Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.

Focusing of the electrostatic potential at EF-hands of calbindin D(9k): titration of acidic residues.

Biological functions for a large class of calmodulin-related proteins, such as target protein activation and Ca(2+) buffering, are based on fine-tuned binding and release of Ca(2+) ions by pairs of coupled EF-hand metal binding sites. These are abundantly filled with acidic residues of so far unknown ionization characteristics, but assumed to be essential for protein function in their ionized forms. Here we describe the measurement and modeling of pK(a) values for all aspartic and glutamic acid residues in apo calbindin D(9k), a representative of calmodulin-related proteins. We point out that while all the acidic residues are ionized predominantly at neutral pH, the onset of proton uptake by Ca(2+) ligands with high pK(a) under these conditions may have functional implications. We also show that the negative electrostatic potential is focused at the bidental Ca(2+) ligand of each site, and that the potential is significantly more negative at the N-terminal binding site.

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k).

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.

Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k).

Calbindin D(28k) is a member of a large family of intracellular Ca(2+) binding proteins characterized by EF-hand structural motifs. Some of these proteins are classified as Ca(2+)-sensor proteins, since they are involved in transducing intracellular Ca(2+) signals by exposing a hydrophobic patch on the protein surface in response to Ca(2+) binding. The hydrophobic patch serves as an interaction site for target enzymes. Other members of this group are classified as Ca(2+)-buffering proteins, because they remain closed after Ca(2+) binding and participate in Ca(2+) buffering and transport functions. ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca(2+)-free and Ca(2+)-loaded form of calbindin D(28k) have exposed hydrophobic surfaces. Since exposure of hydrophobic surface is unfavorable in the aqueous intracellular milieu, calbindin D(28k) most likely interacts with other cellular components in vivo. A Ca(2+)-induced conformational change was readily detected by several optical spectroscopic methods. Thus, calbindin D(28k) shares some of the properties of Ca(2+)-sensor proteins. However, the Ca(2+)-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin. The data also shows that calbindin D(28k) undergoes a rapid and reversible conformational change in response to a H(+) concentration increase within the physiological pH range. The pH-dependent conformational change was shown to reside mainly in EF-hands 1-3. Urea-induced unfolding of the protein at pH 6, 7, and 8 showed that the stability of calbindin D(28k) was increased in response to H(+) in the range examined. The results suggest that calbindin D(28k) may interact with targets in a Ca(2+)- and H(+)-dependent manner.

Fragment complementation of calbindin D28k.

Calbindin D28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF-hands each (residues 1-132 and 133-261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133-261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1-132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1-132 and 133-261 fragments. The affinity between these two fragments is 2 x 10(7) M(-1), with association and dissociation rate constants of 2.7 x 10(4) M(-1) s(-1) and 1.4 x 10(-3) s(-1), respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual alpha-helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The alpha-helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D28k.

EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X.

Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca(2+)-binding consensus sequence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca(2+)-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca(2+) affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca(2+) affinity, we expressed the Ca(2+)-binding EGF pair 3-4 from human protein S. Correct folding was shown by (1)H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by (1)H-(15)N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca(2+) binding were 1.0x10(-6) M and 4. 8x10(-3) M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca(2+) affinity of EGF 4, in the pair 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.

Ionization behavior of acidic residues in calbindin D(9k).

The ionization state of seven glutamate residues, one aspartate, and the C-terminal alpha-COOH group in bovine apo calbindin D(9k) has been studied by measurement and modeling of the pH titration curves and apparent pK(a) values. The observed pK(a) ranged from 3.0 to 6.5. Most of the observed acidic groups were half-ionized at lower pH values than those in unstructured proteins. As a rule, the ionization equilibria extended over a wider pH range than in the case of unperturbed single titrations, indicating a complex influence of protein charges on the charge state of each individual residue. Glu17, which is a backbone Ca(2+)-ligand in the N-terminal binding loop of calbindin D(9k), was half-protonated at pH 3.6 but manifested biphasic titration with apparent pK(a) values of 3.2 and 6.5. Complementary Monte Carlo simulations of the titration process and pK(a) values of the acidic groups in calbindin D(9k) reproduce the experimentally observed titration features, except for the pronounced double titration of Glu17. Discrepancies between the results from direct measurement and from modeling may be partly caused by changes in the protein structure when the net charge changes from -8 to +11 over the isoelectric point at pH 5. Proteins 1999;37:106-115.

