Calcium-binding Proteins

Beat Schwaller, University of Fribourg, Fribourg, Switzerland

Calcium ions act as important second messengers for many intracellular processes and the information contained in this calcium signal is modulated by specific calcium-binding proteins. According to well-conserved structural elements these proteins can be grouped into different families including annexins, C2 domain proteins and EF-hand proteins.

Secondary article
Article Contents
. Physiological Calcium Concentrations and the Calcium Ion as Ubiquitous Cytosolic Second Messenger . Overview of Calcium-binding Proteins . Families of Calcium-binding Proteins . Transport of Calcium

Physiological Calcium Concentrations and the Calcium Ion as Ubiquitous Cytosolic Second Messenger
The extracellular concentration of Ca2 1 ions is approximately 2.5 mmol L 2 1 and is tightly regulated by the two hormones calcitonin and parathyroid hormone (PTH) in an antagonistic way. Calcitonin produced in the thyroid gland lowers the Ca2 1 concentration by favouring formation of hydroxyapatite, while PTH stimulates the activity of osteoclasts resulting in an increased concentration of Ca2 1 . Secretion of PTH is regulated by levels of extracellular Ca2 1 sensed by calcium receptor protein (CaSR, a G protein-coupled receptor) which is expressed on the surface of parathyroid cells (Brown et al., 1993). A similar yet unidentied receptor on osteoblasts is suggested to transmit the extracellular Ca2 1 signal to stimulate osteoblastic proliferation resulting in increased bone formation. In addition, 1,25 dihydroxyvitamin D3 aects the serum Ca2 1 concentration by increasing the resorption of Ca2 1 from the intestine and the kidney. The intracellular concentration of Ca2 1 in a resting cell is approximately four orders of magnitude lower (50 100 nmol L 2 1) than in the serum and its regulation is extremely sophisticated. All cells have in their plasma membranes extrusion systems to remove intracellular Ca2 1 , the most important being the Ca2 1 -ATPase and the Na 1 /Ca2 1 exchanger which either use ATP or the sodium gradient to drive Ca2 1 out of the cell, respectively. On the other hand, inux of Ca2 1 via opening of Ca2 1 channels in the plasma membrane or from activation of intracellular Ca2 1 -release channels such as inositol-1,4,5trisphosphate (IP3) and ryanodine receptors of intracellular stores (e.g. endoplasmic reticulum) leads to an abrupt increase in [Ca2 1 ]i which is used in the cell as a cytosolic second messenger. The information contained in this signal is modulated by specic Ca2 1 -binding proteins (CaBP). The kinetics and geometry of Ca2 1 inux or release from internal stores, the characteristics of soluble Ca2 1 -binding proteins and the eciency of Ca2 1 extrusion and/or reuptake into organelles determine the amplitude and shape of the Ca2 1 transients. This in turn determines which systems are activated by the second messenger Ca2 1 .

Overview of Calcium-binding Proteins

One of the advantages of Ca2 1 as a second messenger is its ability to tightly bind to proteins. Oxygen ligands, as found in carboxylic side-chain groups of aspartate or glutamate or carbonyl oxygen atoms of the main-chain (or side-chain oxygen from the carbonyl group from asparagine and glutamine), bind well to Ca2 1 ions. A special ligand is gamma-carboxyglutamate (g-Gla, Figure 5) with two carboxylic side-chains present in some specic serum proteins (see below). Ca2 1 in a complex with oxygen atoms has a coordination of 68 ligands. This allows the formation of bonds with oxygen groups from dierent amino acids of a protein, thus generating a particular threedimensional complex structure. The Ca2 1 -induced conformational change of a protein can lead to the activation or inhibition of enzymatic activities or the binding to specic ligands either in the presence or absence of bound Ca2 1 . According to their location (extracellular, cytosolic, inside of organelles), proteins capable of binding Ca2 1 ions face a variety of completely dierent environments. For example, the free Ca2 1 concentration is high in the extracellular space and in organelles, but extremely low in the cytosol, and the pH and ionic composition of the milieu vary. Ca2 1 -binding proteins therefore have anities for Ca2 1 that are nely tuned to exert their precise biological functions. Lately it has become clear that not only anities for Ca2 1 (thermodynamic properties), but also the kinetics of binding and releasing of Ca2 1 ions are important parameters and it is necessary to understand these before we can explain how a Ca2 1 signal can control events of such extremely dierent time scales, such as neurotransmitter release (10 2 5 s), muscle contraction (10 2 3 s), metabolic reactions (10 2 1 s) and cell cyclerelated reactions (up to hours). To date the metal-binding properties of only a minority of the known CaBPs have been explored, and even fewer kinetic studies have been performed. There is no one way in which proteins specically bind Ca2 1 , but based on sequence and structure homology, Ca2 1 -binding proteins can be grouped into dierent families. The ones discussed in detail in this chapter
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include the annexins, the C2 domain proteins, the EF-hand family of CaBPs, the pentraxins and the vitamin Kdependent proteins. Furthermore, some low-anity, highcapacity CaBPs present in intracellular organelles and serum will be described.

