A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase.

The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy. In this complex, the CaMKK peptide forms a fold comprising an alpha-helix and a hairpin-like loop whose C-terminus folds back on itself. The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM-target complexes determined so far. The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively. This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes. The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins.     

The Ca2+-binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes.

To gain insight into the molecular mechanisms underlying cutaneous wound repair, we performed a large scale screen to identify novel injury-regulated genes. Here we show a strong up-regulation of the RNA and protein levels of the two Ca(2+)-binding proteins S100A8 and S100A9 in the hyperthickened epidermis of acute murine and human wounds and of human ulcers. Furthermore, both genes were expressed by inflammatory cells in the wound. The increased expression of S100A8 and S100A9 in wound keratinocytes is most likely related to the activated state of the keratinocytes and not secondary to the inflammation of the skin, since we also found up-regulation of S100A8 and S100A9 in the epidermis of activin-overexpressing mice, which develop a hyperproliferative and abnormally differentiated epidermis in the absence of inflammation. Furthermore, S100A8 and S100A9 expression was found to be associated with partially differentiated keratinocytes in vitro. Using confocal microscopy, both proteins were shown to be at least partially associated with the keratin cytoskeleton. In addition, cultured keratinocytes efficiently secreted the S100A8/A9 dimer. These results together with previously published data suggest that S100A8 and S100A9 are novel players in wound repair, where they might be involved in the reorganization of the keratin cytoskeleton in the wounded epidermis, in the chemoattraction of inflammatory cells, and/or in the defense against microorganisms.     

Crystal structure of Ca2+ -free S100A2 at 1.6-A resolution

S100A2 is an EF hand-containing Ca^2 +-binding protein of the family of S100 proteins. The protein is localized exclusively in the nucleus and is involved in cell cycle regulation. It attracted most interest by its function as a tumor suppressor via p53 interaction. We determined the crystal structure of homodimeric S100A2 in the Ca^2 +-free state at 1.6-Å resolution. The structure revealed structural differences between subunits A and B, especially in the conformation of a loop that connects the N- and C-terminal EF hands and represents a part of the target-binding site in S100 proteins. Analysis of the hydrogen bonding network and molecular dynamics calculations indicate that one of the two observed conformations is more stable. The structure revealed Na⁺ bound to each N-terminal EF hand of both subunits coordinated by oxygen atoms of the backbone carbonyl and water molecules. Comparison with the structures of Ca^2 +-free S100A3 and S100A6 suggests that Na⁺ might occupy the S100-specific EF hand in the Ca^2 +-free state.     

The crystal structure at 2A resolution of the Ca2+ -binding protein S100P

S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins. Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro. The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction. A flexible linker connects the two EF-hand motifs. The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P. Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium. The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively. Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system. The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.     

Identification of regions responsible for the open conformation of S100A10 using chimaeric S100A11-S100A10 proteins

S100A11 is a dimeric EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2) and facilitate membrane vesiculation events. In contrast with other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an 'open' conformation that is very similar to S100A11 in its calcium-bound state. To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimaeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimaeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'disulfonic acid, dipotassium salt) fluorescence and phenyl-Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimaeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target binding site. In contrast, S100A11 chimaeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and, consequently, unobservable annexin A2 binding. The present study represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10.     

The three-dimensional solution structure of Ca(2+)-bound S100A1 as determined by NMR spectroscopy

