- Email: [email protected]
Calcium
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Calcium Johan Even is, Anders Malmendal and Sture Forsen* Ca 2+ is involved in an intriguing variety of different biological events. The rapid development of techniques such as region- or organelle-directed fluorescent probes and laser scanning confocal microscopy for studying cellular biological events at a molecular level provides us with a rich daily intake of new results. While detailed three-dimensional structures of many intracellular and extracellular Ca2+-binding proteins have become available, structural information on key membrane proteins is still lacking. An integrated picture of the molecular events behind the multifunctional roles of Ca 2+ in biological systems is still pending.
Addresses Physical Chemistry 2, Lund University, P.O. Box 124, S-22100 Lund, Sweden *e-mail: [email protected] Current Opinion in Chemical Biology 1998, 2:293-302 http://biomednet.com/elecref/1367593100200293 © Current Biology Ltd ISSN 1367-5931
Abbreviations ATP adenosine triphosphate c-ADPR cyclic ADP ribose CaM calmodulin [Ca2+] i cytoplasmic free Ca2+ concentration [Ca2+] n nuclear free Ca2+ concentration [Ca2+]o extracellular free Ca2+ concentration EGF epidermal growth factor EGTA ethylene glycol-bis(J]-aminoethylester) ER endoplasmic reticulum Gla y carboxy-glutamic acid IP3 inositol-1,4,5-trisphosphate IP3 R IP3 receptor LDL low-density lipoprotein LDLR LDL receptor NAADP nicotinic acid adenine dinucleotide phosphate NMR nuclear magnetic resonance NPC nuclear pore complex RyR ryanodine receptor SR sarcoplasmic retioulum TnC troponin C
Introduction
Ca z+ ions are major players in an intracellular signaling system that translates extracellular stimuli into the regulation of a bewildering number of phenomena such as muscle contraction, neurotransmitter release and other secretion processes, cell proliferation, gene expression and cell death. In order to accomplish such varying feats, Ca 2+ concentration transients must be well defined in both time and space [1°], and the target molecules must be finely tuned for the demands at each location. In addition the target's response may differ depending on the history of the cell [2°]. Our present understanding of the CaZ+ signaling system is limited but has greatly increased during the past few years, on one hand through the
combined use of region- or organelle-directed fluorescent probes and laser scanning confocal microscopy [3,4], and on the other through structural biological studies of the target proteins. Ca 2+ also plays an important role in extracellular environments, being involved in cell-cell interactions and as a prerequisite for tissue repair and for controlling macrostructure and shape through exoskeletons or endoskeletons in higher organisms. Few, if any, ions in biological systems have such an extensive biological role as Ca2+. This is reflected in the number of publications in which calcium is involved in one way or the other. A M E D L I N E search carried out in November 1997 produced close to 24,000 articles published during the past two years alone. Needless to say a brief review like this one can in no way capture the developments in the entire calcium research field--nor would we feel competent to even try. What we have settled for is to highlight a few areas - - intracellular and extracellular--and to take a look at recent developments in structural biology and try to connect these with the biological function of calcium. C a 2+ a n d i n t r a c e l l u l a r
signaling
In eukaryotic cells the cytoplasmic calcium concentration ([Ca2+]i) ranges from 100nM in a 'resting' cell to 1-101aM in an activated cell. Usuall,~; both intracellular and extracellular CaZ+ sources are available for signaling. As the exracellular free CaZ+ concentration ([Ca2+]o) in higher organisms is around 1.2 mM, the extracellular pool may be considered effectively infinitely large. The flow of Ca 2+ can be regulated through voltage-, receptor-, and store-operated channels. The existence of store-operated channels, operationally defined as being able to restore the Ca 2+ levels of depleted intracellular stores [S], has been demonstrated in Xenopus oocytes and, recentl'6 in a sympathetic neuronal cell line [6]. For many years the Ca2+-signaling field has been focused on CaZ+ stores in the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR), where IP3R and RyR were discovered. Recently mitochondria have been shown to be active and important participants in the signaling system [7"°]. In addition to these three stores, there is some evidence pointing to another type of high capacity storage pool in mammalian cells [8]. Two major Ca2+-release channels in the ER and SR are welt established--the inositol-l,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine receptor (RyR). Ca 2+ release by IP3R is induced by IP3, while RyR is regulated by cyclic ADP-ribose (c-ADPR) [9*]. Both receptors are also modulated by the cytoplasmic Ca 2+ concentration ([Ca2+]i), calmodulin (CAM), phosphorylation and possibly a number of other factors, for example caffeine. Low
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[Ca2+]i (10nM-1 btM) acts synergistically with IP 3 while high [Ca2+]i (>3 btM) counteracts the stimulatory effect of IP 3 [9°,10]. The Ca 2+ concentration in the lumen ([CaZ+]o] also seems to regulate the flow of CaZ+ through IP3R and RyR channels [11,12]. There is some evidence for a Ca2+-release channel, which is activated by nicotinic acid adenine dinucleotide phosphate (NAADP) [13,14]. While IP3R and RyR display a graded response towards their activating factors, the NAADP-dependent channel is reported to display an 'all-or-none' type of activation [15,16]. The response of ligand-gated ion channels to agonist stimuli is usually very rapid. It is therefore striking that several studies--using either rapid photolytic release of caged IP?, in intact cells [17] or clamped permeabilized hepatocytes [18°]--give evidence of a latency period of some 30 ms between IP 3 binding and the onset of Cae+ release through the [P3R channel. Unless there is some yet undetected technical snag in these experiments, we may witness the result of a slow conformation change in IP3R--possibly a safeguard against spontaneous firing of the channel [12]. The complexity of cytoplasmic CaZ+ signaling, with a variety of observed CaZ+ oscillations such as localized bursts and global waves, is a consequence of ligand triggered chemical events, regulatory feedback mechanisms and diffusive processes [19]. ER membranes seem to be, at least locally, highly populated with I P 3 R s - - a s are SR membranes with R y R s - - a n d synchronized triggering of initially localized Ca2+ release events, resulting in regenerative CaZ+ waves, may take place [1"]. In an elegant study by Home and Meyer [20 °°] the spatial and temporal characteristics of IP3-induced Ca 2+ release in rat leukemia cells were determined. The existence of individual brief (<3 ms) and spatially localized (<2 btm) release events was observed in the presence of the specific CaZ+ chelator EGTA, preventing the Ca2+ diffusion induced triggering of more distant IP3R channels. In the absence of EGTA, global Ca 2+ release was seen as a consequence of synchronized triggering [1",20°°]. Several theoretical models have been put forward to account for the complex cellular events observed in CaZ+ signaling [21,22]. To reduce [Ca2+]i back to resting levels, cell membranes, ER and SR are provided with ATP-driven Ca2+ pumps, CaATPases [23]. When [Ca2+]i rises, mitochondria also rapidly absorb CaZ+ and then dispense it at a slower pace, permitting CaATPases to restore CaZ+ in ER and SR compartments to former levels without disturbing [CaZ+]i [7"°]. The cytoplasmic level of IP 3 is rapidly reduced by enzymatic degradation and the estimated lifetime of IP 3 in living cells is -1 s. Also important for our understanding of the spatio-temporal properties of the Ca 2+ signaling system are the effective diffusion rates of Ca z+ and IP 3 in the cytoplasm. As shown in Xenopus oocytes, the diffusion of IP 3 is largely independent of [IP3], while the Ca -~+
diffusion rate decreases with decreased [Ca2+]i as is typical for transport of ions in a medium filled with slowly diffusing, ion-buffering molecules [24]. The diffusion rates and the effective lifetimes make the effective action range of IP 3 several tens of microns and thus useful for triggering events at some distance, whereas the action range of Ca -)+ is of the order of a few microns at the most, and more suitable for triggering local events.
Figure 1
CytopLasm
i
@0 tOOt Current Opinion in Chemical Biology
The IP3 receptor. Cross-section of the tetrameric IP3 receptor model in the membrane of an intracellular Ca2+ store. As indicated below, only two monomers are shown. Each monomer contains an IP3-binding domain (dark gray), a regulatory domain (light gray) and six membrane-spanning helices (shown as cylinders) [9°,26°]. Ca 2+ sites, located using fusion proteins, are indicated [27]. C and N indicate the carboxyl and amino termini, respectively, of the protein.
At least three isoforms (type I, II and Ill) of both IP3R and RyR have been characterized and their tissue-specific distribution mapped [9°,Z5]. Functional IP3R and RyR arc tetrameric complexes but no high resolution structure of either receptor is as yet available. A structural model of IP3R has been proposed based on cryoelectron microscopy studies [9",26"] (Figure 1). Each IP3R subunit appears to have three functional d o m a i n s - - a n amino-terminal IP 3binding domain, a regulatory domain with binding sites for CaM and other factors and a membrane-spanning domain. Fusion protein studies have localized CaZ+-binding sites on both the cytoplasmic and luminal domains of mouse IP3R-type I [27]. Neither site is of the EF hand type (see below). A similar domain-like structure has been proposed for human cardiac muscle RyR (type I) [28]. At least
Calcium Events, Malmendal and Fors6n
in the case of IP3 R, the tetramer subunits may belong to different isoforms - - most likely allowing for cell type dependent fine tuning of the functional properties [9",10]. It would appear that, to date, most studies of the functional properties of IP3R release channels have been performed on type I. Recent comparisons of IP 3 affinities of full length type I and type III human IP3Rs indicate, however, that generalizations from one isoform to another may be unreliable [29].
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This complicates the interpretation of some experiments addressing the question of the interdependence of [CaZ+]i and [CaZ+]n. T h e complex behavior of [CaZ+]n is further illustrated by rat leukemia cells in which [CaZ+]i gradients are transmitted to the nucleus with a time delay of about 200ms [32]. In cultured neuronal cells, small changes in [Cae+]i are faithfully transmitted to the nucleus, while larger changes (>300 nM) were clearly attenuated [33]; this is difficult to understand unless the nuclear pores close at
high [CaZ+]. Ca 2+ in t h e cell nucleus After many false starts due, to some degree, to the difficulties of localizing [CaZ+]-reporting probes to the nucleus, evidence is mounting for a signaling role of CaZ+ in the nucleus (L Santella, E Carafoli, personal communication; see Note added in proof.) As in the case of cytoplasmic studies, new organdie-directed fluorescent probes and laser scanning confocal microscopy have been of great importance. A schematic picture illustrating the main features of the eukauotic cell nucleus is provided in Figure 2a. Ca 2+ and small proteins (of molecular weight <20 kDa) have been observed to diffuse relatively freely through the nuclear pore complex (NPC) [30]. Recent studies, however, indicate that pore opening is regulated by the
nuclear CaZ+ concentration, [Cae+] n, and/or [CaZ+]i [31].
The discovery of IP3R on the inner nuclear membrane [34"] suggests that findings interpreted as Cae+ diffusion from the cytoplasm might instead have been an IP 3triggered event. This idea is supported by the finding of IP 3 and c-ADPR triggered release of Ca2+ from the Xenopus nuclear envelope space to the nuclear space [35]. Ca 2+ transport pathways in the nuclear envelope are shown in Figure 2b. In the nuclear space, in contrast to the cytoplasm, Cae+ propagation seems to occur through simple diffusion since no Ca2+-induced Cae+ release has been established [36"]. T h e absence in the nucleus of powerful pumping mechanisms to reduce [Ca2+]n results in a larger effective life time and range of action of Ca2+ in the nucleus than in the cytoplasm. This may be critical for the many gene transcription processes dependent on the prolonged presence of CaM-activated proteins, such as CaM-dependent protein kinase IV [37], which presumably
Figure 2
(a)
NPC
ER
(b) Ca 2+
Cytoplasm INM
N
~
~
.""
K+ ATP
I11.
Ca 2+
NES
"" " "" "" "" " . , . ""
c ~ A ' ~ D pR
Pa Nuclear space
Current Opinion in Chemical Biology
The cell nucleus. (a) Components of the nucleus and its environment. NS, nuclear space; INM, inner nuclear membrane; ONM, outer nuclear membrane; NES, nuclear envelope space; NPC, nuclear pore complexes; ER, endoplasmic reticulum. (b) is an expanded version of (a). Ca2+ transport pathways in the ONM and the INM of the nucleus [95]. The ONM is continuous with the ER and is rich in ATP-driven Ca 2+ pumps that pump Ca 2+ into the NES. In contrast, the INM, although connected with the ONM at NPCs, has properties quite distinct from those of the ER membrane [31,96].
