Mapping of three unique Ca(2+)-binding sites in human annexin II.

Site-directed mutagenesis was employed to map and characterize Ca(2+)-binding sites in annexin II, a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14,732-14,739] that a Ca(2+)-binding site is present in the third of the four repeat segments which comprise the 33-kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca(2+)-binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., R?misch, J. and Paques, E.-P. (1990) FEBS Lett. 275, 15-21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C-terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high-affinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca(2+)-binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2+ site(s) of presumably different architecture.     

Annexins--a new family of Ca(2+)-binding proteins

Annexins, the Ca(2+)- and phospholipid-binding proteins, are able to induce Ca(2+)-dependent aggregation of biomembranes. All the representatives of this family contain four or eight tandem repeats, 60-80 amino acids each. All these repeats include a highly conservative 17-member amino acid consensus sequence (an endonexin fold). The central domain comprises all these repeats and contains, in addition, the site(s) with a binding affinity for Ca2+ and phospholipids. Annexins are devoid of the classical "EF-hand" Ca(2+)-binding domain and can therefore be assigned to a new family of Ca(2+)-binding proteins.     

Calcium-activated endonexin II forms calcium channels across acidic phospholipid bilayer membranes

Human endonexin II (annexin V) and recombinant human endonexin II can be activated by Ca2+ to interact with acidic phospholipid bilayers formed at the tip of a patch pipette. Once associated with the bilayer, endonexin II forms voltage-gated channels which are selective for divalent cations according to the following series Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+. However, endonexin II also expresses a selective affinity for Ca2+ which is manifest by an observed reduced current through the open channel when Ca2+ is the charge carrier. La3+ blocks endonexin II channels, as it does synexin (annexin VII) and other types of Ca2+ channels. However, as with synexin, the dihydropyridine Ca2+ channel antagonist nifedipine does not affect endonexin II channel activity. Endonexin II channels are also permeant to Li+, Cs+, Na+, and to a lesser extent, K+, resembling in this manner Ca2+ release channels from sarcoplasmic reticulum. Indeed, the low affinity of endonexin II channels for such ions as Cs+ or Li+ have allowed us to use these cations for measurement of the kinetic properties of the channel, with minimal concerns for the ion/channel interactions observed with the physiological substrate, Ca+. Finally, we observed that endonexin II channel activity always occurred in bursts, making necessary the use of two exponential functions to fit open- and closed-time histograms. We conclude from these data that the domain responsible for endonexin II channel activity, first observed by ourselves in the homologue synexin, is probably the C-terminal tetrad repeat common to both molecules.     

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.     

The C terminus of annexin II mediates binding to F-actin

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains the binding sites for anionic phospholipids, heparin, and F-actin, whereas the p11 subunit provides a regulatory function. The F-actin-binding site is presently unknown. In the present study we have utilized site-directed mutagenesis to create annexin II mutants with truncations in the C terminus of the molecule. Interestingly, a mutant annexin II lacking its C-terminal 16, 13, or 9 amino acids was unable to bind to F-actin but still retained its ability to interact with both anionic phospholipids and heparin. Recombinant AIIt, composed of wild-type p11 subunits and the mutant annexin II subunits, was also unable to bundle F-actin. This loss of F-actin bundling activity was directly attributable to the inability of mutant AIIt to bind F-actin. These results establish for the first time that the annexin II C-terminal amino acid residues, LLYLCGGDD, participate in F-actin binding.     

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.     

Predicted structure for the calcium-dependent membrane-binding proteins p35, p36, and p32

A new family of proteins (annexins) that bind to membranes at micromolar free Ca2+ has been recognized. Its members include an EGF-receptor kinase substrate (p35), a retroviral tyrosine kinase substrate (p36), the liver protein endonexin (p32) and an electric ray protein, calelectrin. Each protein contains four sequence repeats with a further 2-fold internal homology. Using the predicted secondary structure and pattern of conserved hydrophobic residues in each repeat, we have built a three-dimensional model that is largely isostructural with the known molecular conformation of bovine intestinal calcium-binding protein. The final (energy-refined) model had a core formed from the conserved hydrophobic residues. It differed from ICaBP principally in the length of the two Ca2+-binding loops with only one loop being able to bind. The model suggests a mechanism for interaction of these new Ca2+-binding proteins with phospholipid bilayers.     