Families of Calcium-binding Proteins

Annexins
The annexins are a family of widely distributed Ca2 1 - and phospholipid-binding proteins, all of which possess a conserved repeated domain called the annexin fold (Burgoyne and Geisow, 1989). Family members contain four domains of 7075 amino acids making up the protein core, with the exception of annexin VI in which eight such domains are present (Figure 1). The core domain contains the binding sites for Ca2 1 and negatively charged phospholipids (phosphatidylserine). Each annexin fold consists of ve a-helical stretches joined by small linker regions. Most of the initial structural information of annexins was obtained from the determination of the crystal structure of annexin V (Huber et al., 1990). Most recent data on the Ca2 1 -binding stochiometry suggest that annexin V binds 10 Ca2 1 ions as determined from the Xray structure of this protein co-crystallized with phospholipid head groups (Swairjo et al., 1995). Dierent annexins are distinguished from one another by N-terminal domains of variable length that may confer specic functions. To date 10 mammalian annexins have been fully sequenced (I, II, III, IV, V, VI, VII, VIII, XI and XIII). Of over 20 members known, at least one can be detected in nearly every eukaryotic cell. The Ca2 1 -binding anities of the annexins are greatly increased in the presence of phospholipids and vary widely between family members.

Annexins were initially considered to be intracellular cytosolic proteins involved in intracellular vesicular trac, exocytosis and endosomeendosome fusion. The high anity of annexin II for Ca2 1 (in the presence of phospholipids) made it an interesting candidate for involvement in exocytosis, but annexins I and VII also have strong vesicle-aggregating activities. The ability of annexin II to associate with cytoskeletal proteins has suggested that annexins may regulate cytoskeletal interactions with membranes. Several annexins undergo transient translocation to membranes (plasma membrane, secretory granules) when cells are activated and [Ca2 1 ]i is elevated. Puried annexins (e.g. V and VII) can form pores in synthetic bilayers. Most annexins (with the exception of annexin V) are also substrates for protein kinases C (PKC), family members of the C2 family of Ca2 1 -binding proteins. The activity of cytosolic PKCs and of phospholipase A2 seems to be modulated by annexin V, suggesting that this protein could play a role in Ca2 1 and phospholipid signalling pathways. Although annexins have no apparent signal sequences for secretion and the mechanism by which annexins are transported out of cells is as yet unknown, some annexin members (I and II) have been detected in the serum and on the external surface of plasma membranes. This is indicative of specic extracellular functions. The annexin II heterotetramer, consisting of two molecules of annexin II and two molecules of S100A10 (a member of the EFhand family), binds tissue-type plasminogen activator (tPA), plasminogen and plasmin. It accelerates the activation of the clot-dissolving protease plasmin by complexing with plasminogen and tPA and the formation of this complex suggests that the annexin II heterotetramer is the key physiological receptor for plasminogen on the extracellular surface of endothelial cells. Both up- and downregulation of annexins has been observed under pathological conditions. These proteins can have a suppressive action on some tumours while

Figure 1 Structure of the annexins. (a) Annexins consist of an N-terminal domain of variable length and a protein core composed of four annexin folds which contain the binding sites for Ca2 1 and phospholipids. Boxes (a e) represent a helices as observed in the crystal structure of annexin V. Annexin VI is made up from eight annexin folds. Shown here for annexin II, in the N-terminal region, phosphorylation sites and binding sites for targets (S100A10) are present. (b) Structure of annexin V, which in the presence of phospholipids binds 10 molecules of Ca2 1 . Modified from Huber et al. (1990) and Swairjo et al. (1995). (Brookhaven Data Base, PDB entry code: 1A8A.)