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require [Ca2+] n o f - l l a M Ca2+-bound state [38"].
to keep CaM in the active
Intracellular Ca2+-binding proteins Prime targets for Ca 2+ released in a cell are Ca2+-binding proteins, many of which are players in intracellular regulatory processes and come in many sizes and shapes. Here, we will restrict the discussion to two major types: proteins with the EF-hand motif and those with the C2 domain. EF hand proteins
T h e ubiquitous EF hand CaZ+-binding motif appears in many proteins that exert Ca2+-dependent actions in the nucleus or the cytoplasm. This motif exhibits helix-loop-helix architecture, and is normally paired with another EF hand motif with the two Ca2+-binding loops coupled through a short antiparallel 13 sheet [39,40]. An example of EF hand Ca2+ coordination is given in Figure 3a. The binding of two Ca 2+ ions within an EF hand pair is usually highly cooperative. EF hand proteins cover a wide range of Ca 2+ affinities (Kd = 10-9-10 -5 M). Although numerous studies have been performed during the past fifteen years (reviewed in [41,42]), questions about the molecular parameters tuning the affinity, specificity and cooperativity still remain. To aw)id the complexity inferred by paired EF hands, Esc/let~ice coli galactose-binding protein, which has one EF hand, has been used as a basis for mutational studies aimed to elucidate the contributions of residues in different loop positions [43-45]. The impact of 'acid pairs' [46], that is, two charged amino acid residues in antipodal positions in the coordinating sphere, as well as the impact of the context of the two surrounding helices on Ca 2+ affinity have been demonstrated [47]. Intracellular Cae+-binding proteins in higher organisms operate in an environment with a 100-f0,000-fold excess of Mg 2+. T h e issue of the impact of Mg2+ on Ca 2+ and target peptide binding has been raised [48]. Evidence of negative binding cooperativity between MgZ+ and CaZ+ has been found for the Cae+-buffering protein calbindin D9k [49"]. T h e regulatory proteins troponin C (TnC), CaM and a number of homologous proteins consist of two independently folded domains connected by a flexible helical segment. Each domain contains one EF hand pair. Upon Cae+ binding, rearrangements of the helices occur, resulting in exposure of hydrophobic patches, which are poised for binding target proteins (Figure 3); [50] and references therein). Little is known about the molecular mechanism of these conformational changes. Mutational studies on CaM [51"] and T n C [52,53"] have suggested a critical role of a bidentate glutamic acid in this process (Figure 3b,c). CaZ+-saturated cardiac muscle TnC, with only one functional Ca2+-binding site in the
amino-terminal domain, exposes a smaller hydrophobic surface than that observed in CaM and skeletal muscle T n C [53",54}. CaM-(Ca2+)4 interacts with more than 100 different target proteins [55"]. Typically CaM binds to an autoinhibitory peptide segment of the target enzyme, target peptides [56"]. T h e first structure of a complex of CaM and an isolated target peptide was reported in 1992 [57], showing the peptide adopting an amphiphilic helical conformation and effectively enclosed by the two CaM domains. Since then, a deeper understanding has evolved. In CaM complexes with isolated target peptides, the Ca 2+ affinit'/ of CaM is increased 10-1000-fold due to slower off-rates, particularly in the lower affinity amino-terminal domain [38"',58,59]. This increase in affinity may be explained by the higher stability of the calcium saturated state when a target peptide is bound. In contrast, the interactions between both CaM and target peptide with the rest of the protein may result in considerably smaller increases in Cae+ affinity [38"]. Recent studies of target enzyme activation by CaM involve several kinases [60,61], G-protein-coupled kinase receptor 2 [62] and nitric oxide synthase [63,64]. Activation of nitric oxide synthase is required for refilling Ca2* stores [6]. A special sequence in neuromodulin and unconventional muscle myosins, called the 'IQ motif', has been shown to bind Ca2+-frcc CaM. At higher Ca2+ levels, the activity of these molecules is regulated through changes in the mode of interaction with CaM [65",66]. Calpain is a ubiquitous Ca2+-regulated cysteine protease and a heterodimer of two subunits. T h e smaller st, bunit has two domains (dE dVI) and the larger has four domains (dI-dlV), with the active site residues located within domain dlI. Crystal structures of dVI reveal five EF hands (EFI-EF5) sequentially arranged in two pairs plus a singlc EF hand (EFS), which pairs up with a corresponding EF5 of another dVI-domain-containing molecule, to form a homodimer [67",68",69]. This suggests, together with strong homology between dVI and dlV from the large subunit, a naturally occurring interdomain EF hand pair, responsible for the in vi~'o heterodimerization of the two subunits. Only three EF hands (EF1-EF3) of dVI bind Ca ?.+ at physiological intracellular Ca 2+ levels. EF2 and EF3 are canonical EF-hands, while EF1 exhibits a coordination pattern not seen before in an EF hand pair. Upon Cae+-binding, CaM-like structural changes in the first EF hand pair seem to enable proteolytic activit>
The C2 domain The C2 domain reviewed in [70"] is an eight-stranded [~ sandwich Ca2+-binding protein module, first observed in protein kinase C, a protein involved in phospholipid signaling, and subsequently in a wide range of proteins. The crystal structure of phospholipase C8 reveals a C2 domain coupled to a CaM-like domain and a catal'y-tic TIM-barrel structural motif [71"]. C2 domains interact
Calcium Even~.s, Malmendal and Fors6n
Figure 3
297
with small molecules, proteins and cellular membranes, sometimes in a CaZ+-dependent manner. Ca2+-dependent C2 domain participation in two different processes was recently reported: first, binding of the C2 domain in cytoplasmic phospholipase As to phosphatidylcholine vesicles [72°], which is a step in the inflammatory response of the cell; second, binding of the first C2 domain of the synaptic vesicle protein synaptotagmin to the plasma membrane protein syntaxin [73"], which a step in Ca2+-triggered synaptic vesicle exocytosis. In both cases binding of two Ca2+ ions is important for the interaction, and the substrates are suggested to provide Ca 2+ ligands.
(a)
Asp22
Extracellular
(b)
lix B
C a 2+
Besides its central role in regulating intracellular processes, CaZ+ also plays an important role in extracellular processes, such as bone formation, cell adhesion, and blood clotting, and in receptor structures. In mammals the extracellular free CaZ+ concentration, [CaZ+]o, is tightly regulated at -1.2 raM. Many extracellular CaZ+-binding proteins bind Ca 2+ so strongly that they will be essentially saturated with Ca2+ when they arrive at the extracellular space. A characteristic of extracellular proteins is their multidomain nature with numerous independently folded, but often interacting, modules [74°']. The major function of CaZ+ binding to extracellular proteins was long thought to be enhancement of the thermal stability of these proteins and improvement of their resistance to proteases. During the past few years, new crystal structures and NMR-based solution structures have provided a revised and more complex picture. Ca2+ binds either to single domains, or to the interface between domains mediating domain-domain interactions. Ca 2+ may also play a direct role in ligand interaction, as in the C-type lectins (carbohydrate-recognition proteins that depend on [Ca2+]o for their activity) [75], or in membrane association, as in the Gla-module (see below). More commonly Cae+ binding is crucial for inducing or stabilizing protein conformations appropriate for enzymatic activity and/or ligand binding.
Current Opinion in Chemical Biology
Ca 2+ binding to an EF hand motif, and the Ca2+-induced structural changes exemplified by the amino-terminal EF hand of calmodulin. (a) Ca 2+ is coordinated by seven ligands, provided either directly by the protein or indirectly via a water molecule. The overall geometry of the Ca 2+ coordination can be described as a pentagonal bipyramid. (b) The apo state, that is, with no calcium bound [97]. The backbone trace of amino acid residues 5 - 4 0 and the sidechain of the bidentate Glu31 (glutamic acid, residue 31 in the protein) are shown, with the two ligating carboxylate oxygens colored black. (c) The Ca2+-saturated state [98]. To allow for Ca 2+ ligation, the helices (A and B) rearrange to a more perpendicular orientation, thereby opening up a cleft between the helices, with many hydrophobic sidechains exposed. The molecular graphics in this paper were generated using Midas Plus [99], University of California at San Francisco software.
An example of a Cae+-stabilized protein structure is the ligand-binding module of the low-density lipoprotein (LDL) receptor (LDLR) [76"]. This module consists of approximately 40 amino acid residues. T h e mammalian form of L D L R consists of an extracellular domain formed by seven characteristic L D L modules and one E G F precursor domain (see below), one transmembrane helix and a cytoplasmic domain. L D L R is responsible for the uptake of cholesterol-containing lipoprotein particles. In this process, 10 minutes in total, the lipoprotein is bound, transported to an intracellular acidic endosome and discharged from the receptor; the 'empty' receptor is then transported to the surface. Exposed to a multitude of acid baths in the endosome, extreme protein stability is required. Recently, the crystal structure of the fifth ligand-binding module of the L D L receptor was published
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[76",77"]. In addition to confirming three cysteine bridges in this module, the study reveals a significant structural role of a CaZ+ ion, octahedrally coordinated by acidic sidechains. This acidic site was previously thought to bind the basic lipoproteins, an interaction that is now assumed to take place at another site of the module and to be primarily of hydrophobic character. Naturally occurring point mutations, resulting in impaired CaZ+ binding, cause pathologically elevated blood cholesterol levels and premature heart disease [77"',78]. Another CaZ+-binding domain is the small Gla module found as the amino-terminal domain of proteins involved in the blood coagulation cascade. T h e module contains 10-12 y-carboxy-glutamic acid (Gla) residues and binds 6-12 CaZ+ ions with low affinity (Kd -10-3M). Upon Ca z+ binding, the Gla module interacts with phosphatidylserine-rich phospholipid membranes; it has also been suggested that it is involved in direct protein interactions. NMR-derived structures show that Ca 2+ binding to the Gla module induces a sizeable conformational change in the Gla module, resulting in exposure of hydrophobic residues; these hydrophobic residues are largely responsible for the interaction with the membranes [79-81 ]. Epidermal growth factor (EGF)-Iike domains are small but widely distributed modules found in functionally diverse extracellular proteins [74"]. One subfamily of these domains contains a bipartite consensus sequence indicative of a Caa+-binding site [82,83]. In several proteins, point mutations in the genes coding for Cae+-
binding EGF-like domains have been shown to cause human disorders; for example bleeding disorders arc caused by mutations of Ca2+-binding residues in the blood coagulation proteins factor IX and protein S [841. Another example is the Marfan syndrome and other related disorders that are caused by mutations in the major building blocks of extracellular microfibrils, fibrillins 1 and 2 [85]. The solution structure of a pair of EGF-Iike domains of fibrillin-1 reveals that these domains arc arranged in a rigid, rod-like structure that is stabilized by Ca2+ and hydrophobic interactions (Figure 4a) [86"1. The Marfan syndrome is linked to mutations that dcstroy Cae+-binding capability and, therefore, the interdomain interactions. T h e importance of CaZ+ binding for the relative orientations of domains is also demonstrated in studies of the two amino-terminal domains of blood coagulation factor X - - a G l a - E G F p a i r - - w h e r e [Ca2+l could be adjusted so that only the Ca2+-binding site in the EGF domain was populated (Figure 4b) [87"]. A more complex role for extracellular Ca 2+ has been %und in the cadherin family of cell-cell adhesion proteins. This family of proteins usually consists of five homologous extracellular domains, a single membrane-spanning segment and a cytoplasmic region [74",88]. T h e crystal structure of domains 1 and 2 of epithelial (E) cadhcrin shows that it forms parallel directs with the dimer interface found between the two domain junctions where six Ca 2+ ions arc located (Figure 5a) [89"]. Small [Cae+] o fluctuations modulate the number of ions bound and thus the relative orientation of the domains, dimer formation and cell adhesion [89"°,90"*,91]. In addition, each E-cadherin
Figure 4 (a)
(b)
O
,,[
- - : ..... N
EGF c
Current Opinion in Chemical Biology
Examples of Ca2+-stabilized domain interactions. Ca 2+ ions are colored black. (a) NMR-derived structure of the dimer of EGF-like domains 32 (light gray) and 33 (dark gray) of fibfillin-1 [86"]. (b) NMR-derived structure of the dimer of the Gla domain (light gray) and EGF domain 1 (dark gray) of factor X [8?'], The location of Ca 2+ in (b) is an approximation, since the Ca 2+ was not included in the structure calculations.
Calcium Even#& Malmendal and Fors6n
molecule binds six Ca 2+ ions strongly to the remaining domain junctions. These Ca2+ ions are likely to be important for the rod-like arrangement of these domains, resulting in the formation of V-shaped dimers, as indicated by Figure 5b [90"]. In the crystal structure of domain 1 from neural (N) cadherin, the crystal packing provides a model for the interaction of cadherin dimers with cadherin dimers from adjacent cells through antiparallel dimer interactions of each domain 1 with a domain 1 from an adjacent cell cadherin [92], also shown in Figure 5b. Figure 5
299
Conclusion Through the combined use of biochemical, molecular biological and biophysical methods considerable advances have been made to unravel the complex mechanisms behind the Ca2+-dependent signaling systems in eukaryotic cells. The field is, however, presently still in a fairly descriptive state. Detailed structural data on key receptors, ion channels and pumps are lacking. Regulation of Ca 2+ levels in the nucleus is poorly understood. Furthermore, generalizations are made difficult by the individualistic nature of cells from different organisms or with different functions within any given organism. New structural and biophysical studies of CaZ+-binding proteins have provided important information on the roles of Cae+ at a molecular level. In particular, studies of extracellular proteins have revealed an unexpected variety of CaZ+-binding sites and affinities, indicating new, and perhaps not yet imagined, CaZ+ functions. Much remains, to be done, however, to establish links between structure, Ca z+ levels and biological function.
