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Home , Grancalcin, Neurocalcin, Neuronal calcium sensor, Protein superfamily, S100 protein

PERSPECTIVE

Genetic polymorphism and conformational plasticity in the superfamily: Two ways to promote multifunctionality

Mitsuhiko Ikura†‡ and James B. Ames§ †Division of Signaling Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, ON, Canada M5G 2M9; and §Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850

Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 2, 2005 (received for review October 31, 2005)

Calcium signaling pathways control a variety of cellular events such as transcription, protein phosphorylation, nucleotide me- tabolism, and ion transport. These pathways often involve a large number of -binding collectively known as the cal- modulin or EF-hand . Many EF-hand proteins undergo a large upon binding to Ca2؉ and target proteins. All members of the superfamily share marked sequence and similar structural features required to sense Ca2؉. Despite such structural similarities, the functional diversity of EF-hand calcium-binding proteins is extraordinary. Calmodulin itself can bind >300 different proteins, and the many members of the and families collectively recognize a largely different set of target proteins. Recent biochemical and structural studies of many different EF-hand proteins highlight remarkable similarities and variations in conformational responses to the common ligand Ca2؉ and their respective cellular targets. In this review, we examine the essence of molecular recognition activities and the mechanisms by which calmodulin super- .family proteins control a wide variety of Ca2؉ signaling processes

