ion is surrounded closely by the side chains of Glu²³, Asp²⁴, Glu²⁷, and the backbone carbonyl of Asp²¹.

Mutation at Ca²⁺-binding Site I Abolishes CcbP Function in Vivo

Because site I of CcbP binds Ca²⁺ significantly stronger than site II, it appears that site I may contribute prominently to Ca²⁺ sequestration in vivo. To assess the functional significance of this site in heterocyst differentiation regulation, in vivo functional assays were performed.

We constructed plasmids carrying ccbP genes encoding the wild type protein or mutants at site I under the control of the petE promoter (32). The plasmids were used to transform Anabaena sp., so that the expression of ccbP was inducible with copper. During nitrogen step down, and in the absence of added copper, the Anabaena strain transformed with plasmid carrying wild type ccbP gene was able to form heterocysts with slightly decreased heterocyst frequency (10). When the expression of wild type CcbP was induced with copper under nitrogen limiting conditions, the free Ca²⁺ was sequestered and the heterocyst formation was suppressed completely as reported previously (Fig. 3F) (10). However, overexpressing a mutant protein CcbP-D38A failed to suppress heterocyst formation (Fig. 3G). The fact that the point mutation D38A abolishes the Ca²⁺-binding ability of CcbP in vivo demonstrates the biological importance of site I in heterocyst differentiation regulation.

DISCUSSION

Anabaena CcbP Represents a Novel Family of Ca²⁺-binding Proteins

Ca²⁺ binds to a variety of proteins, including those that mediate cell adhesion, enzymes that need Ca²⁺ to be activated, as well as Ca²⁺ buffers and Ca²⁺ sensors. The Ca²⁺-binding protein CcbP from Anabaena displays unique structural characteristics compared with other known families of Ca²⁺-binding proteins. The structure of CcbP shows an overall new fold containing a triangular shaped α-helical region packed tightly onto a β-barrel subdomain. Multiple sequence alignment of CcbP proteins from different cyanobacteria species and from proteobacterium Polaromonas naphthalenivorans CJ2 (Fig. 4A) indicates that the sequence conservation is much higher in the three helices than in the β-barrel region. Because the three α-helices of CcbP together form the highly acidic surface containing the two Ca²⁺-binding sites (Fig. 4, B and C), the amino acid conservation in these regions might be critical for maintaining the structural scaffold in CcbP for Ca²⁺ binding. Although binding of Ca²⁺ ions does not result in significant conformational changes of the protein, it alters the local charge at the two sites and also partially neutralizes the nearby area, which might influence the protein-protein interaction pattern of CcbP (supplemental Fig. 11).

FIGURE 4.

Sequence similarity of the α-helical region of Anabaena CcbP suggests a conserved Ca²⁺-binding scaffold. A, multiple sequence alignment of CcbP proteins from different cyanobacteria species and proteobacterium P. naphthalenivorans CJ2 by ClustalW (46). The secondary structural elements of CcbP from Anabaena sp. PCC 7120 are shown at the top. Strictly conserved residues are shown in red boxes, and highly conserved residues are shown in white boxes. The figure was generated using ESPript (47). B, mapping of the conserved residues onto the ribbon diagram of CcbP. Strictly conserved residues are depicted in red, and highly conserved residues are shown in pink. The figure was prepared by MOLMOL (25). C, surface electrostatic potential representation of Ca²⁺-free CcbP generated by GRASP2 (48). The calculation was performed under the salt concentration of 0.22 m. Red represents negative charges, and blue represents positive charges. The drawing in C is oriented as in B. The Ca²⁺-binding sites are indicated in B and C.

Ca²⁺-binding Sites of CcbP

The Ca²⁺-binding site I of CcbP locates at a short α-turn-β region, which is distinct from previously reported Ca²⁺-binding sites. In vivo functional assays showed that a single amino acid mutation at this site abolished the activity of CcbP in regulating heterocyst formation, demonstrating its biological importance. Furthermore, the amino acids at this site are conserved highly among the CcbP family (Fig. 4A). In particular, residues Tyr³⁵, Leu³⁶, Leu⁴⁰, Pro⁴³, and Phe⁴⁴ are strictly conserved. In addition, residues 37, 38, and 41, whose side chains may participate in Ca²⁺ binding, are restricted to carbonyl-containing Asp, Glu, Asn, or Gln. The in vivo and in vitro experimental results, together with bioinformatics analysis, strongly suggest that this novel Ca²⁺-binding site plays essential role in Ca²⁺ sequestration in Anabaena.

On the other hand, Ca²⁺-binding site II of CcbP consists of an α-loop-α region, which is similar to a single EF-hand motif. Interestingly, the 12-residue sequence of Glu¹⁵–Glu²⁶ (ETEIIVDAEDKE) fits well to an EF-hand Ca²⁺-binding loop. According to the classical EF-hand, the residue Glu²⁶ in position 12 is the most critical and strictly conserved in the EF-hand motif. The acidic residues Glu¹⁵ and Glu¹⁷ at positions 1 and 3 and the water mediating Glu²⁴ at position 9 are ideal for Ca²⁺ binding, whereas the amino acids at positions 5 and 6 show deviations from canonical EF-hands (33). However, the NMR titration experiments and mutagenesis results indicate that unlike canonical EF-hands, the acidic side chains of residues Glu²³, Asp²⁴, and Glu²⁷ in CcbP appear important in Ca²⁺ binding.