(b)~
Note added in proof The paper referred to in the text as (L Santella, E Carafoli, personal communication) has now been published [100].
Acknowledgements We thank Michael J Bcrridgc, Thomas Grundstr6m and Stcn Orrcnius for helpful discussions and Pctra Nybcrg for critical reading of the manuscript. Cell surface
l __
CurrentOpinioninChemicalBiology
Cadherins and cell adhesion. (a) Ribbon representation of the crystal structure of the parallel homodimer of domains 1 (top) and 2 of E-cadherin [89"°]. Monomers are colored light and dark gray, respectively, and the calcium ions are black. (b) A model of the extracellular parts of cadherins involved in cell adhesion [89°°]. Cadherin dimers from adjacent cells interact to form a zipper like structure. Individual domains are represented by cylinders numbered from amino to carboxyl terminus. Plausible Ca 2+ positions are indicated by gray dots.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • =.
1.
of special interest of outstanding interest
BerridgeM.J: Elementary and global aspects of calcium signalling. J Physio/(Lond) 1997, 499:290-306.
Avery readable overview by one of the pioneers of the Ca 2+ signalling field, with an integration of a vast amount of independent experimental data into a coherent model. 2.
•
Dolmetsch RE, Lewis RS, Goodnow CC, Healy Jl: Differential activation of transcription factors induced by Ca 2+ response amplitude and duration. Nature 1997, 386:855-858.
This paper demonstrates that differential gene transcription in B lymphocytes is regulated by, or at least connected with, the level and time course of cytoplasmic Ca 2+ concentrations.
The first identified extracellular EF hand pair was recently observed in BM-40, a small secreted glycoprotein, presumed to take part in bone mineralization, tissue remodeling and cell growth [93°]. One canonical EF hand pair is located in the extracellular CaZ+-binding (EC) domain, which is the second of three domains. This domain has a structure similar to that of a Cae+-loaded CaM domain with an amino-terminal segment taking the place of the CaM target peptide. The observation of EF hands in extracellular proteins raises the question as to whether Ca 2+ could in fact have a role as an cxtracellular messenger. This idea goes against the notion of a constant [Cae+] o, but the local concentration may well vary with time under certain conditions [94].
3.
Sako Y, Sekihata A, Yanagisawa Y, Yamamoto M, Shimada Y, Ozaki K, Kusumi A: Comparison of two-photon excitation
laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-l. J Microsc 1997, Pt 1:9-20. 4.
Miyawaki A, /Iopis J, Helm R, McCafferty JM, Adams JA, Ikura M, Tsien RY: Fluorescent indicators for Ca 2÷ based on green
fluorescent proteins and calmodulin. Nature 1997, 388:882-887. 5.
Putney JW: Capacitive calcium entry revisited. Ceil Calcium 1990, 11:611-624.
6.
Harrington MA, Thompson SH: Activation of the nitric oxide/cGMP pathway is required for refilling intracellular Ca2+ stores in a sympathetic neuron cell line. Cell Calcium 1996, 19:399-407.
7.
•,
Babcock DF, Herrington J, Goodwin PC, Park YB, Hille B:
Mitochondrial participation in the intracellular Ca 2+ network. J Ceil Bio/1997, 136:833-844.
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Bio-inorganic chemistry
Through careful monitoring of changes in [Ca2+] in rat chromaffin cells using organelle-specific fluorescent Ca 2+ probes, it was demonstrated that even a modest elevation of the [Ca2+]i resulted in rapid Ca2+ uptake by the mitochondria.
27.
Sienaert I, De Smedt H, Parys JB, Missiaen L, Vanlingen S, Sipma H, Casteels R: Characterization of a cytosolic and a luminal Ca2+ binding site in the type I inositol 1,4,5trisphosphate receptor. J Biol Chem 1996, 271:27005-27012.
8.
28.
TunwellRE, Wiokenden C, Bertrand BM, Shevchenko VI, Walsh MB, Allen PD, Lai FA: The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis. Biochem J 1996, 318:477487.
29.
Yoneshima H, Miyawaki A, Michikawa T, Furuichi T, Mikoshiba K: Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors. Bioohem J 1997, 322:591-596.
30.
Brown GR, Kbhler M, Berggren P-O: Parallel changes in nuclear and cytosolic calcium in mouse pancreatic [3-cells. Bioohem J 1997, 1997:771-778.
31.
Perez-TerzicC, Jaconi M, Clapham D: Nuclear calcium and the regulation of the nuclear pore complex. Bioessays 1997, 19:787-792.
32.
AIIbritton NL, Oancea E, Kuhn MA, Meyer T: Source of nuclear calcium signals. Proo Nat/Acad Sci USA 1994, 91:1245812462.
33.
Al-Mohanna FA, Caddy KW, Bolsover SR: The nucleus is insulated from large cytosolic calcium ion changes. Nature 1994, 367:745-750.
Pizzo P, Fasolato C, Pozzan T: Dynamic properties of an inositol 1,4,5-trisphosphate- and thapsigargin-insensitive calcium pool in mammalian cell lines. J Ceil Biol 1997, 136:355-366.
9.
Mikoshiba K: The InsP3 receptor and intracellular Ca2+ signaling. Curr Opln Neurobiol 1997, 7:339-345. A brief summary of biochemical and structural information of the inositoF 1,4,5-trisphosphate receptor covering the period up to the end of 1996. 10.
Yoshida Y, Imai S: Structure and function of inositol 1,4,5trisphosphate receptor. Jpn J Pharmacol 1997, 74:125-137.
11.
Missiaen L, Taylor CW, Berridge MJ: Luminal Ca2+ promoting spontaneous Ca2+ release from inositol trisphosphatesensitive stores in rat hepatocytes. J Physio/(Lond) 1992, 455:623-640.
12.
Sitsapesan R, Williams AJ: Regulation of current flow through ryanodine receptors by luminal Ca 2+. J Membr Bio/1997, 159:179-185.
13.
Lee HC, Aarhus R: A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADPribose. J Bio/Chem 1995, 270:2152-2157.
14.
Genazzani AA, Mezna M, Summerhill RJ, Michelangeli F: Kinetic properties of nicotinic acid adenine dinucleotide phosphateinduced Ca2+ release. J Bio/Chem 1997, 272:7669-7675.
15.
Aarhus R, Dickey DM, Graeff RM, Gee KR, Walseth TF, Lee HC: Activation and inactivation of Ca 2+ release by NAADP+. J Bio/ Chem 1996, 271:8513-8516.
16.
Genazzani AA, Empson RM, Galione A: Unique inactivation properties of NAADP-sensitive Ca 2+ release. J Bio/Chem 1996, 271:11599-11602.
1 "Z
Parker I, Yao Y, ilyin V: Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate. Biophys J 1996, 70:222-237.
Marchant JS, Taylor CW: Cooperative activation of IP3 receptors by sequential binding of IP3 and Ca 2+ safeguards against spontaneous activity. Curr Bio/ 1997, 7:510-518. The study presents evidence that cooperative binding of four IP3 molecules is needed for rat hepatocyte IP3R-type II channels to reach full Ca2+-release activity. Furthermore a time lag of some 30 ms is observed between IP3 binding and Ca2+ release onset.
34.
Humbert JP, Matter N, Artault JC, Koppler P, MaMya AN: Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signalling by inositol 1,4,5-trisphosphate. Discrete distribution of inositol phosphate receptors to inner and outer nuclear membranes. J Biol Chem 1996, 271:478-485. The separation of outer and inner nuclear membranes reveals the prevalence of IP3Rs on the inner nuclear membrane. •
35.
18.
•
19.
Thomas AP, Bird GSJ, Hajnbczky G, Robb-Gaspers LD, Putney JW Jr: Spatial and temporal aspects of cellular calcium signaling. FASEB J 1996, 10:1505-1517.
20. Home JH, Meyer T: Elementary calcium-release units induced •• by inositol trisphosphate. Science 1997, 276:1690-1693. This is a landmark paper where the spatial and temporal characteristics of IP3-induced Ca2+ release was studied in rat leukemia cells using a combination of a slowly diffusing fluorescent Ca2+ chelator, photochemical release of caged IP3 and confocal laser scanning microscopy. 21.
Li XY, Stojilkovic SS, Keizer J, Rind'el J: Sensing and refilling calcium stores in an excitable cell. Biophys J 1997, 72:10801091.
Gerasimenko OV, Gerasimenko iV, Tepikin AV, Petersen OH: ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope. Cell 1995, 80:439-444.
36.
Fox JL, Burgstahler AD, Nathanson MH: Mechanism of long-range Ca 2+ signalling in the nucleus of isolated rat hepatocytes. Biochem J 1997, 326:491-495. High speed line scanning confocal laser microscopy is used to follow the spatio-temporal development of Ca 2+ waves in rat hepatocyte nuclei. •
37.
Means AR, Ribar TJ, Kane CD, Hook S, Anderson KA: Regulation and properties of the rat Ca2+/calmodulin-dependent protein kinase IV gene and its protein products. Recent Prog Horm Res 1997, 52:389-407.
38. ••
Peersen OB, Madsen TS, Falke JJ: Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca2+-binding and implications for kinase activation. Protein Science 1997, 6:794-807. A careful study of Ca2+-binding constants and dissociation rates of calmodulin in complexes with peptides and intact proteins. 39.
Kretsinger RH: EF-hands reach out. Nat Struct Biol 1996, 3:1215.
22.
Laurent M, Claret M: Signal-induced Ca 2+ oscillations through the inositol 1,4,5-trisphosphate-gated Ca 2+ channel: an allosteric model, J Theor Biol 1997, 186:307-326.
40.
23.
Carafoli E: Plasma membrane calcium pump: Structure, function and relationships. Basic Res Cardiol 1997, 92:59-61.
Strynadka NCJ, -lames MNG: Crystal structures of the helixloop-helix calcium-binding proteins. Annu Rev Biochem 1989, 58:951-998.
41.
24.
Allbritton NL, Meyer T, Stryer L: Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 1992, 258:1812-1815.
Falke ,lJ, Drake SK, Hazard AL, Peersen O: Molecular tuning of ion binding to calcium signaling proteins. Q Rev Biophys 1994, 27:219-290.
42.
25.
Mackrill JJ, Challiss RAJ, O'Connell DA, Lai FA, Nahorski SR: Differential expression and regulation of ryanodine receptor and myo-inositol 1,4,5-trisphosphate receptor Ca 2+ release channels in mammalian tissues and cell lines. Biochem J 1997, 327:251-258.
Linse S, Forsen S: Determinants that govern high-affinity calcium binding. Adv Second Messenger Phosphoprotein Res 1995, 30:89-151.
43.
Drake SK, Lee KL, Falke JJ: Tuning the equilibrium ion affinity and selectivity of the EF-hand calcium binding motif: Substitutions at the gateway positions. Biochemistry 1996, 35:6697-6705.
44.
Drake SK, Zimmer MA, Miller CL, Falke JJ: Optimizing the metal binding parameters of an EF-hand-like calcium chelation loop: Coordinating side chains play a more important tuning role than chelation flexibility. Biochemistry 1997, 36:9917-9926.
45.
Drake SK, Zimmer MA, Kundrot C, Falke JJ: Molecular tuning of an EF-hand-like calcium binding loop: Contributions of the
26. •
Katayama E, Funahashi H, Michikawa T, Shiraishi T, Ikemoto T, Iino M, Mikoshiba K: Nativ~ structure and arrangement of inositol-l,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy. EMBQ J 1996, 15:4844-4851. This describes the first electron microscopic picture of a crystal of IPsR on the membrane surface of bovine cerebrellar Purkinje cells. The tetrameric arrangement ol the subunits is clearly demonstrated.
Calcium Events, Malmendal and Forsen
coordinating side chain at loop position 3. J Gen Physiol 1997, 110:173-184. 46.
Wu X, Reid RE: Structure/calcium affinity relationships of site III of calmodulin: Testing the acid pair hypothesis using calmodulin mutants. Biochemistry 1997, 36:8649-8656.
4'7.
George SE, Su Z, Fan D, Wang S, Johnson JD: The fourth EF-hand of calmodulin and its helix-loop-helix components: Impact on calcium binding and enzyme activation. Biochemistry 1996, 35:830'7-8313.