Annexin XXI (ANX21) of Giardia lamblia has sequence motifs uniquely sdhared by giardial annexins and is specifically localized in the flagella

We have identified a novel annexin, ANX21, in trophozoites of Giardia lamblia. The nucleotide sequence encoding this protein deviated from a published sequence in predicting an additional endonexin fold in the fourth annexin domain. In addition, several motifs exclusively shared by other annexins of G. lamblia in their predicted fourth repeat and predicted to be localized on the opposite (concave) surface of the molecule became apparent. Western blots of trophozoite fractions probed with antiserum against the recombinant protein indicated that this annexin, like the other giardial annexins ANX19 and ANX20, associates with phospholipids in the presence of Ca(2+). Finally, confocal laser scanning of trophozoites showed that the protein, apart from the median body, was exclusively localized in the eight flagella. Together, these data suggest that ANX21 may function as a Ca(2+)-regulated structural element linking phospholipid bilayer and underlying axoneme.     

The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1

ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca(2+)-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca(2+) with FLAG-tagged Alix or FLAG-tagged Alix Delta EBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged Alix Delta ABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca(2+)-dependent fashion, whereas FLAG-tagged ALG-2(E47A/E114A), a Ca(2+)-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca(2+). Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCT Delta ABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca(2+)-dependent interaction with Alix.     

Characterization of the Ca2+-binding sites of annexin II tetramer

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains three type II and two type III Ca(2+)-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin. In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca(2+)-binding sites (TCM AIIt). Surprisingly, we found that in the presence of Ca(2+), the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca(2+)-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca(2+)-dependent binding to F-actin, the binding of the TCM AIIt was Ca(2+)-independent. These results suggest that the type II or type III Ca(2+)-binding sites do not directly participate in anionic phospholipid or F-actin binding. We therefore propose that in the absence of Ca(2+), the type II and type III Ca(2+)-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin. Ca(2+) binding to these sites, or the inactivation of these Ca(2+)-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca(2+) for binding to heparin, we also propose that novel Ca(2+)-binding sites regulate this binding event.     

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.     

Structural basis for Ca(2+)-induced activation of human PAD4

Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Its gene is a susceptibility locus for rheumatoid arthritis. Here we present the crystal structure of Ca(2+)-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an alpha/beta propeller structure. Five Ca(2+)-binding sites, none of which adopt an EF-hand motif, were identified in the structure of a Ca(2+)-bound inactive mutant with and without bound substrate. These structural data indicate that Ca(2+) binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca(2+) ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of rheumatoid arthritis.     

A polymorphism in thrombospondin-1 associated with familial premature coronary heart disease causes a local change in conformation of the Ca2+-binding repeats

A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca2+-binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca2+-binding repeats alone (Ca) or with the third epidermal growth factor-like module (E3Ca), without (Asn-700) or with (Ser-700) the disease-associated polymorphism. The intrinsic fluorescence of a single tryptophan (Trp-698) adjacent to the polymorphic residue was quenched cooperatively by adding Ca2+. The third epidermal growth factor-like repeat dramatically altered the Ca2+-dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC50) of Ca Asn-700 was 390 microM, and the EC50 of E3Ca Asn-700 was 70 microM. The Ser-700 polymorphism shifted the EC50 to higher Ca2+ concentrations (Ca Ser-700 EC50 of 950 microM and E3Ca Ser-700 EC50 of 110 microM). This destabilizing effect is due to local conformational changes, as the Ser-700 polymorphism did not influence the secondary structure of E3Ca or Ca as assessed by far UV circular dichroism. At 200 microM Ca2+, in which both E3Ca Asn-700 and Ser-700 are in the Ca2+-replete conformation at 37 degrees C, the fluorescence of E3Ca Ser-700 reverted to the Ca2+-depleted spectrum at 50 degrees C compared with 65 degrees C for E3Ca Asn-700. These findings indicate that the Ser-700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca2+ and thermal denaturation.     

Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

A series of point mutations reveal interactions between the calcium-binding sites of calmodulin.

Calmodulin is a member of the "EF-hand" family of Ca(2+)-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca(2+)-binding sites. To examine the contribution of individual Ca(2+)-binding sites to the Ca(2+)-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca(2+)-binding loops has been changed to a glutamine. One-dimensional 1H-NMR has been used to monitor Ca(2+)-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca(2+)-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca(2+)-binding loops greatly decreases the Ca(2+)-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca(2+)-binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca(2+)-binding affinities of the partner C-terminal site. In both the N- and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site III or site IV are strikingly different.     

Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association

A series of N-terminal calmodulin (CaM) mutants was generated to probe the relationship between the N-terminal Ca(2+) affinity and the number of paired, negatively charged Ca(2+) chelating residues in the N-terminal Ca(2+)-binding sites of CaM. When the number of acid pairs [negatively charged residues at positions +x and -x (X-axis), +y and -y (Y-axis), and +z and -z (Z-axis)] was increased from zero to one and then to two, a progressive increase was seen in the N-terminal Ca(2+) affinities. The maximal ranges of the increases observed in the N-terminal Ca(2+) affinity were approximately 8-8.5-fold for site I, approximately 4.5-5-fold for site II, and approximately 11-fold for both sites, in comparison to the mutants containing no acid pairs. The maximal values of N-terminal Ca(2+) affinity were bestowed by the presence of five acidic chelating residues in site I or II, individually. Addition of the sixth acidic chelating residue (third acid pair) to both N-terminal Ca(2+)-binding sites reduced the N-terminal Ca(2+) affinity. The increases in Ca(2+) affinity observed were caused by an increase in the Ca(2+) association rates for the Y- and Z-axis acid pairs, while the X-axis acid pair caused a reduction in the Ca(2+) dissociation rates.     

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.     

Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).     

Calcium binding to the photosystem II subunit CP29

We have identified a Ca(2+)-binding site of the 29-kDa chlorophyll a/b-binding protein CP29, a light harvesting protein of photosystem II most likely involved in photoregulation. (45)Ca(2+) binding studies and dot blot analyses of CP29 demonstrate that CP29 is a Ca(2+)-binding protein. The primary sequence of CP29 does not exhibit an obvious Ca(2+)-binding site therefore we have used Yb(3+) replacement to analyze this site. Near-infrared Yb(3+) vibronic side band fluorescence spectroscopy (Roselli, C., Boussac, A., and Mattioli, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12897-12901) of Yb(3+)-reconstituted CP29 indicated a single population of Yb(3+)-binding sites rich in carboxylic acids, characteristic of Ca(2+)-binding sites. A structural model of CP29 presents two purported extra-membranar loops which are relatively rich in carboxylic acids, one on the stromae side and one on the lumenal side. The loop on the lumenal side is adjacent to glutamic acid 166 in helix C of CP29, which is known to be the binding site for dicyclohexylcarbodiimide (Pesaresi, P., Sandonà, D., Giuffra, E. , and Bassi, R. (1997) FEBS Lett. 402, 151-156). Dicyclohexylcarbodiimide binding prevented Ca(2+) binding, therefore we propose that the Ca(2+) in CP29 is bound in the domain including the lumenal loop between helices B and C.     

The role of Ca2+-coordinating residues of herring antifreeze protein in antifreeze activity

The type II antifreeze protein of Atlantic herring (Clupea harengus harengus) requires Ca(2+) as a cofactor to inhibit the growth of ice crystals. On the basis of homology modeling with Ca(2+)-dependent lectin domains, five residues of herring antifreeze protein (hAFP) are predicted to be involved in Ca(2+) binding: Q92, D94, E99, N113, and D114. The role of E99, however, is less certain. A previous study on a double mutant EPN of hAFP suggested that the Ca(2+)-binding site of hAFP was the ice-binding site. However, it is possible that Ca(2+) might function distantly to affect ice binding. Site-directed mutagenesis was performed on the Ca(2+)-coordinating residues of hAFP in order to define the location of the ice-binding site and to explore the role of these residues in antifreeze activity. Properties of the mutants were investigated in terms of their structural integrity and antifreeze activity. Equilibrium dialysis analysis demonstrated that E99 is a Ca(2+)-coordinating residue. Moreover, proteolysis protection assay revealed that removal of Ca(2+) affected the conformation of the Ca(2+)-binding loop rather than the core structure of hAFP. This finding rules out the possibility that Ca(2+) might act at a distance via a conformational change to affect the function of hAFP. Substitutions at positions 99 and 114 resulted in severely reduced thermal hysteresis activity. These data indicate that the ice-binding site of hAFP is located at the Ca(2+)-binding site and the loop region defined by residues 99 and 114 is important for antifreeze activity.