he Ca2ϩ ion is a highly versatile by stimulating or suppressing different interhelical loop region contains several intracellular signal regulating intracellular signaling pathways (5–7). amino acids essential to the coordina- ϩ many different cellular func- The calmodulin superfamily is a major tion of a single Ca2 ion. Typically, a ϩ tions, including fertilization, cell class of Ca2 sensor proteins, which col- pair of EF-hand motifs in tandem array T lectively play a crucial role in various cel- constitutes a stable structural unit, to- cycle, apoptosis, muscle contraction, vi- sion, and memory (1). In eukaryotic lular signaling cascades through regulation gether generating in the 2ϩ 2ϩ cells, cytoplasmic Ca2ϩ entry and out- of numerous target proteins in a Ca - binding of Ca ions (62, 63). Many EF- flow are governed by two sources: intra- dependent manner (Table 1). It has been hand proteins, such as calmodulin and cellular stores such as the endoplasmic reported that there are nearly 600 mem- members of the NCS family, consist of ϩ bers in this superfamily (60), all of which reticulum and extracellular Ca2 that four EF-hand motifs. This results in two contain one or more Ca2ϩ binding motifs enters the cell through various trans- globular structural units in a single pro- known as the EF-hand, first identified in tein. We will discuss the importance of porters on the plasma membrane (2). by Kretsinger and coworkers this feature to the multifunctionality of Ca2ϩ entry into the cytoplasm is tightly (7). Calmodulin contains four EF-hand these proteins. The S100 family, on the regulated by a variety of components of motifs, with highly conserved amino acid 2ϩ other hand, consists of only one globular the Ca signaling toolkit, which were sequences in all eukaryotes. In fact, this structural unit comprised of two EF- elegantly summarized by Berridge et al. sequence is ranked fifth in an amino acid hand motifs. However, members of this 2ϩ (3). The Ca flux machinery, consisting conservation contest in proteomes after family all form a stable homo- or het- of ion channels, pumps, and exchangers, the H4 and H3, actin B, and erodimer, which contains four EF-hand gives rise to highly localized and tran- ubiquitin (61). In contrast, the motifs in a single structural entity. ϩ sient Ca2 signals that are, in turn, family, which also contains four EF-hand The direct interaction with Ca2ϩ en- transduced by calcium-binding proteins motifs, has two isoforms in the human ables these Ca2ϩ sensor proteins to acting on various and down- (skeletal and cardiac muscles) and many change their conformation from the in- stream effector proteins. isoforms in invertebrates, all diverse in active state (P) to the intermediate state A central question in the field of amino acid sequence (6). Similarly, the (Ca2ϩ-P*), which is a prerequisite to the Ca2ϩ signaling is how different Ca2ϩ neuronal calcium sensor (NCS) and S100 formation of an active conformation in ϩ signaling systems control so many diver- proteins are diverse in sequence and func- complex with a target (Ca2 -P**-E*) gent cellular processes (3)? Such control tion. Recent advances in the structural required to transform the target protein and biochemical understanding of these from its inactive state (E) to the active is achieved at both the cellular and mo- 2ϩ lecular level. The spatial and temporal Ca sensor proteins unveiled two emerg- state (E*) 2ϩ ing themes that explain the vast multifunc- variation of Ca signals, known as ϩ 2ϩ ϩ 2ϩ tionality of the calmodulin superfamily. Ca E Ca waves, spikes, and puffs, are re- ϩ ϩ These molecular themes are genetic poly- P |9 =Ca2 -P*|9=Ca2 -P**-E*. sponsible for generating diverse output morphism and protein conformational required for different physiological con- plasticity, and are likely to be relevant to ditions (4). Also, cell-specific expression other protein superfamilies with diverse of a unique set of components from the functions. Conflict of interest statement: No conflicts declared. 2ϩ Ca signaling toolkit (3) is required for Abbreviations: KChIP, Kϩ channel-interacting protein; NCS, 2؉ generating cell-specific responses to EF-Hand as a Building Block of Ca neuronal calcium sensor. 2ϩ Ca signals. At the molecular level, a Sensor Proteins ‡To whom correspondence should be addressed. E-mail: ϩ variety of Ca2 sensor proteins provide The EF-hand motif consists of a simple [email protected]. totally different physiological responses helix-loop-helix architecture in which the © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508640103 PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1159–1164 Downloaded by guest on September 27, 2021 Table 1. EF-hand Ca2؉-binding proteins and their functional target proteins (35, 36) and (74). Frequenin EF-hand protein Functional targets also is expressed in invertebrates, includ- ing flies (73), worms (75) and yeast (29, Calmodulin light chain (8) 76). Yeast and mammalian frequenins Calmodulin-dependent protein kinases (9) bind and activate a particular phosphati- (10) dylinositol 4-OH kinase isoform (Pik1 Myristoylated C substrates (11) -coupled receptor kinases (GRKs) (12) gene in yeast) (29, 77) required for vesicu- (13) lar trafficking in the late secretory path- Adenylate cyclases (14) way (78). Mammalian frequenin (NCS-1) 2ϩ (15) also regulates voltage-gated Ca channels ϩ Nitric oxide (16) (31) and K channels (30). The KChIP (17, 18) proteins regulate the gating kinetics of Plasma membrane Ca2ϩ ATPase pump (19) Shaker Kϩ channels (34). DREAM͞calse- Cyclic-nucleotide gated ion channels (20) nilin binds to specific DNA sequence ele- SK channels (21) 2ϩ ments in the prodynorphin and c-fos Voltage-gated Ca channels (22) (35) and serves as a transcriptional Inositol 1,4,5-trisphosphate receptors (23) Ryanodine receptors (24) repressor for pain modulation (79, 80). Troponin C Hence, the physiological functions of Skeletal (100%) Skeletal muscle Troponin I (25) the NCS proteins are highly diverse and Cardiac (65%) Cardiac muscle Troponin I (26, 27) nonoverlapping. Invertebrate (37%) Invertebrate Troponin I (28) The S100 proteins also have diverse NCS family physiological functions involved in regulat- ϩ 2ϩ Frequenin (100%) PI4-kinase (29), K channels (30), Ca channels (31) ing cell cycle control, transcription, and ␦ - (55%) Nicotinic acetylcholine receptors (32) secretion (Table 1). controls car- (43%) (33) diac contractility and is associated with a KChIP1–4 (40%) Shaker channels (34) DREAM͞calensilin Dynorphin DRE (35), presenilin (36) number of cardiomyopathies (81). GCAPs (35%) Retinal guanylate cyclases (retGCs) (37) is localized to the nucleus where it regu- Calcineurin B (32%) Calcineurin A-subunit (38) lates transcription of various genes and CIB (24%) Integrin (39), presenilins (40) acts as a tumor suppressor in can- CaBP1 (22%) Inositol 1,4,5-trisphosphate receptors (41), voltage-gated Ca2ϩ channels (42) cer cells (82). In contrast, (83) S100 family and (47) are both tumor promot- S100A1 (100%) (43), SERCA2a (44), (45) ers. acts extracellularly and is S100A4 (53%) Methionine 2 (46) implicated in epidermal inflammatory S100A6 (48%) Ubiquitin (47) diseases such as psoriasis (84). The (47%) II (48) ͞ (43%) Annexin I (49) A9 heterodimer acts extracellu- (40%) RAGE (50) larly as a chemotactic molecule in inflam- (60%) p53 (51), NDR kinase (52), CapZ (53), retinal guanylate cylcase (54) mation (85). S100A10 and S100A11 bind Penta EF-hand family to distinct annexin proteins and regulate Grancalcin (100%) L-plastin (55) annexin trafficking to cell membranes (48, Sorcin (54%) Ca2ϩ channels (56) 49). S100A12 is expressed in phagocytes, ALG-2 (32%) Alix͞AIP1 (57), (58) where it regulates secretion of proinflam- (29%) (59) matory mediators (50). Perhaps the best Relative amino acid sequence identities within each subfamily are indicated in parentheses (%). characterized S100 protein, S100B is References are provided after each target in parentheses. highly expressed in the , where it is implicated in various neurodegenerative diseases such as Alzheimer’s disease, The first conformational transition is key tinct target protein and performs a highly Down syndrome, and multiple sclerosis. ϩ to the Ca2 sensory function and is uni- specialized physiological task. S100B is multifunctional and binds to ϩ versal to all Ca2 sensor proteins. The multiple target proteins, including p53, an second conformational change plays a Functional Diversity of NCS and interaction that is involved in regulating critical role in the activation and recogni- S100 Proteins transcription (51). S100B also interacts tion of specific targets. The NCS proteins, as their name implies, with NDR kinase (52), CapZ (53), and are expressed almost exclusively in the retinal (86) to control Genetic Polymorphism in NCS and central nervous system. The common fea- neuronal excitability. In summary, the S100 Proteins tures of NCS proteins are an Ϸ200-resi- physiological functions of S100 proteins A growing number of EF-hand proteins due chain containing four EF-hand motifs are even more wide ranging and diverse exist as splice variants or multiple iso- and an amino-terminal than those of the NCS family. forms that exhibit diverse biological func- consensus sequence (65). The best charac- tions. This evolutionary diversity is best terized NCS protein is recoverin, a calci- Structure and Target Recognition illustrated by members of the NCS (64 um-myristoyl switch protein in retinal rod of NCS and S100 Proteins and 65) and S100 (63 and 65) families. A cells (68, 69) that controls desensitization The three-dimensional structures of vari- total of 24 human genes code for S100 of rhodopsin (33) by regulating rhodopsin ous NCS proteins [recoverin (69, 87), neu- isoforms (66), and Ͼ15 genes code for kinase activity (70). Recoverin was also rocalcin (88) and frequenin (89)] reveal NCS isoforms and Ͼ10 splice variants identified as the antigen in cancer-associ- that their main-chain globular folds are (67). The S100 genes are typically 40– ated retinopathy (71). Other NCS proteins nearly identical, which is not surprising 60% identical in sequence and the NCS include neurocalcin (72), frequenin given the sequence conservation. In all genes range from 22–55% identity (Table (NCS-1) (73), Kϩ channel-interacting pro- NCS proteins, four EF-hands form a 1). Each protein isoform binds to a dis- teins (KChIPs) (34), DREAM͞calsenilin highly compact, globular fold, in contrast