48.
Ohki S, Ikura M, Zhang M: Identification of Mg2+-binding sites and the role of Mg 2+ on target recognition by calmodulin. Biochemistry 1997, 36:4309-4316.
49. •
Andersson M, Malmendal A, Linse S, Ivarsson I, Forsen S, Svensson LA: Structural basis for the negative allostery between Ca 2+- and Mg2+-binding in the intracellular Ca 2+receptor calbindin Dgk. Protein Sci 1997, 6:1139-114'7. Calbindin D9k shows negative allostery between Ca 2+ and Mg 2+ binding, in interesting agreement with the structural differences between the Mg 2+- and the Ca2+-saturated states. 50.
Chazin WJ: Releasing the calcium trigger. Nat Struct Biol 1995, 2:707-710.
51.
Events J, Thulin E, Malmendal A, Forsen S, CarlstrSm G: NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state. Biochemistry 1997, 36:3448-345'7. This paper shows that mutation of the bidentate Ca 2+ ligand Glu140 lowers the Ca2+ affinity significantly and disturbs the structural rearrangements induced by Ca 2+ binding. •
52.
Gagne SM, Li MX, Sykes BD: Mechanism of the direct coupling between binding and induced structural change in regulatory calcium binding proteins. Biochemistry 1997, 36:4386-4392.
53. •
Li MX, Gagn6 SM, Spyracopoulos L, KIcks CPAM, Audette G, Chandra M, Solaro PJ, Smillie LB, Sykes BD: NMR studies of Ca 2+ binding to the regulatory domains of cardiac and E41A skeletal muscle troponin C reveal the importance of site I to energetics of the induced structural changes. Biochemistry 199'7, 36:12519-12525. Cardiac muscle troponin C and the Glu41Ala mutant of skeletal muscle troponin C do not open upon Ca 2+ binding. The critical role of this bidentate ligand in site l is emphasized. 54.
Spyracopoulous L, Li MX, Sia SK, Gagne SM, Chandra M, Solaro RL, Sykes BD: Calcium-induced structural transition in the regulatory domain of human cardiac troponin C. Biochemistry 1997, 36:12138-12146.
301
64.
Stevens-Truss R, Beckingham K, Marietta MA: Calcium binding sites of calmodulin and electron transfer by neuronal nitric oxide synthase. Biochemistry 1997, 36:12337-12345.
65.
Houdusse A, Silver M, Cohen C: A model of Ca2+-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. Structure 1996, 4:1475-
•
1490. A model of Ca2+-free CaM binding to the IQ motif of unconventional myosin is presented. Changes in Ca2+ level is suggested to module the activity through CaM-activation. 66.
Houdusse A, Cohen C: Structure of the regulatory domain of scallop myosin at 2~, resolution: Implications for regulation. Structure 1996, 4:21-32.
67 •
Blanchard H, Grochulski P, Li Y, Arthur JSC, Davies PL, Elce JS, Cyg~er M: Structure of a calpain Ca2+-binding domain reveals a novel EF-hand and Ca2+-induced conformational changes. Nat Struct Biol 199?, 4:532-538. The crystal structure of domain VI of rat m-calpain is determined, both with and without bound Ca2+. A model for subunit dimerization in calpain is presented. 68. •
Lin GD, Chattopadhyay D, Maki M, Wang KKW, Carson M, Jin L, Yuen PW, Takano E, Hatanaka M, DeLucas LJ et aL: Crystal structure of calcium bound domain VI of calpain at 1.9A resolution and its role in enzyme assembly, regulation, and inhibitor binding. Nat Struet Biol 1997, 4:539-547. The crystal structure of Ca2+-bound domain VI of porcine calpain is presented. A model for subunit dimerization in calpain is given. 69.
Kretsinger RH: EF-hands embrace. Nat Struct Biol 1997, 4:514516.
70. ,,
Nalefski EA, Falke JJ: The C2 domain calcium-binding motif: Structural and functional diversity. Protein Sci 1996, 5:23752390. An ambitious compilation of the knowledge so far collected on C2 domains. 71. ••
Essen LO, Perisic O, Cheung R, Katan M, Williams RL: Crystal structure of a mammalian phosphoinositide-specific phospholipase C& Nature 1996, 380:595-602. The structure described here gives important clues to the function of a multidomain protein, phospholipase C8, involved in phospholipid signaling. 72. •
Nalefski EA, Slazas MM, Falke JJ: Ca2+-signaling cycle of a membrane docking C2 domain. Biochemistry 1997, 36:12011 12018. See annotation to [73"].
55.
Shao X, Li C, Fernandez I, Zhang X, Sydhof TC, Rizo J: Synaptotagmin-syntaxin interaction: the C2 domain as a Ca 2+dependent electrostatic switch. Neuron 1997, 18:133-142. These papers [73",74"] describe two nice studies on Ca2+-dependent interactions of two different C2 domains.
56. •
'74. ••
Cello MR, Pauls T, Schwaller B (Eds): Guidebook to the calciumbinding proteins. Oxford: Oxford University Press; 1996. he book presents information on more than eighty Ca2+-binding proteins.
Goldberg J, Nairn AC, Kuriyan J: Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I. Ce//1996, 84:875-887 The paper presents a crystal structure of Ca2+/CaM-dependent protein kinase I in the autoinhibited form.
73.
•
Maurer P, Hohenester E: Structural and functional aspects of calcium binding in extracellular matrix proteins. Matrix Biol 1997, 15:569-580. Comprehensive review about extracellular Ca 2+ binding proteins. '75.
Kishore U, Eggleton P, Reid KD: Modular organization of carbohydrate recognition domains in animal lectins. Matrix Biol 1997, 15:583-592.
57.
Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A: Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 1992, 256:632-638.
58.
Brown SE, Martin SR, Bayley PM: Kinetic control of the dissociation pathway of calmodulin-peptide complexes. J Biol Chem 1997, 272:3389-3397.
59.
Martin SR, Bayley PM, Brown SE, Porumb T, Zhang M, Ikura M: Spectroscopic characterization of a high-affinity calmodulintarget peptide hybride molecule. Biochemistry 1996, 35:35083517.
60.
Chin D, Means AR: Methionine to glutamine substitutions in the C-terminal domain of calmodulin impair the activation of three protein kinases. J Bio/Chem 1996, 271:30465-304'71.
61.
Browne JP, Strom M, Martin SR, Bayley PM: The role of !3-sheet interactions in domain stability, folding, and target recognition reactions of calmodulin. Biochemistry 1997, 36:9550-9561.
78.
Blacklow SC, Kim PS: Protein folding and calcium binding defects arising from familial hypercholesterolemia mutations of the LDL-receptor. Nat Struct Bio11996, 3:758-762.
62.
Haga K, Tsuga H, Haga T: Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin. Biochemistry 1997, 36:1315-1321.
79.
63.
Matsubara M, Hayashi N, Titani K, Taniguchi H: Circular dichroism and 1H NMR studies on the structures of peptides from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and complex with calmodulin. J Biol Chem 199'7, 272:23050-23056.
Sunnerhagen M, Forsen S, Hoffren AM, Drakenberg T, Teleman O, Stenflo J: Structure of the Ca2+-free GLA domain sheds light on membrane binding of blood coagulation proteins. Nat Struct Biol 1995, 6:504-509.
80.
Freedman SJ, BIo.stein MD, Baleja JD, Jacobs M, Furie BC, Furie B: Identification of the phospholipid binding site in the vitamin K-dependent blood coagulation protein factor IX. J Biol Chem 1996, 271:16227-16236.
76. he
Brown MS, Herz .J, Goldstein JL: Calcium cages, acid baths and recycling receptors. Nature 1997, 388:629-630. authors give a pleasant introduction to the low-density lipoprotein receptor. 77. ••
Fass D, Blacklow SC, Kim PS, Berger JM: Molecular basis of familial hypercholesterolemia from structure of LDL-receptor module. Nature 199'7, 388:691-693. The crystal structure of an LDL module is presented, showing that naturally occurring point mutations found in patients with the disease familial hypercholesterolaemia alter residues that directly coordinate Ca 2+ or that serve as scaffolding residues.
302
Bio-inorganic chemistry
81.
Li L, Darden TA, Freedman SJ, Furie BC, Furie B, Baleja JD, Smith H, Hiskey RG, Pedersen LG: Refinement of the NMR solution structure of the gamma-carboxyglutamic acid domain of coagulation factor IX using molecular dynamics simulation with initial Ca 2+ positions determined by a genetic algorithm. Biochemistry 199?, 36:2132-2138.
82.
Handford P, Mayhew M, Baron M, Winship PR, Campbell ID, Brownlee GG: Key residues involved in Ca 2+ binding motifs in EGF-like domains. Nature 1991,351:164-16?.
83.
Stenflo J, Oehlin AK, Persson E, Valcare C, Astermark J, Drakenberg T, Selander M, Bj6rk I: Epidermal growth factorlike domains in the vitamin K-dependent clotting factors. Some structure-function relationships. Ann NY Acad Sci 1991, 614:11-29.
84.
85.
Rao Z, Handford P, Mayhew M, Knott V, Brownlee GG, Stuart D: • • epidermal growth factor-like The structure of a Ca2+-banding domain: its role in protein-protein interactions. Cell 1995, 82:131-141. Dietz HC, Pyeritz RE: Mutations in the human gene for fibrillin-1 (FBN1) in the Marfan syndrome and related disorders. Hum Mol Gen 1995, 4:1 fl99-1809.
86. •
Downing AK, Knott V, Werner JM, Cardy CM, Campbell ID, Handford PA: Solution structure of a pair of calcium-binding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders. Oe//1996, 85:597-605. Two general modes of interaction between consecutive EGF-like domains are proposed here: one with a rodlike arrangement of the domains and another with a kinked orientation. The interaction is in both cases stabilized by a Ca2+ ion. 87 •
Sunnerhagen M, Olah G, Stenflo J, Forsen S, Drakenberg T, Trewhella J: The relative orientation of GLA and EGF domains in coagulation factor X is altered by Ca 2+ binding to the first EGF domain. A combined NMR-small angle X-ray scattering study. Biochemistry 1996, 35:11647-11559. The study shows a Ca2+-stabilized Gla-EGF junction. 88.
Chothia C, Jones EY: The molecular structure of cell adhesion molecules. Annu Rev Biochem 199"7, 66:823-862.
89. •.
Nagar B, Overduin M, Ikura M, Rini JM: Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 1996, 380:360-364. This paper describes an elegant structure determination giving important clues to the mechanism of cell-cell adhesion. 90. =,
Koch AW, Pokutta S, Lustig A, Engel J: Calcium binding and homoassociation of E-cadherin domains. Biochemistry 199'7, 36:7697-??05. The data obtained on the Ca2+-binding characteristics and Ca2+-dependent dimerization of E-cadherin fragments contribute significantly to the understanding of these molecules. 91.
Lewis JE, Jensen PJ, Johnson KR, Wheelock MJ: E-cadherin mediates adherens junction organization through protein kinase C. J Cell Sci 1994, 107:3615-3621.
92.
Shapiro L, Fannon AM, Kwong PD, Thompson A, Lehmann MS, Grebel G, Legrand J-F, AIs-Nielsen J, Colman DR, Hendrickson WA: Structural basis of cell-cell adhesion by cadherins. Nature 1995, 374:32?-337.
93. •
Hohenester E, Maurer P, Hohenadl C, Timpl R, Jansonius JN, Engel J: Structure of a novel extracellular calcium-binding module in BM-40. Nat Struct Bio/1996, 3:67-73. The first extracellular EF hand to be discovered is described. 94.
Brown EM, Vassilev PM, Hebert SC: Calcium ions as extracellular messengers. Cell 1995, 83:679-682.
95.
Gerasimenko OV, Gerasimenko .IV, Petersen OH, Tepikin AV: Calcium transport pathways in the nucleus. Pf/i~gers Arch 1996, 432:1-6.
96.
Santella L: The cell nucleus: an Eldorado to future calcium research? J Membr Bio/1996, 153:83-92.
9?.
Kubinowa H, Tjandra N, Grzesiek S, Ren H, Klee CB, Bax A: Solution structure of calcium-free calmodulin. Nat Struct Biol 1995, 2:?68-7?6.
98.
Chattopadhyaya R, Meador WE, Means AR, Quiocho FA: Calmodulin structure refined at 1.7 &ngstr6ms. J Mol Biol 1992, 228:1177-1192.
99.
FerrinTE, Huang CC, Jarvis LE, Langridge R: The MIDAS display system. J Mol Graphics 1988, 6:13-27.
100.
Santella L, Carafoli E: Calcium signalling in the cell nucleus. FASEB J 1997, 11:1091-1109.