S100A1 is an EF-hand-containing Ca(2+)-binding protein that undergoes a conformational change upon binding calcium as is necessary to interact with protein targets and initiate a biological response. To better understand how calcium influences the structure and function of S100A1, the three-dimensional structure of calcium-bound S100A1 was determined by multidimensional NMR spectroscopy and compared to the previously determined structure of apo. In total, 3354 nuclear Overhauser effect-derived distance constraints, 240 dihedral constraints, 160 hydrogen bond constraints, and 362 residual dipolar coupling restraints derived from a series of two-dimensional, three-dimensional, and four-dimensional NMR experiments were used in its structure determination (>21 constraints per residue). As with other dimeric S100 proteins, S100A1 is a symmetric homodimer with helices 1, 1', 4, and 4' associating into an X-type four-helix bundle at the dimer interface. Within each subunit there are four alpha-helices and a short antiparallel beta-sheet typical of two helix-loop-helix EF-hand calcium-binding domains. The addition of calcium did not change the interhelical angle of helices 1 and 2 in the pseudo EF-hand significantly; however, there was a large reorientation of helix 3 in the typical EF-hand. The large conformational change exposes a hydrophobic cleft, defined by residues in the hinge region, the C terminus, and regions of helix 3, which are important for the interaction between S100A1 and a peptide (TRTK-12) derived from the actin-capping protein CapZ.     

The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.     

Solution structure and dynamics of S100A5 in the apo and Ca2+-bound states

S100A5 is a calcium binding protein of the S100 family, with one canonical and one S100-specific EF-hand motif per subunit. Although its function is still unknown, it has recently been reported to be one of the S100 proteins able to interact with the receptor for advanced glycation end products. The homodimeric solution structures of S100A5 in both the apo and the calcium(II)-loaded forms have been obtained, and show a conformational rearrangement upon calcium binding. This rearrangement involves, in particular, the hinge loop connecting the N-terminal and the C-terminal EF-hand domains, the reorientation of helix III with respect to helix IV, as common to several S100 proteins, and the elongation of helix IV. The details of the structural changes are important because they must be related to the different functions, still largely unknown, of the different members of the S100 family. For the first time for a full-length S100 protein, relaxation measurements were performed on both the apo and the calcium-bound forms. A quite large mobility was observed in the hinge loop, which is not quenched in the calcium form. The structural differences resulting upon calcium binding change the global shape and the distribution of hydrophobic and charged residues of the S100A5 homodimer in a modest but significantly different manner with respect to the closest homologues S100A4 and S100A6.     

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

Resonance assignments of Ca2+-bound human S100A11

The S100 family belongs to the EF-hand calcium-binding proteins regulating a wide range of important cellular processes via protein–protein interactions. Most S100 proteins adopt a conformation of non-covalent homodimer for their functions. Calcium binding to the EF-hand motifs of S100 proteins is essential for triggering the structural changes, promoting exposure of hydrophobic regions necessary for target protein interactions. S100A11 is a protein found in diverse tissues and possesses multiple functions upon binding to different target proteins. RAGE is a multiligand receptor binding to S100A11 and the interactions at molecular level have not been reported. However, the three-dimensional structure of human S100A11 containing 105 amino acids is still not available for further interaction studies. To determine the solution structure, for the first time we report the ¹H, ¹⁵N and ¹³C resonance assignments and protein secondary structure prediction of human S100A11 dimer in complex with calcium using a variety of triple resonance NMR experiments and the chemical shift index (CSI) method, respectively.     

Zinc-binding site of an S100 protein revealed. Two crystal structures of Ca2+-bound human psoriasin (S100A7) in the Zn2+-loaded and Zn2+-free states

The crystal structure of human psoriasin (S100A7) in the native, calcium-bound form has been determined from two crystal forms of the protein crystallized with and without divalent zinc. The overall structures of the dimeric protein closely resemble the previously determined holmium-substituted structure. The structures also reveal a zinc-binding site of the protein, which is formed by three histidines and an aspartate residue. Together, these residues coordinate the zinc ion in a way similar to the pattern seen in certain metalloproteases and in particular the collagenase family of proteins. Sequence comparison suggests that this zinc site is present in a number of the remaining members of the S100 family. The structure of S100A7 crystallized in the absence of zinc further shows that loss of zinc results in a reorganization of the adjacent empty and distorted EF-hand loop, causing it to resemble a calcium-loaded EF-hand.     

Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100 target specificity and activation of the kinase

NDR, a nuclear serine/threonine kinase, belongs to the subfamily of Dbf2 kinases that is critical to the morphology and proliferation of cells. The activity of NDR kinase is modulated in a Ca(2+)/S100B-dependent manner by phosphorylation of Ser281 in the catalytic domain and Thr444 in the C-terminal regulatory domain. S100B, which is a member of the S100 subfamily of EF-hand proteins, binds to a basic/hydrophobic sequence at the junction of the N-terminal regulatory and catalytic domains (NDR(62-87)). Unlike calmodulin-dependent kinases, regulation of NDR by S100B is not associated with direct autoinhibition of the active site, but rather involves a conformational change in the catalytic domain triggered by Ca(2+)/S100B binding to the junction region. To gain further insight into the mechanism of activation of the kinase, studies have been carried out on Ca(2+)/S100B in complex with the intact N-terminal regulatory domain, NDR(1-87). Multidimensional heteronuclear NMR analysis showed that the binding mode and stoichiometry of a peptide fragment of NDR (NDR(62-87)) is the same as for the intact N-terminal regulatory domain. The solution structure of Ca(2+)/S100B and NDR(62-87) has been determined. One target molecule is found to associate with each subunit of the S100B dimer. The peptide adopts three turns of helix in the bound state, and the complex is stabilized by both hydrophobic and electrostatic interactions. These structural studies, in combination with available biochemical data, have been used to develop a model for calcium-induced activation of NDR kinase by S100B.     

The Zn2+ and Ca2+‐binding S100B and S100A1 proteins: beyond the myths

The S100 genes encode a conserved group of 21 vertebrate‐specific EF‐hand calcium‐binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium‐ and zinc‐binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn²⁺. The Ca²⁺ and Zn²⁺‐dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate‐specific proteins and propose a new model for stable interactions of S100B dimers with full‐length target proteins. A chaperone‐associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.     

Effect of Ca2+ on conformation of actin in the F-actin--heavy meromyosin complex

The polarized fluorescence of intrinsic tryptophan residues and the birefringence of ghost muscle fibres of rabbit were measured during thin filaments binding to heavy meromyosin containing 5,5'-dithiobis [2-nitrobenzoic acid] light chains and to those devoid of them with a view of investigating conformational changes in F-actin. Ca2+ binding to heavy meromyosin containing 5,5'-dithiobis [2-nitrobenzoic acid] light chains was shown to affect the character of these changes during the formation of the F-actin - heavy meromyosin complex.     

Ca2+-Binding S100 Proteins in the Central Nervous System

A number of neurodegenerative disorders have been attributed to abberrations of intracellular Ca²⁺ homeostasis regulated by Ca²⁺-binding proteins. This chapter will focus on the S100B and S100A6 proteins, which are highly expressed in the central nervous system. Their protein structures, localizations, and association with brain pathology as well as their potential use in clinical diagnostics will be discussed.     

Ca(2+)-dependent inhibition of actin-activated myosin ATPase activity by S100C (S100A11), a novel member of the S100 protein family

S100C (S100A11, calgizzarin) inhibits the actin-activated myosin Mg(2+)-ATPase activity of smooth muscle in a dose-dependent manner: its half-maximal effect occurs at a S100C/actin molar ratio of 0.05 and its maximal effect occurs at a ratio of 0.20. Furthermore, S100C was found to bind to actin with a stoichiometry of 1:6-7 in the presence of Ca(2+), with an affinity of 1 x 10(-6) M determined by cosedimentation assays. Other Ca(2+)-binding proteins such as S100A1, S100A2, S100B, and calmodulin did not inhibit actin-activated myosin Mg(2+)-ATPase activity. Calmodulin, S100A1, and S100B reversed the inhibitory effect of calponin in a Ca(2+)-dependent manner, S100A2 had no effect, and S100C had additional inhibitory effects. The results suggest that S100C might be involved in the regulation of actin-activated myosin Mg(2+)-ATPase activity through its Ca(2+)-dependent interaction with actin filaments.     

The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.