Expression and purification of human calbindin D28k.

Calbindin D28k is a protein abundant in the mammalian central nervous system and in epithelial tissue involved in Ca2+ transport. Human calbindin D28k was cloned into a Pet3a vector and expressed in Escherichia coli. The protein was purified in three steps: (i) heat precipitation of bacterial proteins, (ii) ion-exchange chromatography on a DEAE-cellulose column in the presence of calcium, and (iii) ion-exchange chromatography on a DEAE-Sephacel column in the presence of EDTA. The protein was then supplemented with calcium and dialyzed against neutral water. The final yield was 20-50 mg of pure, homogeneous calcium-loaded calbindin D28k per liter of bacterial culture. The identity and purity of the protein were confirmed by immunoblotting, SDS-polyacrylamide gel electrophoresis, and agarose gel electrophoresis in the absence and presence of calcium and 1H NMR spectroscopy. The entire expression and purification protocol takes only 3 days and is easy to scale up and down. It was designed to minimize degradation and deamidation.

NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.

When size is important. Accommodation of magnesium in a calcium binding regulatory domain.

The accommodation of Mg2+ in the N-terminal domain of calmodulin was followed through amide 1H and 15N chemical shifts and line widths in heteronuclear single-quantum coherence spectroscopy NMR spectra. Mg2+ binds sequentially to the two Ca2+-binding loops in this domain, with affinities such that nearly half of the loops would be occupied by Mg2+ in resting eukaryotic cells. Mg2+ binding seems to occur without ligation to the residue in the 12th loop position, previously proven largely responsible for the major rearrangements induced by binding of the larger Ca2+. Consequently, smaller Mg2+-induced structural changes are indicated throughout the protein. The two Ca2+-binding loops have different Mg2+ binding characteristics. Ligands in the N-terminal loop I are better positioned for cation binding, resulting in higher affinity and slower binding kinetics compared with the C-terminal loop II (koff = 380 +/- 40 s-1 compared with approximately 10,000 s-1 at 25 degreesC). The Mg2+-saturated loop II undergoes conformational exchange on the 100-microseconds time scale. Available data suggest that this exchange occurs between a conformation providing a ligand geometry optimized for Mg2+ binding and a conformation more similar to that of the empty loop.

Ca2+ binding and conformational changes in a calmodulin domain.

Calcium activation of the C-terminal domain of calmodulin was studied using 1H and 15N NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop IV. The study involves determination of Ca2+ binding constants, assignments, and structural characterizations of the apo, (Ca2+)1, and (Ca2+)2 states of the E104Q mutant and comparisons to the wild-type protein and the E140Q mutant [Evenäs et al. (1997) Biochemistry 36, 3448-3457]. NMR titration data show sequential Ca2+ binding in the E104Q mutant. The first Ca2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both mutants, the major structural changes occur upon Ca2+ binding to loop IV, which implies a different response to Ca2+ binding in the N- and C-terminal EF-hands. Spectral characteristics show that the (Ca2+)1 and (Ca2+)2 states of the E104Q mutant undergo global exchange on a 10-100 micros time scale between conformations seemingly similar to the closed and open structures of this domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of the open conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a single structure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensemble of structures the precision is good. However, the clearly dynamic nature of the state, a large number of violated distance restraints, ill-defined secondary structural elements, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of the E104Q mutant but rather represents the distance-averaged structure of at least two distinct different conformations.

Hydrophobic core substitutions in calbindin D9k: effects on stability and structure.