Calcium Johan Even is, Anders Malmendal and Sture Forsen* Ca 2+ is involved in an intriguing variety of different biological events. The rapid development of techniques such as region- or organelle-directed fluorescent probes and laser scanning confocal microscopy for studying cellular biological events at a molecular level provides us with a rich daily intake of new results. While detailed three-dimensional structures of many intracellular and extracellular Ca2+-binding proteins have become available, structural information on key membrane proteins is still lacking. An integrated picture of the molecular events behind the multifunctional roles of Ca 2+ in biological systems is still pending.
Addresses Physical Chemistry 2, Lund University, P.O. Box 124, S-22100 Lund, Sweden *e-mail: [email protected] Current Opinion in Chemical Biology 1998, 2:293-302 http://biomednet.com/elecref/1367593100200293 © Current Biology Ltd ISSN 1367-5931
Abbreviations ATP adenosine triphosphate c-ADPR cyclic ADP ribose CaM calmodulin [Ca2+] i cytoplasmic free Ca2+ concentration [Ca2+] n nuclear free Ca2+ concentration [Ca2+]o extracellular free Ca2+ concentration EGF epidermal growth factor EGTA ethylene glycol-bis(J]-aminoethylester) ER endoplasmic reticulum Gla y carboxy-glutamic acid IP3 inositol-1,4,5-trisphosphate IP3 R IP3 receptor LDL low-density lipoprotein LDLR LDL receptor NAADP nicotinic acid adenine dinucleotide phosphate NMR nuclear magnetic resonance NPC nuclear pore complex RyR ryanodine receptor SR sarcoplasmic retioulum TnC troponin C
Introduction
Ca z+ ions are major players in an intracellular signaling system that translates extracellular stimuli into the regulation of a bewildering number of phenomena such as muscle contraction, neurotransmitter release and other secretion processes, cell proliferation, gene expression and cell death. In order to accomplish such varying feats, Ca 2+ concentration transients must be well defined in both time and space [1°], and the target molecules must be finely tuned for the demands at each location. In addition the target's response may differ depending on the history of the cell [2°]. Our present understanding of the CaZ+ signaling system is limited but has greatly increased during the past few years, on one hand through the
combined use of region- or organelle-directed fluorescent probes and laser scanning confocal microscopy [3,4], and on the other through structural biological studies of the target proteins. Ca 2+ also plays an important role in extracellular environments, being involved in cell-cell interactions and as a prerequisite for tissue repair and for controlling macrostructure and shape through exoskeletons or endoskeletons in higher organisms. Few, if any, ions in biological systems have such an extensive biological role as Ca2+. This is reflected in the number of publications in which calcium is involved in one way or the other. A M E D L I N E search carried out in November 1997 produced close to 24,000 articles published during the past two years alone. Needless to say a brief review like this one can in no way capture the developments in the entire calcium research field--nor would we feel competent to even try. What we have settled for is to highlight a few areas - - intracellular and extracellular--and to take a look at recent developments in structural biology and try to connect these with the biological function of calcium. C a 2+ a n d i n t r a c e l l u l a r
signaling
In eukaryotic cells the cytoplasmic calcium concentration ([Ca2+]i) ranges from 100nM in a 'resting' cell to 1-101aM in an activated cell. Usuall,~; both intracellular and extracellular CaZ+ sources are available for signaling. As the exracellular free CaZ+ concentration ([Ca2+]o) in higher organisms is around 1.2 mM, the extracellular pool may be considered effectively infinitely large. The flow of Ca 2+ can be regulated through voltage-, receptor-, and store-operated channels. The existence of store-operated channels, operationally defined as being able to restore the Ca 2+ levels of depleted intracellular stores [S], has been demonstrated in Xenopus oocytes and, recentl'6 in a sympathetic neuronal cell line [6]. For many years the Ca2+-signaling field has been focused on CaZ+ stores in the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR), where IP3R and RyR were discovered. Recently mitochondria have been shown to be active and important participants in the signaling system [7"°]. In addition to these three stores, there is some evidence pointing to another type of high capacity storage pool in mammalian cells [8]. Two major Ca2+-release channels in the ER and SR are welt established--the inositol-l,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine receptor (RyR). Ca 2+ release by IP3R is induced by IP3, while RyR is regulated by cyclic ADP-ribose (c-ADPR) [9*]. Both receptors are also modulated by the cytoplasmic Ca 2+ concentration ([Ca2+]i), calmodulin (CAM), phosphorylation and possibly a number of other factors, for example caffeine. Low
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[Ca2+]i (10nM-1 btM) acts synergistically with IP 3 while high [Ca2+]i (>3 btM) counteracts the stimulatory effect of IP 3 [9°,10]. The Ca 2+ concentration in the lumen ([CaZ+]o] also seems to regulate the flow of CaZ+ through IP3R and RyR channels [11,12]. There is some evidence for a Ca2+-release channel, which is activated by nicotinic acid adenine dinucleotide phosphate (NAADP) [13,14]. While IP3R and RyR display a graded response towards their activating factors, the NAADP-dependent channel is reported to display an 'all-or-none' type of activation [15,16]. The response of ligand-gated ion channels to agonist stimuli is usually very rapid. It is therefore striking that several studies--using either rapid photolytic release of caged IP?, in intact cells [17] or clamped permeabilized hepatocytes [18°]--give evidence of a latency period of some 30 ms between IP 3 binding and the onset of Cae+ release through the [P3R channel. Unless there is some yet undetected technical snag in these experiments, we may witness the result of a slow conformation change in IP3R--possibly a safeguard against spontaneous firing of the channel [12]. The complexity of cytoplasmic CaZ+ signaling, with a variety of observed CaZ+ oscillations such as localized bursts and global waves, is a consequence of ligand triggered chemical events, regulatory feedback mechanisms and diffusive processes [19]. ER membranes seem to be, at least locally, highly populated with I P 3 R s - - a s are SR membranes with R y R s - - a n d synchronized triggering of initially localized Ca2+ release events, resulting in regenerative CaZ+ waves, may take place [1"]. In an elegant study by Home and Meyer [20 °°] the spatial and temporal characteristics of IP3-induced Ca 2+ release in rat leukemia cells were determined. The existence of individual brief (<3 ms) and spatially localized (<2 btm) release events was observed in the presence of the specific CaZ+ chelator EGTA, preventing the Ca2+ diffusion induced triggering of more distant IP3R channels. In the absence of EGTA, global Ca 2+ release was seen as a consequence of synchronized triggering [1",20°°]. Several theoretical models have been put forward to account for the complex cellular events observed in CaZ+ signaling [21,22]. To reduce [Ca2+]i back to resting levels, cell membranes, ER and SR are provided with ATP-driven Ca2+ pumps, CaATPases [23]. When [Ca2+]i rises, mitochondria also rapidly absorb CaZ+ and then dispense it at a slower pace, permitting CaATPases to restore CaZ+ in ER and SR compartments to former levels without disturbing [CaZ+]i [7"°]. The cytoplasmic level of IP 3 is rapidly reduced by enzymatic degradation and the estimated lifetime of IP 3 in living cells is -1 s. Also important for our understanding of the spatio-temporal properties of the Ca 2+ signaling system are the effective diffusion rates of Ca z+ and IP 3 in the cytoplasm. As shown in Xenopus oocytes, the diffusion of IP 3 is largely independent of [IP3], while the Ca -~+
diffusion rate decreases with decreased [Ca2+]i as is typical for transport of ions in a medium filled with slowly diffusing, ion-buffering molecules [24]. The diffusion rates and the effective lifetimes make the effective action range of IP 3 several tens of microns and thus useful for triggering events at some distance, whereas the action range of Ca -)+ is of the order of a few microns at the most, and more suitable for triggering local events.
Figure 1
CytopLasm
i
@0 tOOt Current Opinion in Chemical Biology
The IP3 receptor. Cross-section of the tetrameric IP3 receptor model in the membrane of an intracellular Ca2+ store. As indicated below, only two monomers are shown. Each monomer contains an IP3-binding domain (dark gray), a regulatory domain (light gray) and six membrane-spanning helices (shown as cylinders) [9°,26°]. Ca 2+ sites, located using fusion proteins, are indicated [27]. C and N indicate the carboxyl and amino termini, respectively, of the protein.
At least three isoforms (type I, II and Ill) of both IP3R and RyR have been characterized and their tissue-specific distribution mapped [9°,Z5]. Functional IP3R and RyR arc tetrameric complexes but no high resolution structure of either receptor is as yet available. A structural model of IP3R has been proposed based on cryoelectron microscopy studies [9",26"] (Figure 1). Each IP3R subunit appears to have three functional d o m a i n s - - a n amino-terminal IP 3binding domain, a regulatory domain with binding sites for CaM and other factors and a membrane-spanning domain. Fusion protein studies have localized CaZ+-binding sites on both the cytoplasmic and luminal domains of mouse IP3R-type I [27]. Neither site is of the EF hand type (see below). A similar domain-like structure has been proposed for human cardiac muscle RyR (type I) [28]. At least
Calcium Events, Malmendal and Fors6n
in the case of IP3 R, the tetramer subunits may belong to different isoforms - - most likely allowing for cell type dependent fine tuning of the functional properties [9",10]. It would appear that, to date, most studies of the functional properties of IP3R release channels have been performed on type I. Recent comparisons of IP 3 affinities of full length type I and type III human IP3Rs indicate, however, that generalizations from one isoform to another may be unreliable [29].
295
This complicates the interpretation of some experiments addressing the question of the interdependence of [CaZ+]i and [CaZ+]n. T h e complex behavior of [CaZ+]n is further illustrated by rat leukemia cells in which [CaZ+]i gradients are transmitted to the nucleus with a time delay of about 200ms [32]. In cultured neuronal cells, small changes in [Cae+]i are faithfully transmitted to the nucleus, while larger changes (>300 nM) were clearly attenuated [33]; this is difficult to understand unless the nuclear pores close at
high [CaZ+]. Ca 2+ in t h e cell nucleus After many false starts due, to some degree, to the difficulties of localizing [CaZ+]-reporting probes to the nucleus, evidence is mounting for a signaling role of CaZ+ in the nucleus (L Santella, E Carafoli, personal communication; see Note added in proof.) As in the case of cytoplasmic studies, new organdie-directed fluorescent probes and laser scanning confocal microscopy have been of great importance. A schematic picture illustrating the main features of the eukauotic cell nucleus is provided in Figure 2a. Ca 2+ and small proteins (of molecular weight <20 kDa) have been observed to diffuse relatively freely through the nuclear pore complex (NPC) [30]. Recent studies, however, indicate that pore opening is regulated by the
nuclear CaZ+ concentration, [Cae+] n, and/or [CaZ+]i [31].
The discovery of IP3R on the inner nuclear membrane [34"] suggests that findings interpreted as Cae+ diffusion from the cytoplasm might instead have been an IP 3triggered event. This idea is supported by the finding of IP 3 and c-ADPR triggered release of Ca2+ from the Xenopus nuclear envelope space to the nuclear space [35]. Ca 2+ transport pathways in the nuclear envelope are shown in Figure 2b. In the nuclear space, in contrast to the cytoplasm, Cae+ propagation seems to occur through simple diffusion since no Ca2+-induced Cae+ release has been established [36"]. T h e absence in the nucleus of powerful pumping mechanisms to reduce [Ca2+]n results in a larger effective life time and range of action of Ca2+ in the nucleus than in the cytoplasm. This may be critical for the many gene transcription processes dependent on the prolonged presence of CaM-activated proteins, such as CaM-dependent protein kinase IV [37], which presumably
Figure 2
(a)
NPC
ER
(b) Ca 2+
Cytoplasm INM
N
~
~
.""
K+ ATP
I11.
Ca 2+
NES
"" " "" "" "" " . , . ""
c ~ A ' ~ D pR
Pa Nuclear space
Current Opinion in Chemical Biology
The cell nucleus. (a) Components of the nucleus and its environment. NS, nuclear space; INM, inner nuclear membrane; ONM, outer nuclear membrane; NES, nuclear envelope space; NPC, nuclear pore complexes; ER, endoplasmic reticulum. (b) is an expanded version of (a). Ca2+ transport pathways in the ONM and the INM of the nucleus [95]. The ONM is continuous with the ER and is rich in ATP-driven Ca 2+ pumps that pump Ca 2+ into the NES. In contrast, the INM, although connected with the ONM at NPCs, has properties quite distinct from those of the ER membrane [31,96].