The affinity of Ca²⁺ binding by CcbP was determined in the previous study with the Ca²⁺ electrode method, which identified a Ca²⁺-binding site with K_d of 200 nm and a second Ca²⁺-binding site with K_d of 12.8 μm (11). In this study, we also determined K_d values of CcbP with NMR titration at both high (∼0.5 mm) and low (0.05 mm) protein concentrations. The results show that site I binds Ca²⁺ with a dissociation constant K_d ∼ 40–100 μm, whereas site II shows much weaker Ca²⁺-binding affinity (K_d in the millimolar range). Thus, both methods clearly identified the Ca²⁺-binding site with a K_d at low micromolar range, demonstrating that CcbP is a Ca²⁺-binding protein. However, there is an unexpected result from our present study; the high affinity site (K_d ∼ 200 nm) was not observed in NMR titrations. Based on the facts that the Scatchard plot used to determine K_d values in Ca²⁺ electrode method could be affected by certain errors (34), we speculate that the apparent “high affinity binding site” of CcbP observed in the Ca²⁺-electrode method could be introduced artificially, and the much weaker Ca²⁺-binding site II observed in NMR titration was not observable by the Scatchard plot. This was further supported by the results from ITC experiments, which demonstrated that CcbP contains a Ca²⁺-binding site I with a K_d value of 39 ± 9 μm and a Ca²⁺-binding site II with a K_d value in the millimolar range (Fig. 5). Therefore, CcbP binds Ca²⁺ with micromolar and millimolar range affinities. This calcium-binding ability was shown to be functionally important in vivo and underlines the biological role of CcbP in Anabaena Ca²⁺ signaling during heterocyst differentiation.

FIGURE 5.

Isothermal titration calorimetric analysis of the Ca²⁺ binding to CcbP. Trace of the calorimetric titration of 34 × 8-μl aliquots of 2.9 mm CaCl₂ into the cell containing 100 μm Ca²⁺-free CcbP (upper panel) and integrated binding isotherm (lower panel). The binding isotherm in the lower panel was fit using a two-site binding model where N₁ = 1 (fixed), K₁ = (2.57 ± 0.49) × 10⁴ [M⁻¹], ΔH₁ = −(2.02 ± 0.12) × 10³ [cal·mol⁻¹]; N₂ = 1 (fixed), K₂ = 565 ± 63 [M⁻¹], ΔH₂ = −(5.79 ± 0.36) × 10³ [cal·mol⁻¹]. N is the stoichiometry of binding, K is the Ca²⁺-binding association constant (1/K_d), and ΔH is the heat change per mol.

Previous studies demonstrated that CcbP is directly associated with calcium sequestration in cyanobacterial cells and acts as a negative regulator in heterocyst differentiation. The detailed mechanism of the function of CcbP in calcium sequestration in vivo, however, is yet unclear. Our structural study of CcbP demonstrated that it is indeed a calcium binding protein with a novel calcium-binding motif (Ca²⁺-binding site I), which has biological significance. A Ca²⁺-buffering function was suggested previously based on functional studies (11). Because the Ca²⁺ binding affinity of site I is in the micro-molar range, whereas the intracellular concentrations of free Ca²⁺ in cyanobacteria are between 100 nm and 200 nm (11), the calcium ions bound by CcbP alone might not be a major pool of bound calcium under nitrogen-replete conditions. CcbP could be more important in regulation of free calcium concentration during the process of heterocyst formation when calcium concentration increased significantly in heterocysts and proheterocysts (10). The increase of calcium concentration could come from an increased influx of calcium and/or a release of bound calcium ions, which remains to be further investigated.

SH3-like Subdomain in CcbP

Apart from the three acidic α-helices that contain the two Ca²⁺-binding sites, the structure of CcbP unexpectedly reveals a β-barrel subdomain topologically and structurally reminiscent of eukaryotic SH3 domain. The SH3 domain is a small module with 55–70 residues commonly found in eukaryotic signaling pathways, and it mediates transient protein-protein interactions with moderate affinity (35, 36). It recognizes specific proline-rich sequences and prefers sequences with a PxxP core motif (where x is any amino acid residue) (35–38). A close inspection found a short proline-rich sequence YPWIPGRSRIP in HetR, which contains the general consensus sequence ΦPxΦPx+ (where Φ is a hydrophobic residue, x is any amino acid, and + is a basic residue, usually arginine) of the class II motif that interacts with the canonical SH3 domains. Moreover, this sequence in HetR also closely resembles the consensus RPxΦPΦR+SxP motif recognized by the p53bp2 SH3 domain (39). Therefore, a likely scenario is that the proline-rich sequence of HetR recognizes and interacts with the SH3-like domain of CcbP, which facilitates the degradation of CcbP and the release of Ca²⁺ ions during heterocyst differentiation.

Other prokaryotic domains that are sequentially unrelated to but topologically reminiscent of eukaryotic SH3 domains have also been discovered in recent years (40–45). Among these, the SH3-like domain in the bacterial histidine kinase CheA mirrors the SH3 domains in mammalian tyrosine kinases and suggests the ubiquitous involvement of this common topology in cell signaling among different life kingdoms (40). Our study reveals the presence of an SH3-like subdomain in Anabaena CcbP, which demonstrates a direct association of the SH3-like domain with a Ca²⁺-binding protein. This represents another paradigm for the coupling of SH3 topology to prokaryotic signaling processes, and in particular, is the first example for the involvement of SH3-like domains in prokaryotic Ca²⁺ signaling.

Conclusions

In summary, the present study reveals that Anabaena CcbP adopts an overall new fold with two Ca²⁺-binding sites. Site I consists of an α-turn-β region unreported previously, whereas site II resembles a single EF-hand motif. Furthermore, CcbP harbors an SH3-like subdomain which might play a role in Ca²⁺ release. Our study provides the structural basis for understanding the function of CcbP in the Ca²⁺ signaling in Anabaena and offers novel insights for future investigations into the molecular mechanism of heterocyst differentiation regulation.