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favouring tumour progression in others. The dierent eects may be linked to the transient expression patterns during development and dierentiation and are similar to the pro- or anti-tumorigenic eects of proteins belonging to the EF-hand family of CaBPs.

C2 domain calcium-binding proteins

The C2 domain is a Ca2 1 -binding motif of approximately 130 amino acid residues in length, which forms a compact eight-stranded antiparallel b sandwich consisting of two four-stranded b sheets (Sutton et al., 1995). Almost 100 C2 domain sequences are known and can be grouped into two dierent classes (topologies) which slightly dier in their connectivity of the eight b strands (Figure 2). Topology I is present in the a, b and g isoforms of Ca2 1 -dependent protein kinase C (PKC), where the C2 domain was originally identied as the second of four conserved domains (C1C4) responsible for the Ca2 1 -dependent regulation of PKC. Ca2 1 -regulated proteins with topology II include phospholipase C (d1) or cytosolic phospholipase A2 (cPLA2). The majority of C2 domain proteins contain one such domain, but several proteins involved in vesicular transport such as synaptotagmins (integral membrane proteins acting as a Ca2 1 sensors on the surface of synaptic vesicles), DOC2 or rabphilin-3A contain two C2 domains. Proteins participating in signal transduction include PLCs, cPLA2 or phosphatidylinositol 3-kinases which are involved in the generation of lipid second messengers. Thus, most of the C2 domain proteins can be characterized as signalling molecules interacting with intracellular membranes. In this respect, they share the ability of the annexins to form complexes with phospholipids (phosphatidylserine) and the C2 domain has also been referred as a Ca2 1 -dependent lipid-binding domain. The anity of synaptotagmin for Ca2 1 is rather low (approx. 0.2 mmol L 2 1), but is signicantly increased in the presence of phospholipids (5 mmol L 2 1). In addition to binding their respective ligands (e.g. phospholipids), some members also bind inositol polyphosphates or other target proteins. It is important to note that not all C2 domains are able to bind Ca2 1 or phospholipids and it is assumed that as the result of divergent evolution, these properties were lost in some C2 domain proteins and thus, interaction with specic proteins is Ca2 1 independent. The Ca2 1 -binding sites of C2 domains have been identied from X-ray diraction patterns and nuclear magnetic resonance (NMR) studies. In all C2 domains, several Ca2 1 ions bind at the top loops connecting the b strands. In the case of the C2A domain of synaptotagmin or the C2 domain of PKCb, three Ca2 1 ions are bound in a similar fashion (Figure 2). Ca2 1 is coordinated mainly by aspartate side-chains that serve as bidentate ligands for two or three Ca2 1 ions. Additionally, serine or threonine

Figure 2 Typical structure of a C2-domain protein. (a) Ribbon diagram showing the topology of the eight strands of the C2A-domain of synaptotagmin I (left) and the PLCd1 (right). Modified from Nalefski and Falke (1996) and Rizo and Sudhof (1998). (Brookhaven Data Base, PDB entry code for PKCb (topology I): 1A25 and for PLCd1 (topology II): 1DJH.) The loop region is depicted with three Ca2 1 ions bound. (b) Schematic representation of the topologies found in synaptotagmin I (topology I) and PLCd1 (topology II). Strand-numbering corresponds to the order as found in the primary sequence. In both cases, eight strands form the domain, but the geometrical arrangement of the strands is different in the two groups.