296
Bio-inorganicchemistry
require [Ca2+] n o f - l l a M Ca2+-bound state [38"].
to keep CaM in the active
Intracellular Ca2+-binding proteins Prime targets for Ca 2+ released in a cell are Ca2+-binding proteins, many of which are players in intracellular regulatory processes and come in many sizes and shapes. Here, we will restrict the discussion to two major types: proteins with the EF-hand motif and those with the C2 domain. EF hand proteins
T h e ubiquitous EF hand CaZ+-binding motif appears in many proteins that exert Ca2+-dependent actions in the nucleus or the cytoplasm. This motif exhibits helix-loop-helix architecture, and is normally paired with another EF hand motif with the two Ca2+-binding loops coupled through a short antiparallel 13 sheet [39,40]. An example of EF hand Ca2+ coordination is given in Figure 3a. The binding of two Ca 2+ ions within an EF hand pair is usually highly cooperative. EF hand proteins cover a wide range of Ca 2+ affinities (Kd = 10-9-10 -5 M). Although numerous studies have been performed during the past fifteen years (reviewed in [41,42]), questions about the molecular parameters tuning the affinity, specificity and cooperativity still remain. To aw)id the complexity inferred by paired EF hands, Esc/let~ice coli galactose-binding protein, which has one EF hand, has been used as a basis for mutational studies aimed to elucidate the contributions of residues in different loop positions [43-45]. The impact of 'acid pairs' [46], that is, two charged amino acid residues in antipodal positions in the coordinating sphere, as well as the impact of the context of the two surrounding helices on Ca 2+ affinity have been demonstrated [47]. Intracellular Cae+-binding proteins in higher organisms operate in an environment with a 100-f0,000-fold excess of Mg 2+. T h e issue of the impact of Mg2+ on Ca 2+ and target peptide binding has been raised [48]. Evidence of negative binding cooperativity between MgZ+ and CaZ+ has been found for the Cae+-buffering protein calbindin D9k [49"]. T h e regulatory proteins troponin C (TnC), CaM and a number of homologous proteins consist of two independently folded domains connected by a flexible helical segment. Each domain contains one EF hand pair. Upon Cae+ binding, rearrangements of the helices occur, resulting in exposure of hydrophobic patches, which are poised for binding target proteins (Figure 3); [50] and references therein). Little is known about the molecular mechanism of these conformational changes. Mutational studies on CaM [51"] and T n C [52,53"] have suggested a critical role of a bidentate glutamic acid in this process (Figure 3b,c). CaZ+-saturated cardiac muscle TnC, with only one functional Ca2+-binding site in the
amino-terminal domain, exposes a smaller hydrophobic surface than that observed in CaM and skeletal muscle T n C [53",54}. CaM-(Ca2+)4 interacts with more than 100 different target proteins [55"]. Typically CaM binds to an autoinhibitory peptide segment of the target enzyme, target peptides [56"]. T h e first structure of a complex of CaM and an isolated target peptide was reported in 1992 [57], showing the peptide adopting an amphiphilic helical conformation and effectively enclosed by the two CaM domains. Since then, a deeper understanding has evolved. In CaM complexes with isolated target peptides, the Ca 2+ affinit'/ of CaM is increased 10-1000-fold due to slower off-rates, particularly in the lower affinity amino-terminal domain [38"',58,59]. This increase in affinity may be explained by the higher stability of the calcium saturated state when a target peptide is bound. In contrast, the interactions between both CaM and target peptide with the rest of the protein may result in considerably smaller increases in Cae+ affinity [38"]. Recent studies of target enzyme activation by CaM involve several kinases [60,61], G-protein-coupled kinase receptor 2 [62] and nitric oxide synthase [63,64]. Activation of nitric oxide synthase is required for refilling Ca2* stores [6]. A special sequence in neuromodulin and unconventional muscle myosins, called the 'IQ motif', has been shown to bind Ca2+-frcc CaM. At higher Ca2+ levels, the activity of these molecules is regulated through changes in the mode of interaction with CaM [65",66]. Calpain is a ubiquitous Ca2+-regulated cysteine protease and a heterodimer of two subunits. T h e smaller st, bunit has two domains (dE dVI) and the larger has four domains (dI-dlV), with the active site residues located within domain dlI. Crystal structures of dVI reveal five EF hands (EFI-EF5) sequentially arranged in two pairs plus a singlc EF hand (EFS), which pairs up with a corresponding EF5 of another dVI-domain-containing molecule, to form a homodimer [67",68",69]. This suggests, together with strong homology between dVI and dlV from the large subunit, a naturally occurring interdomain EF hand pair, responsible for the in vi~'o heterodimerization of the two subunits. Only three EF hands (EF1-EF3) of dVI bind Ca ?.+ at physiological intracellular Ca 2+ levels. EF2 and EF3 are canonical EF-hands, while EF1 exhibits a coordination pattern not seen before in an EF hand pair. Upon Cae+-binding, CaM-like structural changes in the first EF hand pair seem to enable proteolytic activit>
The C2 domain The C2 domain reviewed in [70"] is an eight-stranded [~ sandwich Ca2+-binding protein module, first observed in protein kinase C, a protein involved in phospholipid signaling, and subsequently in a wide range of proteins. The crystal structure of phospholipase C8 reveals a C2 domain coupled to a CaM-like domain and a catal'y-tic TIM-barrel structural motif [71"]. C2 domains interact
Calcium Even~.s, Malmendal and Fors6n
Figure 3
297
with small molecules, proteins and cellular membranes, sometimes in a CaZ+-dependent manner. Ca2+-dependent C2 domain participation in two different processes was recently reported: first, binding of the C2 domain in cytoplasmic phospholipase As to phosphatidylcholine vesicles [72°], which is a step in the inflammatory response of the cell; second, binding of the first C2 domain of the synaptic vesicle protein synaptotagmin to the plasma membrane protein syntaxin [73"], which a step in Ca2+-triggered synaptic vesicle exocytosis. In both cases binding of two Ca2+ ions is important for the interaction, and the substrates are suggested to provide Ca 2+ ligands.
(a)
Asp22
Extracellular
(b)
lix B
C a 2+
Besides its central role in regulating intracellular processes, CaZ+ also plays an important role in extracellular processes, such as bone formation, cell adhesion, and blood clotting, and in receptor structures. In mammals the extracellular free CaZ+ concentration, [CaZ+]o, is tightly regulated at -1.2 raM. Many extracellular CaZ+-binding proteins bind Ca 2+ so strongly that they will be essentially saturated with Ca2+ when they arrive at the extracellular space. A characteristic of extracellular proteins is their multidomain nature with numerous independently folded, but often interacting, modules [74°']. The major function of CaZ+ binding to extracellular proteins was long thought to be enhancement of the thermal stability of these proteins and improvement of their resistance to proteases. During the past few years, new crystal structures and NMR-based solution structures have provided a revised and more complex picture. Ca2+ binds either to single domains, or to the interface between domains mediating domain-domain interactions. Ca 2+ may also play a direct role in ligand interaction, as in the C-type lectins (carbohydrate-recognition proteins that depend on [Ca2+]o for their activity) [75], or in membrane association, as in the Gla-module (see below). More commonly Cae+ binding is crucial for inducing or stabilizing protein conformations appropriate for enzymatic activity and/or ligand binding.
Current Opinion in Chemical Biology
Ca 2+ binding to an EF hand motif, and the Ca2+-induced structural changes exemplified by the amino-terminal EF hand of calmodulin. (a) Ca 2+ is coordinated by seven ligands, provided either directly by the protein or indirectly via a water molecule. The overall geometry of the Ca 2+ coordination can be described as a pentagonal bipyramid. (b) The apo state, that is, with no calcium bound [97]. The backbone trace of amino acid residues 5 - 4 0 and the sidechain of the bidentate Glu31 (glutamic acid, residue 31 in the protein) are shown, with the two ligating carboxylate oxygens colored black. (c) The Ca2+-saturated state [98]. To allow for Ca 2+ ligation, the helices (A and B) rearrange to a more perpendicular orientation, thereby opening up a cleft between the helices, with many hydrophobic sidechains exposed. The molecular graphics in this paper were generated using Midas Plus [99], University of California at San Francisco software.
An example of a Cae+-stabilized protein structure is the ligand-binding module of the low-density lipoprotein (LDL) receptor (LDLR) [76"]. This module consists of approximately 40 amino acid residues. T h e mammalian form of L D L R consists of an extracellular domain formed by seven characteristic L D L modules and one E G F precursor domain (see below), one transmembrane helix and a cytoplasmic domain. L D L R is responsible for the uptake of cholesterol-containing lipoprotein particles. In this process, 10 minutes in total, the lipoprotein is bound, transported to an intracellular acidic endosome and discharged from the receptor; the 'empty' receptor is then transported to the surface. Exposed to a multitude of acid baths in the endosome, extreme protein stability is required. Recently, the crystal structure of the fifth ligand-binding module of the L D L receptor was published
298
Bio-inorganic chemistry
[76",77"]. In addition to confirming three cysteine bridges in this module, the study reveals a significant structural role of a CaZ+ ion, octahedrally coordinated by acidic sidechains. This acidic site was previously thought to bind the basic lipoproteins, an interaction that is now assumed to take place at another site of the module and to be primarily of hydrophobic character. Naturally occurring point mutations, resulting in impaired CaZ+ binding, cause pathologically elevated blood cholesterol levels and premature heart disease [77"',78]. Another CaZ+-binding domain is the small Gla module found as the amino-terminal domain of proteins involved in the blood coagulation cascade. T h e module contains 10-12 y-carboxy-glutamic acid (Gla) residues and binds 6-12 CaZ+ ions with low affinity (Kd -10-3M). Upon Ca z+ binding, the Gla module interacts with phosphatidylserine-rich phospholipid membranes; it has also been suggested that it is involved in direct protein interactions. NMR-derived structures show that Ca 2+ binding to the Gla module induces a sizeable conformational change in the Gla module, resulting in exposure of hydrophobic residues; these hydrophobic residues are largely responsible for the interaction with the membranes [79-81 ]. Epidermal growth factor (EGF)-Iike domains are small but widely distributed modules found in functionally diverse extracellular proteins [74"]. One subfamily of these domains contains a bipartite consensus sequence indicative of a Caa+-binding site [82,83]. In several proteins, point mutations in the genes coding for Cae+-
binding EGF-like domains have been shown to cause human disorders; for example bleeding disorders arc caused by mutations of Ca2+-binding residues in the blood coagulation proteins factor IX and protein S [841. Another example is the Marfan syndrome and other related disorders that are caused by mutations in the major building blocks of extracellular microfibrils, fibrillins 1 and 2 [85]. The solution structure of a pair of EGF-Iike domains of fibrillin-1 reveals that these domains arc arranged in a rigid, rod-like structure that is stabilized by Ca2+ and hydrophobic interactions (Figure 4a) [86"1. The Marfan syndrome is linked to mutations that dcstroy Cae+-binding capability and, therefore, the interdomain interactions. T h e importance of CaZ+ binding for the relative orientations of domains is also demonstrated in studies of the two amino-terminal domains of blood coagulation factor X - - a G l a - E G F p a i r - - w h e r e [Ca2+l could be adjusted so that only the Ca2+-binding site in the EGF domain was populated (Figure 4b) [87"]. A more complex role for extracellular Ca 2+ has been %und in the cadherin family of cell-cell adhesion proteins. This family of proteins usually consists of five homologous extracellular domains, a single membrane-spanning segment and a cytoplasmic region [74",88]. T h e crystal structure of domains 1 and 2 of epithelial (E) cadhcrin shows that it forms parallel directs with the dimer interface found between the two domain junctions where six Ca 2+ ions arc located (Figure 5a) [89"]. Small [Cae+] o fluctuations modulate the number of ions bound and thus the relative orientation of the domains, dimer formation and cell adhesion [89"°,90"*,91]. In addition, each E-cadherin
Figure 4 (a)
(b)
O
,,[
- - : ..... N
EGF c
Current Opinion in Chemical Biology
Examples of Ca2+-stabilized domain interactions. Ca 2+ ions are colored black. (a) NMR-derived structure of the dimer of EGF-like domains 32 (light gray) and 33 (dark gray) of fibfillin-1 [86"]. (b) NMR-derived structure of the dimer of the Gla domain (light gray) and EGF domain 1 (dark gray) of factor X [8?'], The location of Ca 2+ in (b) is an approximation, since the Ca 2+ was not included in the structure calculations.
Calcium Even#& Malmendal and Fors6n
molecule binds six Ca 2+ ions strongly to the remaining domain junctions. These Ca2+ ions are likely to be important for the rod-like arrangement of these domains, resulting in the formation of V-shaped dimers, as indicated by Figure 5b [90"]. In the crystal structure of domain 1 from neural (N) cadherin, the crystal packing provides a model for the interaction of cadherin dimers with cadherin dimers from adjacent cells through antiparallel dimer interactions of each domain 1 with a domain 1 from an adjacent cell cadherin [92], also shown in Figure 5b. Figure 5
299
Conclusion Through the combined use of biochemical, molecular biological and biophysical methods considerable advances have been made to unravel the complex mechanisms behind the Ca2+-dependent signaling systems in eukaryotic cells. The field is, however, presently still in a fairly descriptive state. Detailed structural data on key receptors, ion channels and pumps are lacking. Regulation of Ca 2+ levels in the nucleus is poorly understood. Furthermore, generalizations are made difficult by the individualistic nature of cells from different organisms or with different functions within any given organism. New structural and biophysical studies of CaZ+-binding proteins have provided important information on the roles of Cae+ at a molecular level. In particular, studies of extracellular proteins have revealed an unexpected variety of CaZ+-binding sites and affinities, indicating new, and perhaps not yet imagined, CaZ+ functions. Much remains, to be done, however, to establish links between structure, Ca z+ levels and biological function.