The effects of hydrophobic core mutations on the stability and structure of the four-helix calcium-binding protein, calbindin D9k, have been investigated. Eleven mutations involving eight residues distributed within the hydrophobic core of calbindin D9k were examined. Stabilities were measured by denaturant and thermal induced unfolding monitored by circular dichroism spectroscopy. The mutations were found to exert large effects on the stability with midpoints in the urea induced unfolding varying from 1.8 M for Leu23 --> Gly up to 6.6 M for Val70 --> Leu and free energies of unfolding in the absence of denaturant ranging from 6.6 to 27.4 kJ/mol for the Phe66 --> Ala mutant and the wild-type, respectively. A significant correlation was found between the difference in free energy of unfolding (Delta Delta GNU) and the change in the surface area of the side chain caused by the mutation, in agreement with other studies. Notably, both increases and decreases in side-chain surface area caused quantitatively equivalent effects on the stability. In other words, a correlation between the absolute value of the change in the surface of the side chain and Delta DeltaGNU was observed with a value of approximately 0.14 kJ M-1 A-2. The generality of this observation is discussed. Significant effects on the cooperativity of the unfolding reaction were also observed. However, a correlation between the cooperativity and Delta Delta GNU, which has been reported in other systems as an indication of effects of mutations on the unfolded state, was not observed for calbindin D9k. Despite the large effects on Delta Delta GNU and cooperativity, the structures of the mutants in the native form remained intact as indicated by circular dichroism, NMR, and fluorescence measurements. The structural response to calcium-binding was also conserved. The following paper in this issue [Kragelund, B. B., et al. (1998) Biochemistry 37, 8926-8937] examines the effects of these mutations on the calcium binding properties of calbindin D9k.

NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state.

In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+-induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild-type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 +/- 15% of the open conformation and an exchange rate of (1-7) x 10(4) s(-1) were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2+ titration of the 15N-labeled mutant, the macroscopic binding constants [log(K1) = 4.9 +/- 0.3 and log(K2) = 3.15 +/- 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+-binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.

A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies.

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.

Domain organization of calbindin D28k as determined from the association of six synthetic EF-hand fragments.

Calbindin D28k is an intracellular Ca(2+)-binding protein containing six subdomains of EF-hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic peptide fragments, each corresponding to one EF-hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2+ to allow for the reconstitution of domains. The mixture was compared to native calbindin D28k and to the sum of the properties of the individual peptides using circular dichroism (CD), fluorescence, and 1H NMR spectroscopy, as well as gel filtration and ion-exchange chromatography. It was anticipated that if the peptides associate to form native-like domains, the properties would be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the peptides in the mixture interact with one another. For example, the CD and fluorescence spectra for the mixture are very similar to those of the intact calbindin D28k, suggesting that the mixed EF-hand fragments associate to form a native-like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D28k, a variety of peptide combinations containing two to five EF-hand fragments were studied. The spectral and chromatographic properties of all the mixtures containing less than six peptides were closer to the sum of the properties of the relevant individual peptides than to the mixture of the six peptides. The results strongly suggest that all six EF-hands are packed into one globular domain. The association of the peptide fragments is observed to drive the folding of the individual subdomains. For example, one of the fragments, EF2, which is largely unstructured in isolation even in the presence of high concentrations of Ca2+, is considerably more structured in the presence of the other peptides, as judged by CD difference spectroscopy. The CD data also suggest that the packing between the individual subdomains is specific.

Complexes formed between calmodulin and the antagonists J-8 and TFP in solution.

The binding of the antagonists N-(8-aminooctyl)-5-iodonaphthalene-1-sulfonamide (J-8) and trifluoperazine (TFP) to intact calcium-saturated bovine calmodulin (CaM) and also of J-8 to the C-terminal domain (tr2c) has been investigated. Using a combination of NMR methods, including NOESY data, mobility measurements, and chemical shift and line-shape analysis, we show that the primary interaction between J-8 and tr2c is between the naphthalene ring of the antagonist and the hydrophobic pocket of the protein, similar to the binding of the hydrophobic side-chain residues of calmodulin target peptides. Comparison of the mobility of the drug, the intensity and pattern of intermolecular NOESY cross-peaks, and chemical shift changes shows that there is no significant change in the binding mode in J-8. CaM compared to J-8.tr2c, with one molecule binding to each domain. In particular, we find that the mobility of the aliphatic amino "tail" of J-8 remains highly mobile in both systems. This contrasts with the notion that the tail may bridge between the two domains to give a "globular" form of CaM. We also show that TFP induces very similar shift changes to J-8 and that the stoichiometry of the major binding event in all three cases is one drug molecule per domain. It also appears that secondary binding sites for the drug molecules are present in all three systems.