side-chains and carbonyl oxygen from the backbone are involved in the chelation of the Ca2 1 ions. As opposed to the sensor proteins of the EF-hand family, the C2 domains do not undergo signicant Ca2 1 -dependent conformational changes. The interaction of synaptotagmin with the negatively charged syntaxin is thought to be mediated by the change in electrostatic potential. The chelation of the
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aspartates in the Ca2 1 -binding sites of synaptotagmin then allow the interaction of basic residues surrounding this Ca2 1 -binding site with syntaxin. A similar mechanism has been proposed for the interaction with negatively charged phospholipids, since replacing the same acidic and basic residues in the C2A domain of synaptotagmin inhibited binding of phospholipids. It is proposed that the Ca2 1 -binding sites of C2 domain proteins act as electrostatic switches. This might be important for very fast Ca2 1 -triggered reactions such as exocytosis (e.g. neurotransmitter release) because it does not necessitate large conformational changes of the C2 domain proteins.

EF-hand proteins
The typical canonical EF-hand Ca2 1 -binding site consists of a stretch of 29 amino acids consisting of an a helix (residues 110), a loop around the Ca2 1 ion (residues 10 21), and a second a helix (residues 1929). The second helix is oriented almost perpendicular to the rst one (Kretsinger and Nockolds, 1973). The Ca2 1 ion is coordinated by seven oxygen ligands, derived from side-chains of aspartate and glutamate residues, carbonyl groups of the peptide backbone and a bridging water molecule (Figure 3). The geometrical arrangement of the ligands can be described as a pentagonal bipyramid with the Ca2 1 ion occupying the centre of the pyramid. The name EF-hand stems from the protein structure of parvalbumin, in which six a helices named AF form a total of three EF-hand domains (AB, CD, EF) and the last domain (EF) gave the entire family its name. Although most proteins within this family contain the typical EFhand motif, a variation is present, for example, in the rst Ca2 1 -binding site of the S100 proteins (two EF-hand proteins) where two additional residues occur in the rst Ca2 1 -binding loop. In addition, the Ca2 1 -binding loop contains a b-pleated sheet, which is important for the pairing of two EF-hand domains. This double EF-hand motif appears to be a basic structural feature common to all EF-hand Ca2 1 -binding proteins, and almost all of the family members have an even number of EF-hand domains (2, 4, 6 or 8). In the case of parvalbumin, which consists of three EF-hand domains, only two (CD and EF) are capable of metal binding. This organization into tandem domains seems to be important for correct structural folding during protein synthesis and also strongly aects the functional properties of individual Ca2 1 -binding sites. Based on dierences in selectivity and anity of EFhand proteins for Ca2 1 and Mg2 1 binding, two types of Ca2 1 -binding sites have been characterized: mixed Ca2 1 / Mg2 1 sites and Ca2 1 -specic sites. The mixed Ca2 1 / Mg2 1 sites bind Ca2 1 with high anity and Mg2 1 with moderate anity in a competitive manner (dissociation
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Figure 3 EF-hand motif. (a) The three-dimensional arrangement of the EF-hand motif can be simulated by the right hand, with the index finger representing the E-helix (residues 1 10), the bent middle finger symbolizing the Ca2 1 -binding loop (10 21), and the thumb depicting the F-helix 19 29). The geometrical arrangement of the seven oxygen ligands coordinating the Ca2 1 ion can best be described as a pentagonal bipyramid. Modified from Celio et al. (1996). (b) Crystal structure from EFdomain of parvalbumin. Modified from Kretsinger and Nockolds (1973). (c) Coordination of the Ca2 1 ion in calmodulin with the seven oxygen ligands (five from side-chains, one from a carbonyl group of the backbone and one from a water molecule). (d) Consensus sequence for the canonical EF-hand domain. The symbol n denotes nonpolar side-chains and the positions X, Y, Z, 2 Y, 2 X and 2 Z provide the oxygen ligand for the Ca2 1 binding. At position 2 Y a carbonyl oxygen bonds to the Ca2 1 ion. The 2 X ligand (usually glutamate) binds Ca2 1 with both oxygen atoms from the carboxylate group (Kawasaki et al., 1998). * any amino acid; I, isoleucine.

constants: KCa 5 10 2 710 2 9 mol L 2 1; KMg 5 10 2 310 2 5 mol L 2 1). Under physiological conditions, i.e. at low resting levels of Ca2 1 , they are occupied by Mg2 1 ; when intracellular Ca2 1 concentration is elevated Mg2 1 is replaced by Ca2 1 , a reaction that is often characterized by relatively slow kinetics. In comparison with mixed Ca2 1 / Mg2 1 sites, the anity range spanned for Ca2 1 -specic sites is two orders of magnitude lower with respect to Ca2 1 binding and still further reduced in the case of Mg2 1 (KCa 5 10 2 510 2 7 mol L 2 1; KMg 5 10 2 110 2 2 mol L 2 1). Mg2 1 ions thus have only a minor eect on Ca2 1 binding. In the resting cell, Ca2 1 -specic sites are considered to be in a metal-free state, and hence in readiness to bind Ca2 1 ions directly, with fast kinetics, when intracellular levels of this cation are raised. These sites are believed to be responsible for Ca2 1 -dependent regulatory functions involved in protein activation. Important functions of EF-hand Ca2 1 -binding proteins include the modulation of Ca2 1 signals, direct Ca2 1 dependent enzymatic activities, and the buering of cytosolic Ca2 1 concentration. Members of the rst group

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interact with target proteins in a Ca2 1 -dependent manner, thereby modulating protein activation. For example, the binding of Ca2 1 to calmodulin induces marked conformational changes, which enable it to interact with, and regulate the activity of, a large number of dierent target proteins. In contrast, troponin C, which has a very similar overall structure to calmodulin is involved solely in the Ca2 1 -dependent regulation of skeletal and heart muscle contraction. Proteins within the second group manifest direct Ca2 1 dependent enzymatic activity. Currently, eight subfamilies have been described, including aequorin, Ca2 1 -dependent protein kinase, calcineurin, diacylglycerol-kinase, protein phosphatase, calpain, glycerol phosphate dehydrogenase and calmodulin domain protein kinase. Members of the third group do not interact with other proteins but act as intracellular Ca2 1 buers, thereby modulating the temporal and spatial aspects of Ca2 1 transients. Proteins expressed in specic subpopulation of neurons include calretinin (CR), calbindin D-28k (CB) and parvalbumin. While the former two have ve and four Ca2 1 -specic sites, respectively with presumably fast Ca2 1 -binding kinetics, the latter one with two Ca2 1 / Mg2 1 mixed sites is a slow-onset Ca2 1 buer. This means that CR and CB are fast enough to immediately buer Ca2 1 entering via channels from the outside or released from internal stores and thus to limit the amplitude of Ca2 1 transients. PV on the other hand is too slow to play a role in the rising phase of [Ca2 1 ]i, but helps to shorten transients. The hypothesis on the protective role of these proteins against Ca2 1 overload has been put forward, but several recent ndings in transgenic mice lacking one or more of these proteins do not generally support this function in vivo. Overexpression of Ca2 1 -binding proteins in culture systems can modulate Ca2 1 transients and protect cells from apoptosis due to elevated levels of Ca2 1 . The downregulation of some CaBPs before and during the development of several neurodegenerative diseases has been linked with a failure of these neurons to withstand excitotoxic insults. Therefore, the involvement of these proteins in such a protective mechanism remains to be further investigated. Calmodulin Calmodulin (CaM) is a ubiquitously expressed four EFhand Ca2 1 -binding protein (17 kDa) that appears to be the primary Ca2 1 sensor in eukaryotic cells. It acts as a Ca2 1 signal-transmitting subunit for many proteins in mammalian cells. It consists of two pairs of two EF-hands linked by a relatively exible tether and is dumbbell-shaped in the metal-free form (Figure 4a; Babu et al., 1988). When bound with three or four molecules of Ca2 1 , CaM becomes globular and is involved in the regulation of multiple cellular functions including intermediary metabolism, secretion, motility, signal transduction, cell growth

Figure 4 Structure of calmodulin (CaM). (a) In the Ca2 1 -free form CaM is dumbbell-shaped and the two pairs of EF-hand domains are linked by a flexible tether (Babu et al, 1988). (b) After Ca2 1 binding, CaM becomes more globular and can wrap around target proteins (e.g. myosin lightchain kinase; Ikura et al., 1992). (Brookhaven Data Base, PDB entry codes: 1CLL and 3CLN.)

and division. CaM is essential for the survival of all cells, but expression levels can vary considerably. In vertebrates, a multigene family of three to four dierent, maximally divergent genes encode an identical 148-amino-acid protein. CaM is the best conserved protein detected so far and no naturally occurring mutations have as yet been identied in multicellular organisms. Upon Ca2 1 binding CaM undergoes a conformational change that exposes hydrophobic pockets on the underside of each pair of EF-hands. In the presence of target peptide, a further conformational change results in the exible linker between the two heads being dramatically bent, partially unwound and wrapped around the target peptide (Figure 4b; Ikura et al., 1992). Calmodulin has been shown to interact with well over 100 dierent target proteins and anities for bona de CaM targets are usually in the nanomolar range. CaM is found in the cytosol, associated with membranes and also in the nucleus. The regulated redistribution of CaM between the cytosol and the nucleus appears to play an important role in the proper regulation
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of CaM function. Of the most important targets for CaM are the calmodulin-dependent protein kinases II and IV (CaMKII and IV). CaMKII phosphorylates target proteins implicated in many important cellular processes, including neurotransmitter synthesis and release, modulation of ion channels and energy metabolism. Troponin C Skeletal muscle troponin C (TnC) is the Ca2 1 -sensing component of the troponin complex and contains a pair of high-anity, Ca2 1 /Mg2 1 mixed sites in the C-terminal half and a pair of low-anity Ca2 1 -specic sites in the Nterminal half. The structure of TnC is very similar to that of calmodulin (Figure 4a). The high-anity metal-binding sites are probably saturated with Ca2 1 or Mg2 1 during muscle relaxation and contraction, respectively and allow constitutive association of TnC with the troponin complex. Ca2 1 -binding to the low-anity site(s) with fast kinetics induces a conformational change of the N-terminal half which is transmitted to the other components and is the triggering event of muscular contraction. Parvalbumin Parvalbumin (PV) possesses two high-anity Ca2 1 / Mg2 1 mixed sites and is highly expressed in fast-twitch muscles. Under resting [Ca2 1 ]i, the metal-binding sites are occupied by Mg2 1 ions. The kinetics of Ca2 1 binding is determined by the relatively slow o-rate for Mg2 1 . During muscle contraction PV does not compete with TnC for the binding of Ca2 1 , but helps to increase the initial rate of [Ca2 1 ]i decay. Thus, it shortens the relaxation phase after very brief contractions. In this example, it is evident that Mg2 1 is nearly as important as Ca2 1 ; it not only lowers the Ca2 1 anity to the physiological range, but signicantly aects the kinetics of Ca2 1 binding and release. Structural data is available from Brookhaven Data Base (PDB: entry codes: 1BU3, 1PVA and 3PAT).

Vitamin K-dependent proteins

The most characteristic feature of proteins in this family is the presence of 913 gamma-carboxyglutamate (g-Gla; Figure 5a) in the N-terminal Gla domain (residues 145). This carboxylation reaction is carried out by a vitamin-K dependent enzyme system. g-Gla is a much stronger chelator for Ca2 1 ions than glutamate. Gla domains are present in serum proteins involved in blood coagulation and include prothrombin and factors VIIa, IX and X. As for the annexins, it was postulated that the negatively charged phospholipids exposed on the surface of blood platelets after injury anchor prothrombin to the plasma membrane by the bridging function of Ca2 1 ions. This attachment would allow the interaction with factor Xa (a serine protease) and factor V (a stimulatory protein) which then leads to the cleavage of prothrombin and the newly formed thrombin is released from the membrane. Although the order of reactions taking place during this process is clear, the mode of attachment of prothrombin via electrostatic interactions is less certain. The structure of the prothrombin fragment 1 revealed that most Ca2 1 ions and Gla residues were buried inside the Gla domain (Soriano-Garcia et al., 1992). A similar structure was also found in the Gla domains of factors VIIa and IX. More recent models suggest specic-site binding of these factors to membranes. The interaction may involve specic binding to one or more phospholipid head groups, but also a cluster of hydrophobic residues in the Gla domains seems to be important for membrane binding (Figure 5). At the moment it is not known whether these residues are directly involved in the actual membrane contact.

Pentraxins
Like annexins, the pentraxins belong to the family of peripheral membrane-binding proteins (Schwalbe et al., 1992). Members are divided into two subfamilies: serum amyloid P (SAP) and C-reactive proteins (CRP). As for annexins or some C2-domain proteins, human SAP has a high anity for negatively charged phospholipids. The protein consists of a dimer of pentamers and each pentamer can bind 10 Ca2 1 ions (Brookhaven Data Base, PDB entry code: 1SAC). The mechanism of binding to membranes is similar to that of annexins, whereby the Ca2 1 ions form a complex with the protein and the acidic phospholipids. The exact biological functions of pentraxins are not known, but recently an interaction of SAP with calumenin, a secreted Ca2 1 -binding protein, has been demonstrated.
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Figure 5 Vitamin K-dependent proteins. (a) Structure of the modified amino acid g-glutamate (Gla). (b) Picture of prothrombin fragment 1. The peptide is in wire-frame form except for Ala1, Phe5, Leu6 and Val9. Modified from Nelsestuen and Ostrowski (1999). (Brookhaven Data Base, PDB entry code: 2PF2.)

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Low-affinity, high-capacity CaBPs

The Ca concentration in the lumen of organelles (endoplasmic reticulum, mitochondria) is in the millimolar range, like the extracellular milieu, and thus the anities of CaBPs in these organelles are adapted to these conditions. Proteins belonging to this group are calnexin, calmegin, calreticulin and calsequestrin. They are all associated with endo(sarco)plasmic reticulum membranes and are thought to play a role in a variety of cellular functions (e.g. Ca2 1 homeostasis, protein folding, adhesion, gene expression, etc.). The proteins can be divided into distinct structural and functional domains which sometimes show strong homologies. A proline-rich domain (P domain) is present in calreticulin, calnexin and calmegin. The C domains implicated in Ca2 1 -binding are very acidic and bind Ca2 1 , Mg2 1 , Zn2 1 with low anity and high capacity (for Ca2 1 : Kd 5 0.52 mmol L 2 1; Bmax 4 2550 moles of Ca2 1 per mol). In some cases they can also bind Fe3 1 or K 1 . Calnexin is one of the major Ca2 1 -binding proteins of the endoplasmic reticulum membrane and binds transiently to newly synthesized membrane and soluble proteins that are destined for the secretory pathway. This membrane-bound chaperone is thought to be directly involved in the quality-control mechanism of the endoplasmic reticulum (ER). Stable, if not permanent, complex formation has been reported with unassembled multisubunit and misfolded proteins. Calreticulin binds to blood clotting factors (factor IX, factor X and prothrombin) and has antithrombotic activity. A chaperone function for the protein has also been suggested. Calsequestrin (CS) is the major Ca2 1 -binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It is localized within the terminal cisternae of the SR, close to the luminal site of the junctional membrane and in potential contact with the Ca2 1 -release channels. Its proposed functions include Ca2 1 storage and an involvement in Ca2 1 release. About 30% of the total amino acids of CS are acidic residues, mainly concentrated within the C-terminus.
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sensing protein (CAS, also named gp330 or megalin) has been implicated in sensing the extracellular concentration of Ca2 1 . 1,25-Dihydroxyvitamin D3 induces absorption of Ca2 1 from the intestinal lumen and also Ca2 1 resorption in the kidney. It upregulates the expression of two EF-hand CaBPs, calbindin D-28k and calbindin D-9k by a mechanism similar to that of other steroid hormones. Calbindin D-9k is present in absorptive epithelial cells of mammalian duodenum and its tissue concentration is correlated with active transcellular, vitamin D-dependent Ca2 1 absorption. Furthermore, it is assumed to play a role in Ca2 1 reabsorption in the kidney distal tubules. In this tissue also calbindin D-28k is abundantly expressed and is thought to participate in Ca2 1 resorption. Calbindin D-9k does not exist in amphibians and birds and instead, calbindin D-28k is expressed in the enterocytes in these species. In both cases, the proteins act as Ca2 1 transporters, shuttling these potentially cytotoxic ions from the luminal to the apical side (blood vessels).