(b)~
Note added in proof The paper referred to in the text as (L Santella, E Carafoli, personal communication) has now been published [100].
Acknowledgements We thank Michael J Bcrridgc, Thomas Grundstr6m and Stcn Orrcnius for helpful discussions and Pctra Nybcrg for critical reading of the manuscript. Cell surface
l __
CurrentOpinioninChemicalBiology
Cadherins and cell adhesion. (a) Ribbon representation of the crystal structure of the parallel homodimer of domains 1 (top) and 2 of E-cadherin [89"°]. Monomers are colored light and dark gray, respectively, and the calcium ions are black. (b) A model of the extracellular parts of cadherins involved in cell adhesion [89°°]. Cadherin dimers from adjacent cells interact to form a zipper like structure. Individual domains are represented by cylinders numbered from amino to carboxyl terminus. Plausible Ca 2+ positions are indicated by gray dots.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • =.
1.
of special interest of outstanding interest
BerridgeM.J: Elementary and global aspects of calcium signalling. J Physio/(Lond) 1997, 499:290-306.
Avery readable overview by one of the pioneers of the Ca 2+ signalling field, with an integration of a vast amount of independent experimental data into a coherent model. 2.
•
Dolmetsch RE, Lewis RS, Goodnow CC, Healy Jl: Differential activation of transcription factors induced by Ca 2+ response amplitude and duration. Nature 1997, 386:855-858.
This paper demonstrates that differential gene transcription in B lymphocytes is regulated by, or at least connected with, the level and time course of cytoplasmic Ca 2+ concentrations.
The first identified extracellular EF hand pair was recently observed in BM-40, a small secreted glycoprotein, presumed to take part in bone mineralization, tissue remodeling and cell growth [93°]. One canonical EF hand pair is located in the extracellular CaZ+-binding (EC) domain, which is the second of three domains. This domain has a structure similar to that of a Cae+-loaded CaM domain with an amino-terminal segment taking the place of the CaM target peptide. The observation of EF hands in extracellular proteins raises the question as to whether Ca 2+ could in fact have a role as an cxtracellular messenger. This idea goes against the notion of a constant [Cae+] o, but the local concentration may well vary with time under certain conditions [94].
3.
Sako Y, Sekihata A, Yanagisawa Y, Yamamoto M, Shimada Y, Ozaki K, Kusumi A: Comparison of two-photon excitation
laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-l. J Microsc 1997, Pt 1:9-20. 4.
Miyawaki A, /Iopis J, Helm R, McCafferty JM, Adams JA, Ikura M, Tsien RY: Fluorescent indicators for Ca 2÷ based on green
fluorescent proteins and calmodulin. Nature 1997, 388:882-887. 5.
Putney JW: Capacitive calcium entry revisited. Ceil Calcium 1990, 11:611-624.
6.
Harrington MA, Thompson SH: Activation of the nitric oxide/cGMP pathway is required for refilling intracellular Ca2+ stores in a sympathetic neuron cell line. Cell Calcium 1996, 19:399-407.
7.
•,
Babcock DF, Herrington J, Goodwin PC, Park YB, Hille B:
Mitochondrial participation in the intracellular Ca 2+ network. J Ceil Bio/1997, 136:833-844.
300
Bio-inorganic chemistry
Through careful monitoring of changes in [Ca2+] in rat chromaffin cells using organelle-specific fluorescent Ca 2+ probes, it was demonstrated that even a modest elevation of the [Ca2+]i resulted in rapid Ca2+ uptake by the mitochondria.
27.
Sienaert I, De Smedt H, Parys JB, Missiaen L, Vanlingen S, Sipma H, Casteels R: Characterization of a cytosolic and a luminal Ca2+ binding site in the type I inositol 1,4,5trisphosphate receptor. J Biol Chem 1996, 271:27005-27012.
8.
28.
TunwellRE, Wiokenden C, Bertrand BM, Shevchenko VI, Walsh MB, Allen PD, Lai FA: The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis. Biochem J 1996, 318:477487.
29.
Yoneshima H, Miyawaki A, Michikawa T, Furuichi T, Mikoshiba K: Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors. Bioohem J 1997, 322:591-596.
30.
Brown GR, Kbhler M, Berggren P-O: Parallel changes in nuclear and cytosolic calcium in mouse pancreatic [3-cells. Bioohem J 1997, 1997:771-778.
31.
Perez-TerzicC, Jaconi M, Clapham D: Nuclear calcium and the regulation of the nuclear pore complex. Bioessays 1997, 19:787-792.
32.
AIIbritton NL, Oancea E, Kuhn MA, Meyer T: Source of nuclear calcium signals. Proo Nat/Acad Sci USA 1994, 91:1245812462.
33.
Al-Mohanna FA, Caddy KW, Bolsover SR: The nucleus is insulated from large cytosolic calcium ion changes. Nature 1994, 367:745-750.
Pizzo P, Fasolato C, Pozzan T: Dynamic properties of an inositol 1,4,5-trisphosphate- and thapsigargin-insensitive calcium pool in mammalian cell lines. J Ceil Biol 1997, 136:355-366.
9.
Mikoshiba K: The InsP3 receptor and intracellular Ca2+ signaling. Curr Opln Neurobiol 1997, 7:339-345. A brief summary of biochemical and structural information of the inositoF 1,4,5-trisphosphate receptor covering the period up to the end of 1996. 10.
Yoshida Y, Imai S: Structure and function of inositol 1,4,5trisphosphate receptor. Jpn J Pharmacol 1997, 74:125-137.
11.
Missiaen L, Taylor CW, Berridge MJ: Luminal Ca2+ promoting spontaneous Ca2+ release from inositol trisphosphatesensitive stores in rat hepatocytes. J Physio/(Lond) 1992, 455:623-640.
12.
Sitsapesan R, Williams AJ: Regulation of current flow through ryanodine receptors by luminal Ca 2+. J Membr Bio/1997, 159:179-185.
13.
Lee HC, Aarhus R: A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADPribose. J Bio/Chem 1995, 270:2152-2157.
14.
Genazzani AA, Mezna M, Summerhill RJ, Michelangeli F: Kinetic properties of nicotinic acid adenine dinucleotide phosphateinduced Ca2+ release. J Bio/Chem 1997, 272:7669-7675.
15.
Aarhus R, Dickey DM, Graeff RM, Gee KR, Walseth TF, Lee HC: Activation and inactivation of Ca 2+ release by NAADP+. J Bio/ Chem 1996, 271:8513-8516.
16.
Genazzani AA, Empson RM, Galione A: Unique inactivation properties of NAADP-sensitive Ca 2+ release. J Bio/Chem 1996, 271:11599-11602.
1 "Z
Parker I, Yao Y, ilyin V: Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate. Biophys J 1996, 70:222-237.
Marchant JS, Taylor CW: Cooperative activation of IP3 receptors by sequential binding of IP3 and Ca 2+ safeguards against spontaneous activity. Curr Bio/ 1997, 7:510-518. The study presents evidence that cooperative binding of four IP3 molecules is needed for rat hepatocyte IP3R-type II channels to reach full Ca2+-release activity. Furthermore a time lag of some 30 ms is observed between IP3 binding and Ca2+ release onset.
34.
Humbert JP, Matter N, Artault JC, Koppler P, MaMya AN: Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signalling by inositol 1,4,5-trisphosphate. Discrete distribution of inositol phosphate receptors to inner and outer nuclear membranes. J Biol Chem 1996, 271:478-485. The separation of outer and inner nuclear membranes reveals the prevalence of IP3Rs on the inner nuclear membrane. •
35.
18.
•
19.
Thomas AP, Bird GSJ, Hajnbczky G, Robb-Gaspers LD, Putney JW Jr: Spatial and temporal aspects of cellular calcium signaling. FASEB J 1996, 10:1505-1517.
20. Home JH, Meyer T: Elementary calcium-release units induced •• by inositol trisphosphate. Science 1997, 276:1690-1693. This is a landmark paper where the spatial and temporal characteristics of IP3-induced Ca2+ release was studied in rat leukemia cells using a combination of a slowly diffusing fluorescent Ca2+ chelator, photochemical release of caged IP3 and confocal laser scanning microscopy. 21.
Li XY, Stojilkovic SS, Keizer J, Rind'el J: Sensing and refilling calcium stores in an excitable cell. Biophys J 1997, 72:10801091.
Gerasimenko OV, Gerasimenko iV, Tepikin AV, Petersen OH: ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope. Cell 1995, 80:439-444.
36.
Fox JL, Burgstahler AD, Nathanson MH: Mechanism of long-range Ca 2+ signalling in the nucleus of isolated rat hepatocytes. Biochem J 1997, 326:491-495. High speed line scanning confocal laser microscopy is used to follow the spatio-temporal development of Ca 2+ waves in rat hepatocyte nuclei. •
37.
Means AR, Ribar TJ, Kane CD, Hook S, Anderson KA: Regulation and properties of the rat Ca2+/calmodulin-dependent protein kinase IV gene and its protein products. Recent Prog Horm Res 1997, 52:389-407.
38. ••
Peersen OB, Madsen TS, Falke JJ: Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca2+-binding and implications for kinase activation. Protein Science 1997, 6:794-807. A careful study of Ca2+-binding constants and dissociation rates of calmodulin in complexes with peptides and intact proteins. 39.
Kretsinger RH: EF-hands reach out. Nat Struct Biol 1996, 3:1215.
22.
Laurent M, Claret M: Signal-induced Ca 2+ oscillations through the inositol 1,4,5-trisphosphate-gated Ca 2+ channel: an allosteric model, J Theor Biol 1997, 186:307-326.
40.
23.
Carafoli E: Plasma membrane calcium pump: Structure, function and relationships. Basic Res Cardiol 1997, 92:59-61.
Strynadka NCJ, -lames MNG: Crystal structures of the helixloop-helix calcium-binding proteins. Annu Rev Biochem 1989, 58:951-998.
41.
24.
Allbritton NL, Meyer T, Stryer L: Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 1992, 258:1812-1815.
Falke ,lJ, Drake SK, Hazard AL, Peersen O: Molecular tuning of ion binding to calcium signaling proteins. Q Rev Biophys 1994, 27:219-290.
42.
25.
Mackrill JJ, Challiss RAJ, O'Connell DA, Lai FA, Nahorski SR: Differential expression and regulation of ryanodine receptor and myo-inositol 1,4,5-trisphosphate receptor Ca 2+ release channels in mammalian tissues and cell lines. Biochem J 1997, 327:251-258.
Linse S, Forsen S: Determinants that govern high-affinity calcium binding. Adv Second Messenger Phosphoprotein Res 1995, 30:89-151.
43.
Drake SK, Lee KL, Falke JJ: Tuning the equilibrium ion affinity and selectivity of the EF-hand calcium binding motif: Substitutions at the gateway positions. Biochemistry 1996, 35:6697-6705.
44.
Drake SK, Zimmer MA, Miller CL, Falke JJ: Optimizing the metal binding parameters of an EF-hand-like calcium chelation loop: Coordinating side chains play a more important tuning role than chelation flexibility. Biochemistry 1997, 36:9917-9926.
45.
Drake SK, Zimmer MA, Kundrot C, Falke JJ: Molecular tuning of an EF-hand-like calcium binding loop: Contributions of the
26. •
Katayama E, Funahashi H, Michikawa T, Shiraishi T, Ikemoto T, Iino M, Mikoshiba K: Nativ~ structure and arrangement of inositol-l,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy. EMBQ J 1996, 15:4844-4851. This describes the first electron microscopic picture of a crystal of IPsR on the membrane surface of bovine cerebrellar Purkinje cells. The tetrameric arrangement ol the subunits is clearly demonstrated.
Calcium Events, Malmendal and Forsen
coordinating side chain at loop position 3. J Gen Physiol 1997, 110:173-184. 46.
Wu X, Reid RE: Structure/calcium affinity relationships of site III of calmodulin: Testing the acid pair hypothesis using calmodulin mutants. Biochemistry 1997, 36:8649-8656.
4'7.
George SE, Su Z, Fan D, Wang S, Johnson JD: The fourth EF-hand of calmodulin and its helix-loop-helix components: Impact on calcium binding and enzyme activation. Biochemistry 1996, 35:830'7-8313.
48.
Ohki S, Ikura M, Zhang M: Identification of Mg2+-binding sites and the role of Mg 2+ on target recognition by calmodulin. Biochemistry 1997, 36:4309-4316.
49. •
Andersson M, Malmendal A, Linse S, Ivarsson I, Forsen S, Svensson LA: Structural basis for the negative allostery between Ca 2+- and Mg2+-binding in the intracellular Ca 2+receptor calbindin Dgk. Protein Sci 1997, 6:1139-114'7. Calbindin D9k shows negative allostery between Ca 2+ and Mg 2+ binding, in interesting agreement with the structural differences between the Mg 2+- and the Ca2+-saturated states. 50.
Chazin WJ: Releasing the calcium trigger. Nat Struct Biol 1995, 2:707-710.
51.
Events J, Thulin E, Malmendal A, Forsen S, CarlstrSm G: NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state. Biochemistry 1997, 36:3448-345'7. This paper shows that mutation of the bidentate Ca 2+ ligand Glu140 lowers the Ca2+ affinity significantly and disturbs the structural rearrangements induced by Ca 2+ binding. •
52.
Gagne SM, Li MX, Sykes BD: Mechanism of the direct coupling between binding and induced structural change in regulatory calcium binding proteins. Biochemistry 1997, 36:4386-4392.
53. •
Li MX, Gagn6 SM, Spyracopoulos L, KIcks CPAM, Audette G, Chandra M, Solaro PJ, Smillie LB, Sykes BD: NMR studies of Ca 2+ binding to the regulatory domains of cardiac and E41A skeletal muscle troponin C reveal the importance of site I to energetics of the induced structural changes. Biochemistry 199'7, 36:12519-12525. Cardiac muscle troponin C and the Glu41Ala mutant of skeletal muscle troponin C do not open upon Ca 2+ binding. The critical role of this bidentate ligand in site l is emphasized. 54.
Spyracopoulous L, Li MX, Sia SK, Gagne SM, Chandra M, Solaro RL, Sykes BD: Calcium-induced structural transition in the regulatory domain of human cardiac troponin C. Biochemistry 1997, 36:12138-12146.
301
64.
Stevens-Truss R, Beckingham K, Marietta MA: Calcium binding sites of calmodulin and electron transfer by neuronal nitric oxide synthase. Biochemistry 1997, 36:12337-12345.
65.
Houdusse A, Silver M, Cohen C: A model of Ca2+-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. Structure 1996, 4:1475-
•
1490. A model of Ca2+-free CaM binding to the IQ motif of unconventional myosin is presented. Changes in Ca2+ level is suggested to module the activity through CaM-activation. 66.
Houdusse A, Cohen C: Structure of the regulatory domain of scallop myosin at 2~, resolution: Implications for regulation. Structure 1996, 4:21-32.
67 •
Blanchard H, Grochulski P, Li Y, Arthur JSC, Davies PL, Elce JS, Cyg~er M: Structure of a calpain Ca2+-binding domain reveals a novel EF-hand and Ca2+-induced conformational changes. Nat Struct Biol 199?, 4:532-538. The crystal structure of domain VI of rat m-calpain is determined, both with and without bound Ca2+. A model for subunit dimerization in calpain is presented. 68. •
Lin GD, Chattopadhyay D, Maki M, Wang KKW, Carson M, Jin L, Yuen PW, Takano E, Hatanaka M, DeLucas LJ et aL: Crystal structure of calcium bound domain VI of calpain at 1.9A resolution and its role in enzyme assembly, regulation, and inhibitor binding. Nat Struet Biol 1997, 4:539-547. The crystal structure of Ca2+-bound domain VI of porcine calpain is presented. A model for subunit dimerization in calpain is given. 69.
Kretsinger RH: EF-hands embrace. Nat Struct Biol 1997, 4:514516.
70. ,,
Nalefski EA, Falke JJ: The C2 domain calcium-binding motif: Structural and functional diversity. Protein Sci 1996, 5:23752390. An ambitious compilation of the knowledge so far collected on C2 domains. 71. ••
Essen LO, Perisic O, Cheung R, Katan M, Williams RL: Crystal structure of a mammalian phosphoinositide-specific phospholipase C& Nature 1996, 380:595-602. The structure described here gives important clues to the function of a multidomain protein, phospholipase C8, involved in phospholipid signaling. 72. •
Nalefski EA, Slazas MM, Falke JJ: Ca2+-signaling cycle of a membrane docking C2 domain. Biochemistry 1997, 36:12011 12018. See annotation to [73"].
55.
Shao X, Li C, Fernandez I, Zhang X, Sydhof TC, Rizo J: Synaptotagmin-syntaxin interaction: the C2 domain as a Ca 2+dependent electrostatic switch. Neuron 1997, 18:133-142. These papers [73",74"] describe two nice studies on Ca2+-dependent interactions of two different C2 domains.
56. •
'74. ••
Cello MR, Pauls T, Schwaller B (Eds): Guidebook to the calciumbinding proteins. Oxford: Oxford University Press; 1996. he book presents information on more than eighty Ca2+-binding proteins.
Goldberg J, Nairn AC, Kuriyan J: Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I. Ce//1996, 84:875-887 The paper presents a crystal structure of Ca2+/CaM-dependent protein kinase I in the autoinhibited form.
73.
•
Maurer P, Hohenester E: Structural and functional aspects of calcium binding in extracellular matrix proteins. Matrix Biol 1997, 15:569-580. Comprehensive review about extracellular Ca 2+ binding proteins. '75.
Kishore U, Eggleton P, Reid KD: Modular organization of carbohydrate recognition domains in animal lectins. Matrix Biol 1997, 15:583-592.
57.
Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A: Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 1992, 256:632-638.
58.
Brown SE, Martin SR, Bayley PM: Kinetic control of the dissociation pathway of calmodulin-peptide complexes. J Biol Chem 1997, 272:3389-3397.
59.
Martin SR, Bayley PM, Brown SE, Porumb T, Zhang M, Ikura M: Spectroscopic characterization of a high-affinity calmodulintarget peptide hybride molecule. Biochemistry 1996, 35:35083517.
60.
Chin D, Means AR: Methionine to glutamine substitutions in the C-terminal domain of calmodulin impair the activation of three protein kinases. J Bio/Chem 1996, 271:30465-304'71.
61.
Browne JP, Strom M, Martin SR, Bayley PM: The role of !3-sheet interactions in domain stability, folding, and target recognition reactions of calmodulin. Biochemistry 1997, 36:9550-9561.
78.
Blacklow SC, Kim PS: Protein folding and calcium binding defects arising from familial hypercholesterolemia mutations of the LDL-receptor. Nat Struct Bio11996, 3:758-762.
62.
Haga K, Tsuga H, Haga T: Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin. Biochemistry 1997, 36:1315-1321.
79.
63.
Matsubara M, Hayashi N, Titani K, Taniguchi H: Circular dichroism and 1H NMR studies on the structures of peptides from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and complex with calmodulin. J Biol Chem 199'7, 272:23050-23056.
Sunnerhagen M, Forsen S, Hoffren AM, Drakenberg T, Teleman O, Stenflo J: Structure of the Ca2+-free GLA domain sheds light on membrane binding of blood coagulation proteins. Nat Struct Biol 1995, 6:504-509.
80.
Freedman SJ, BIo.stein MD, Baleja JD, Jacobs M, Furie BC, Furie B: Identification of the phospholipid binding site in the vitamin K-dependent blood coagulation protein factor IX. J Biol Chem 1996, 271:16227-16236.
76. he
Brown MS, Herz .J, Goldstein JL: Calcium cages, acid baths and recycling receptors. Nature 1997, 388:629-630. authors give a pleasant introduction to the low-density lipoprotein receptor. 77. ••
Fass D, Blacklow SC, Kim PS, Berger JM: Molecular basis of familial hypercholesterolemia from structure of LDL-receptor module. Nature 199'7, 388:691-693. The crystal structure of an LDL module is presented, showing that naturally occurring point mutations found in patients with the disease familial hypercholesterolaemia alter residues that directly coordinate Ca 2+ or that serve as scaffolding residues.
302
Bio-inorganic chemistry
81.
Li L, Darden TA, Freedman SJ, Furie BC, Furie B, Baleja JD, Smith H, Hiskey RG, Pedersen LG: Refinement of the NMR solution structure of the gamma-carboxyglutamic acid domain of coagulation factor IX using molecular dynamics simulation with initial Ca 2+ positions determined by a genetic algorithm. Biochemistry 199?, 36:2132-2138.
82.
Handford P, Mayhew M, Baron M, Winship PR, Campbell ID, Brownlee GG: Key residues involved in Ca 2+ binding motifs in EGF-like domains. Nature 1991,351:164-16?.
83.
Stenflo J, Oehlin AK, Persson E, Valcare C, Astermark J, Drakenberg T, Selander M, Bj6rk I: Epidermal growth factorlike domains in the vitamin K-dependent clotting factors. Some structure-function relationships. Ann NY Acad Sci 1991, 614:11-29.
84.
85.
Rao Z, Handford P, Mayhew M, Knott V, Brownlee GG, Stuart D: • • epidermal growth factor-like The structure of a Ca2+-banding domain: its role in protein-protein interactions. Cell 1995, 82:131-141. Dietz HC, Pyeritz RE: Mutations in the human gene for fibrillin-1 (FBN1) in the Marfan syndrome and related disorders. Hum Mol Gen 1995, 4:1 fl99-1809.
86. •
Downing AK, Knott V, Werner JM, Cardy CM, Campbell ID, Handford PA: Solution structure of a pair of calcium-binding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders. Oe//1996, 85:597-605. Two general modes of interaction between consecutive EGF-like domains are proposed here: one with a rodlike arrangement of the domains and another with a kinked orientation. The interaction is in both cases stabilized by a Ca2+ ion. 87 •
Sunnerhagen M, Olah G, Stenflo J, Forsen S, Drakenberg T, Trewhella J: The relative orientation of GLA and EGF domains in coagulation factor X is altered by Ca 2+ binding to the first EGF domain. A combined NMR-small angle X-ray scattering study. Biochemistry 1996, 35:11647-11559. The study shows a Ca2+-stabilized Gla-EGF junction. 88.
Chothia C, Jones EY: The molecular structure of cell adhesion molecules. Annu Rev Biochem 199"7, 66:823-862.
89. •.
Nagar B, Overduin M, Ikura M, Rini JM: Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 1996, 380:360-364. This paper describes an elegant structure determination giving important clues to the mechanism of cell-cell adhesion. 90. =,
Koch AW, Pokutta S, Lustig A, Engel J: Calcium binding and homoassociation of E-cadherin domains. Biochemistry 199'7, 36:7697-??05. The data obtained on the Ca2+-binding characteristics and Ca2+-dependent dimerization of E-cadherin fragments contribute significantly to the understanding of these molecules. 91.
Lewis JE, Jensen PJ, Johnson KR, Wheelock MJ: E-cadherin mediates adherens junction organization through protein kinase C. J Cell Sci 1994, 107:3615-3621.
92.
Shapiro L, Fannon AM, Kwong PD, Thompson A, Lehmann MS, Grebel G, Legrand J-F, AIs-Nielsen J, Colman DR, Hendrickson WA: Structural basis of cell-cell adhesion by cadherins. Nature 1995, 374:32?-337.
93. •
Hohenester E, Maurer P, Hohenadl C, Timpl R, Jansonius JN, Engel J: Structure of a novel extracellular calcium-binding module in BM-40. Nat Struct Bio/1996, 3:67-73. The first extracellular EF hand to be discovered is described. 94.
Brown EM, Vassilev PM, Hebert SC: Calcium ions as extracellular messengers. Cell 1995, 83:679-682.
95.
Gerasimenko OV, Gerasimenko .IV, Petersen OH, Tepikin AV: Calcium transport pathways in the nucleus. Pf/i~gers Arch 1996, 432:1-6.
96.
Santella L: The cell nucleus: an Eldorado to future calcium research? J Membr Bio/1996, 153:83-92.
9?.
Kubinowa H, Tjandra N, Grzesiek S, Ren H, Klee CB, Bax A: Solution structure of calcium-free calmodulin. Nat Struct Biol 1995, 2:?68-7?6.
98.
Chattopadhyaya R, Meador WE, Means AR, Quiocho FA: Calmodulin structure refined at 1.7 &ngstr6ms. J Mol Biol 1992, 228:1177-1192.
99.
FerrinTE, Huang CC, Jarvis LE, Langridge R: The MIDAS display system. J Mol Graphics 1988, 6:13-27.
100.
Santella L, Carafoli E: Calcium signalling in the cell nucleus. FASEB J 1997, 11:1091-1109.