Measurement and modelling of sequence-specific pKa values of lysine residues in calbindin D9k.

A pH titration study of calbindin D9k was performed using heteronuclear 1H-13C two-dimensional NMR spectroscopy. The protein was produced with carbon-13 label in the side-chain of lysine residues, next to the titrating group. The site-specific pKa values of these lysine residues, ranging from 10.1 to 12.1, were obtained from the analysis of pH-dependent chemical shifts of 13C and 1H resonances. Ionization constants for both the Ca(2+)-free (apo) and Ca(2+)-loaded forms of the protein were determined. The proton uptake by lysine residues in the apo form was shifted up to 1.7 units towards high pH as compared to that for the model compound. The binding of calcium affected the pKa values of all lysine residues. The largest reduction of one pK unit was observed for Lys55, which is also the closest to the calcium binding sites. A threefold increase in protein concentration, from 0.5 to 1.5 mM, reduced the pKa values by 0.1 to 0.4 pK unit in agreement with the screening concept of ionic interactions. All the observed pKa shifts were site-specific, depending on the local electrostatic environment and were reproduced in Monte Carlo simulations based on the three-dimensional structure of calbindin D9k and a dielectric continuum model for the electrostatic interactions.

Ca2+-binding stoichiometry of calbindin D28k as assessed by spectroscopic analyses of synthetic peptide fragments.

Calbindin D28k is an intracellular Ca2+-binding protein noted for its abundance and specific distribution in mammalian brain and sensory neurons. This protein contains six putative Ca2+-binding sites, referred to as EF-hands. Due to the presence of the large number of putative sites, previous studies have been unsuccessful in definitively establishing the stoichiometry of Ca2+ binding. We describe a synthetic approach to identify the number of Ca2+-binding sites in which 6 33-residue peptides, designated EF1-EF6, corresponding to the 6 EF-hand sequences of calbindin D28k, were made. The response of each peptide to Ca2+ addition was assessed by 1H NMR spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis. The Ca2+ binding by CD experiments was performed at two peptide concentrations, 20 and 200 microM, and the NMR studies at peptide concentrations ranging from 20 to 100 microM. The CD and 1H NMR data show that five of the six peptides bind Ca2+ as isolated peptides, namely, EF1, EF3, EF4, EF5, and EF6. The EF6 peptide appears to bind Ca2+ with lower affinity than the other four functional sites. In contrast, EF2 does not appear to bind Ca2+ under any of the spectroscopic conditions tested. The data suggest that at least five of the six putative sites in the native protein bind Ca2+, although their relative affinities cannot be deduced from studies of the isolated peptides.

Calcium-induced structural changes and domain autonomy in calmodulin.

We have determined the solution structures of the apo and (Ca2+)2 forms of the carboxy-terminal domain of calmodulin using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The results show that both forms adopt well-defined structures with essentially equal secondary structure. A comparison of the structures of the two forms shows that Ca2+ binding causes major rearrangements of the secondary structure elements with changes in inter-residue distances of up to 15 A and exposure of the hydrophobic interior of the four-helix bundle. Comparisons with previously determined high-resolution X-ray structures and models of calmodulin indicate that this domain is structurally autonomous.

Characterization of the N-terminal half-saturated state of calbindin D9k: NMR studies of the N56A mutant.

Calbindin D9k is a small EF-hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site (1) is investigated by NMR experiments on the half-saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038-1044). Analysis of calcium-induced changes in chemical shifts, amide proton exchange rates, and NOEs indicates that ion binding to the N-terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calcium-loaded state. These results are similar to those observed for the (Cd2+)1 state of the wild-type protein, a model for the complementary half-saturated state with an ion bound in the C-terminal site (II). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half-saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF-hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation.