Cerebrospinal fluid-induced retardation of amyloid ß aggregation correlates with Alzheimer's disease and the APOE ε4 allele.

Misfolding and aggregation of amyloid ß (Aß) are key features of Alzheimer's disease (AD) pathogenesis, but the molecular events controlling this process are not known in detail. In vivo, Aß aggregation and plaque formation occur in the interstitial fluid of the brain extracellular matrix. This fluid communicates freely with cerebrospinal fluid (CSF). Here, we examined the effect of human CSF on Aß aggregation kinetics in relation to AD diagnosis and carrier status of the apolipoprotein E (APOE) ε4 allele, the main genetic risk factor for sporadic AD. The aggregation of Aß was inhibited in the presence of CSF and, surprisingly, the effect was more pronounced in APOE ε4 carriers. However, by fractionation of CSF using size exclusion chromatography, it became evident that it was not the ApoE protein itself that conveyed the inhibition, since the retarding species eluted at lower volume, corresponding to a much higher molecular weight, than ApoE monomers. Cholesterol quantification and immunoblotting identified high-density lipoprotein particles in the retarding fractions, indicating that such particles may be responsible for the inhibition. These results add information to the yet unresolved puzzle on how the risk factor of APOE ε4 functions in AD pathogenesis.

A peptide from human semenogelin I self-assembles into a pH-responsive hydrogel.

The peptide GSFSIQYTYHV derived from human semenogelin I forms a transparent hydrogel through spontaneous self-assembly in water at neutral pH. Linear rheology measurements demonstrate that the gel shows a dominating elastic response over a large frequency interval. CD, fluorescence and FTIR spectroscopy and cryo-TEM studies imply long fibrillar aggregates of extended ß-sheet. Dynamic light scattering data indicate that the fibril lengths are of the order of micrometers. Time-dependent thioflavin T fluorescence shows that fibril formation by GSFSIQYTYHV is a nucleated reaction. The peptide may serve as basis for development of smart biomaterials of low immunogenicity suitable for biomedical applications, including drug delivery and wound healing.

The effect of nanoparticles on amyloid aggregation depends on the protein stability and intrinsic aggregation rate.

Nanoparticles interfere with protein amyloid formation. Catalysis of the process may occur due to increased local protein concentration and nucleation on the nanoparticle surface, whereas tight binding or a large particle/protein surface area may lead to inhibition of protein aggregation. Here we show a clear correlation between the intrinsic protein stability and the nanoparticle effect on the aggregation rate. The results were reached for a series of five mutants of single-chain monellin differing in intrinsic stability toward denaturation, for which a correlation between protein stability and aggregation propensity has been previously documented by Szczepankiewicz et al. [Mol. Biosyst.20107 (2), 521-532]. The aggregation process was monitored by thioflavin T fluorescence in the absence and presence of copolymeric nanoparticles with different hydrophobic characters. For mutants with a high intrinsic stability and low intrinsic aggregation rate, we find that amyloid fibril formation is accelerated by nanoparticles. For mutants with a low intrinsic stability and high intrinsic aggregation rate, we find the opposite--a retardation of amyloid fibril formation by nanoparticles. Moreover, both catalytic and inhibitory effects are most pronounced with the least hydrophobic nanoparticles, which have a larger surface accessibility of hydrogen-bonding groups in the polymer backbone.

Inhibition of IAPP and IAPP(20-29) fibrillation by polymeric nanoparticles.

The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20-29)) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation as a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied from 50:50 to 100:0. The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP(20-29) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed as a delayed onset of ThT fluorescence increase, plus a reduction of the amount of fibrils formed as indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP(20-29)), the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.

Streptococcal M protein: structural studies of the hypervariable region, free and bound to human C4BP.

Streptococcus pyogenes is a Gram-positive bacterium that causes several diseases, including acute tonsillitis and toxic shock syndrome. The surface-localized M protein, which is the most extensively studied virulence factor of S. pyogenes, has an approximately 50-residue N-terminal hypervariable region (HVR) that plays a key role in the escape of the host immunity. Despite the extensive sequence variability in this region, many HVRs specifically bind human C4b-binding protein (C4BP), a plasma protein that inhibits complement activation. Although the more conserved parts of M protein are known to have dimeric coiled-coil structure, it is unclear whether the HVR also is a coiled coil. Here, we use nuclear magnetic resonance (NMR) to study the conformational properties of HVRs from M4 and M22 proteins in isolation and in complex with the M protein binding portion of C4BP. We conclude that the HVRs of M4 and M22 are folded as coiled coils and that the folded nucleus of the M4 HVR has a length of approximately 27 residues. Moreover, we demonstrate that the C4BP binding surface of M4-N is found within a region of four heptad repeats. Using molecular modeling, we propose a model for the structure of the M4 HVR that is consistent with our experimental information from NMR spectroscopy.

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.

Isolated hypervariable regions derived from streptococcal M proteins specifically bind human C4b-binding protein: implications for antigenic variation.

Antigenic variation in microbial surface proteins represents an apparent paradox, because the variable region must retain an important function, while exhibiting extensive immunological variability. We studied this problem for a group of streptococcal M proteins in which the approximately 50-residue hypervariable regions (HVRs) show essentially no residue identity but nevertheless bind the same ligand, the human complement regulator C4b-binding protein (C4BP). Synthetic peptides derived from different HVRs were found to retain the ability to bind C4BP, implying that the HVR corresponds to a distinct ligand-binding domain that can be studied in isolated form. This finding allowed direct characterization of the ligand-binding properties of isolated HVRs and permitted comparisons between different HVRs in the absence of conserved parts of the M proteins. Affinity chromatography of human serum on immobilized peptides showed that they bound C4BP with high specificity and inhibition experiments indicated that different peptides bound to the same site in C4BP. Different C4BP-binding peptides did not exhibit any immunological cross-reactivity, but structural analysis suggested that they have similar folds. These data show that the HVR of streptococcal M protein can exhibit extreme variability in sequence and immunological properties while retaining a highly specific ligand-binding function.

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.

The first epidermal growth factor-like domain of the low-density lipoprotein receptor contains a noncanonical calcium binding site.

Removal of cholesterol-containing particles from the circulation is mediated by the low-density lipoprotein (LDL) receptor. Upon ligand binding, the receptor-ligand complex is endocytosed, and the ligand is released. The important biological role of the LDL receptor (LDLR) has been highlighted by the identification of more than 400 LDLR mutations that are associated with familial hypercholesterolemia. The extracellular region of the LDLR is modular in nature and principally comprises multiple copies of ligand binding, epidermal growth factor-like (EGF), and YWTD-type domains. This report describes characterization of the calcium binding properties of the tandem pair of EGF domains. While only the C-terminal EGF module contains the consensus sequence associated with calcium binding, a noncanonical calcium binding site in the N-terminal domain has been revealed using solution NMR spectroscopy. The calcium dissociation constants for the N- and C-terminal sites have been measured under physiologically relevant pH and ionic strength conditions using a combination of solution NMR, intrinsic protein fluorescence, and chromophoric chelator methods to be approximately 50 microM and approximately 10-20 microM, respectively. Identification of the novel calcium binding motif in LDLR sequences from other species suggests that it may confer specificity within the LDLR gene family. Comparison of the K(d) for the C-terminal site with the calcium concentration in late vesicles indicates that the binding properties of this module may be tuned to titrate upon endocytosis of the LDL receptor-ligand complex, and thus calcium binding may play a role in the ligand dissociation process.

The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved methionines in a surface-exposed amphipathic alpha-helix.

The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.

An extended hydrophobic core induces EF-hand swapping.

The structure of calbindin D(9k) with two substitutions was determined by X-ray crystallography at 1.8-A resolution. Unlike wild-type calbindin D(9k), which is a monomeric protein with two EF-hands, the structure of the mutated calbindin D(9k) reveals an intertwined dimer. In the dimer, two EF-hands of the monomers have exchanged places, and thus a 3D domain-swapped dimer has been formed. EF-hand I of molecule A is packed toward EF-hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF-hands, promotes the conversion of monomers to 3D domain-swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D(9k). Once formed, the calbindin D(9k) dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D(9k) dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.

Fragment complementation studies of protein stabilization by hydrophobic core residues.

Interactions that stabilize the native state of a protein have been studied by measuring the affinity between subdomain fragments with and without site-specific residue substitutions. A calbindin D(9k) variant with a single CNBr cleavage site at position 43 between its two EF-hand subdomains was used as a starting point for the study. Into this variant were introduced 11 site-specific substitutions involving hydrophobic core residues at the interface between the two EF-hands. The mutants were cleaved with CNBr to produce wild-type and mutated single-EF-hand fragments: EF1 (residues 1--43) and EF2 (residues 44--75). The interaction between the two EF-hands was studied using surface plasmon resonance (SPR) technology, which follows the rates of association and dissociation of the complex. Wild-type EF1 was immobilized on a dextran matrix, and the wild-type and mutated versions of EF2 were injected at several different concentrations. In another set of experiments, wild-type EF2 was immobilized and wild-type or mutant EF1 was injected. Dissociation rate constants ranged between 1.1 x 10(-5) and 1.0 x 10(-2) s(-1) and the association rate constants between 2 x 10(5) and 4.0 x 10(6) M(-1) s(-1). The affinity between EF1 and EF2 was as high as 3.6 x 10(11) M(-1) when none of them was mutated. For the 11 hydrophobic core mutants, a strong correlation (r = 0.999) was found between the affinity of EF1 for EF2 and the stability toward denaturation of the corresponding intact protein. The observed correlation implies that the factors governing the stability of the intact protein also contribute to the affinity of the bimolecular EF1-EF2 complex. In addition, the data presented here show that interactions among hydrophobic core residues are major contributors both to the affinity between the two EF-hand subdomains and to the stability of the intact domain.

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.

Conversion of alpha-lactalbumin to a protein inducing apoptosis.

In this study alpha-lactalbumin was converted from the regular, native state to a folding variant with altered biological function. The folding variant was shown to induce apoptosis in tumor cells and immature cells, but healthy cells were resistant to this effect. Conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells) required partial unfolding of the protein and a specific fatty acid, C18:1, as a necessary cofactor. Conversion was achieved with alpha-lactalbumin derived from human milk whey and with recombinant protein expressed in Escherichia coli. We thus have identified the folding change and the fatty acid as two key elements that define HAMLET, the apoptosis-inducing functional state of alpha-lactalbumin. Although the environment in the mammary gland favors the native conformation of alpha-lactalbumin that serves as a specifier in the lactose synthase complex, the conditions under which HAMLET was formed resemble those in the stomach of the nursing child. Low pH is known to release Ca(2+) from the high-affinity Ca(2+)-binding site and to activate lipases that hydrolyze free fatty acids from milk triglycerides. We propose that this single amino acid polypeptide chain may perform vastly different biological functions depending on its folding state and the in vivo environment. It may be speculated that molecules like HAMLET can aid in lowering the incidence of cancer in breast-fed children by purging of tumor cells from the gut of the neonate.

A folding variant of alpha-lactalbumin with bactericidal activity against Streptococcus pneumoniae.

This study describes an alpha-lactalbumin folding variant from human milk with bactericidal activity against antibiotic-resistant and -susceptible strains of Streptococcus pneumoniae. The active complex precipitated with the casein fraction at pH 4.6 and was purified from casein by a combination of anion exchange and gel chromatography. Unlike other casein components, the active complex was retained on the ion-exchange matrix and eluted only with high salt. The eluted fraction showed N-terminal and mass spectrometric identity with human milk alpha-lactalbumin, but native alpha-lactalbumin had no bactericidal effect. Spectroscopic analysis demonstrated that the active form of the molecule was in a different folding state, with secondary structure identical to alpha-lactalbumin from human milk whey, but fluctuating tertiary structure. Native alpha-lactalbumin could be converted to the active bactericidal form by ion-exchange chromatography in the presence of a cofactor from human milk casein, characterized as a C18:1 fatty acid. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram-negative and other Gram-positive bacteria were resistant. The folding variant of alpha-lactalbumin is a new example of naturally occurring molecules with antimicrobial activity.