References
Babu YS, Bugg CE and Cook WJ (1988) Structure of calmodulin rened at 2.2 A resolution. Journal of Molecular Biology 204: 191204. Brown EM, Gamba G, Riccardi D et al. (1993) Cloning and characterization of an extracellular Ca2 1 -sensing receptor from bovine parathyroid. Nature 366: 575580. Burgoyne RD and Geisow MJ (1989) The annexin family of calciumbinding proteins. Cell Calcium 10: 110. Celio M, Pauls T and Schwaller B (1996) Guidebook to the CalciumBinding Proteins, pp. 1238. Oxford: Oxford University Press. Huber R, Romisch J and Paques EP (1990) The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes. EMBO Journal 9: 38673874. Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB and Bax A (1992) Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256: 632638. Kawasaki H, Nakayama S and Kretsinger RH (1998) Classication and evolution of EF-hand proteins. Biometals 11: 277295. Kretsinger RH and Nockolds CE (1973) Carp muscle calcium-binding protein. II. Structure determination and general description. Journal of Biological Chemistry 248: 33133326. Nalefski EA and Falke JJ (1996) The C2 domain calcium-binding motif: structural and functional diversity. Protein Science 5: 23752390. Nelsestuen GL and Ostrowski BG (1999) Membrane association with multiple calcium ions: vitamin-K-dependent proteins, annexins and pentraxins. Current Opinions in Structural Biology 9: 433437. Rizo J and Sudhof TC (1998) C2-domains, structure and function of a universal Ca2 1 -binding domain. Journal of Biological Chemistry 273: 1587915882. Schwalbe RA, Dahlback B, Coe JE and Nelsestuen GL (1992) Pentraxin family of proteins interact specically with phosphorylcholine and/or phosphorylethanolamine. Biochemistry 31: 49074915. Soriano-Garcia M, Padmanabhan K, de Vos AM and Tulinsky A (1992) The Ca2 1 ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry 31: 25542566. Sutton RB, Davletov BA, Berghuis AM, Sudhof TC and Sprang SR (1995) Structure of the rst C2 domain of synaptotagmin I: a novel Ca2 1 phospholipid-binding fold. Cell 80: 929938.

Transport of Calcium
Human serum albumin is by far the most abundant Ca2 1 binding protein in serum, but a2-macroglobulin, immunoglobulin M (IgM) and haptoglobins bind approximately the same amount of calcium per gram as albumin. Besides these proteins, small molecules (e.g. bicarbonate, phosphate, citrate) and the pH determine the free serum concentration of Ca2 1 . Ca2 1 ions in the serum have been shown to act as a rst messenger by binding to a calciumsensing receptor (CaSR) which was rst detected on the surface of parathyroid cells, but is also present in epithelial and kidney cells. Another protein, a putative calcium-

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Swairjo MA, Concha NO, Kaetzel MA, Dedman JR and Seaton BA (1995) Ca2 1 -bridging mechanism and phospholipid head group recognition in the membrane-binding protein annexin V. Nature Structural Biology 2: 968974.

Further Reading
Brown EM (1999) Physiology and pathophysiology of the extracellular calcium-sensing receptor. American Journal of Medicine 106: 238253. Gewurz H, Zhang XH and Lint TF (1995) Structure and function of the pentraxins. Current Opinions in Immunology 7: 5464.

McDonald JF, Evans TC, Jr, Emeagwali DB et al. (1997) Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency. Biochemistry 36: 1558915598. Persson E and Petersen LC (1995) Structurally and functionally distinct Ca2 1 binding sites in the gamma-carboxyglutamic acid-containing domain of factor VIIa. European Journal of Biochemistry 234: 293 300. Steno J (1999) Contributions of Gla and EGF-like domains to the function of vitamin K-dependent coagulation factors. Critical Review on Eukaryotic Gene Expression 9: 5988.

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