An EF-hand phage display study of calmodulin subdomain pairing.

The interaction between the two EF-hands, EF3 and EF4, in the C-terminal domain of vertebrate calmodulin is addressed using an EF-hand phage display library. Significant specificity is observed in the presence of Ca(2+), as EF3-EF4 heterodimers are favored over EF3-EF3 and EF4-EF4 homodimers. Primarily EF4-type (and not EF3-type) amino acids are selected when an EF3 peptide is used as the target and vice versa. The results show that this specificity is promoted by several factors. There are three positions, corresponding to Phe89, Ala102, and Leu105, that are strongly selected as EF3-type hydrophobic residues with an EF4 target. When EF3 is the target peptide, EF4-type residues, Ile125, Tyr138 and Phe141, are selected. Remarkably, this subset consists of the same three residue positions in EF3 or EF4 and seems to be involved in specifying the heterodimer preference in both cases. In addition, electrostatic repulsion between the acidic monomers in an EF4 homodimer may further influence the preferred stability of heterodimers. This hypothesis is based on the observation that positively charged residues are strongly selected at four positions when EF4 is the target. A survey of EF-hand pairs suggests that charge separation is a common way to achieve efficient attraction of Ca(2+) without causing electrostatic repulsion between the subdomains. No significant specificity of binding is observed in the ion free state or in the presence of magnesium as no sequence is preferentially selected. The residues at the interface between the two EF-hands are thus highly optimized for the Ca(2+) bound state. At some residue positions, EF3-type amino acids are chosen with EF3-target in the presence of Ca(2+). These residues are not involved in the preference for heterodimer over homodimer formation, but represent key positions to mutate in the intact domain to stabilize its Ca(2+)-bound state.

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.

Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.

Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.

Battle for the EF-hands: magnesium-calcium interference in calmodulin.

The ubiquitous Ca(2+)-regulatory protein calmodulin activates target enzymes as a response to submicromolar Ca(2+) increases in a background of millimolar Mg(2+). The potential influence of Mg(2+)/Ca(2+) competition is especially intriguing for the N-terminal domain of the protein which possesses the sites with the lowest Ca(2+) specificity. The interdependence of Ca(2+) and Mg(2+) binding in the N-terminal domain of calmodulin was therefore studied using (43)Ca NMR, (1)H-(15)N NMR, and fluorescent Ca(2+) chelator techniques. The apparent affinity for Ca(2+) was found to be significantly decreased at physiological Mg(2+) levels. At Ca(2+) concentrations of an activated cell the (Ca(2+))(2) state of the N-terminal domain is therefore only weakly populated, indicating that for this domain Ca(2+) binding is intimately associated with binding of target molecules. The data are in good agreement with a two-site model in which each site can bind either Ca(2+) or Mg(2+). The Mg(2+)-Ca(2+) binding interaction is slightly positively allosteric, resulting in a significantly populated (Mg(2+))(1)(Ca(2+))(1) state. The Ca(2+) off-rate from this state is determined to be at least one order of magnitude faster than from the (Ca(2+))(2) state. These two findings indicate that the (Mg(2+))(1)(Ca(2+))(1) state is structurally and/or dynamically different from the (Ca(2+))(2) state. The (43)Ca quadrupolar coupling constant and the (1)H and (15)N chemical shifts of the (Mg(2+))(1)(Ca(2+))(1) state were calculated from titration data. The values of both parameters suggest that the (Mg(2+))(1)(Ca(2+))(1) state has a conformation more similar to the "closed" apo and (Mg(2+))(2) states than to the "open" (Ca(2+))(2) state.