1160 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508640103 Ikura and Ames Downloaded by guest on September 27, 2021 gated patch. By contrast, recoverin has mostly charged residues on the surface of the C-terminal half (highlighted blue and pink), whereas neurocalcin and KChIP1 have mostly uncharged and polar residues shown in white. We propose that these different patterns of charge distribution and hydrophobicity on the surface of the C-terminal half of NCS proteins are im- portant structural determinants for con- ferring target specificity. A similar contrast in surface charge distribution is also apparent in representa- tive structures of S100 proteins (Fig. 1B). S100B contains an abundance of nega- tively charged residues (Asp and Glu shaded pink) located on the protein sur- face balanced by a nearly equal number of positively charged surface residues (blue). By contrast, S100A10 and S100A12 have fewer charged residues on their protein surfaces and a much higher percentage of hydrophobic residues (yellow). Two target helices derived from p53 (shown dark red in Fig. 1B) are bound to the S100B ho- modimer in a symmetrical fashion (51). Target helices derived from annexin II bind to related binding sites on S100A10 (48). Both the p53 and annexin II target helices contain an amphipathic hydropho- bic face that interacts with a complemen- tary hydrophobic crevice on S100B and S100A10, respectively. The orientation of the p53 target helix with respect to S100B is quite different from that of the annexin Fig. 1. Space-filling structures of NCS (A) and S100 (B) proteins. Positive, negative, and hydrophobic target helix bound to S100A10 (Fig. 1B). residues on the protein surface are highlighted in blue, pink, and yellow, respectively. Ribbon diagram of The different target orientations are ex- bound target helices are shown in red. Conserved hydrophobic residues implicated in target recognition plained, in part, by differences in the sur- are labeled. Atomic coordinates are available from the : 1rec (recoverin), 1bjf (neuro- face contour properties at the respective calcin), 1g8i (frequenin), 1s6c (KChIP1), 1bt6 (S100A10), 1dt7 (S100B), and 1e8a (S100A12). binding sites. Another important factor is the distribution of charged residues on the to the dumbbell arrangement of two inde- dues in this region are conserved and cor- surface near the target-. The pendent domains found in calmodulin respond to residues of recoverin that in- highly charged environment on S100B (90) and troponin C (91). The three- teract with the myristoyl group in the may exert local forces that influence the 2ϩ 2ϩ disposition of the target helix by forming dimensional structures of various S100 Ca -free state (69, 95, 96). Upon Ca ͞ isoforms are also very similar to one an- binding, these exposed residues in the hy- electrostatic and or hydrogen-bonding other and contain a symmetrical dimer of drophobic patch have been implicated in contacts. By contrast, the corresponding residues on S100A10 are mostly neutral EF-hands arranged in a compact globular target recognition from mutagenesis stud- and do not interact strongly with the tar- fold (51, 92–94). If the overall main-chain ies (97–100), and these residues form in- get helix. The characteristic surface charge conformation of the various NCS and termolecular contacts with target proteins properties of the various S100 isoforms S100 isoforms is so similar, how can one as observed in the recent crystal structure help explain the unique target recognition explain the functional differences? An of KChIP1 (101). The structural interac- of each isoform that is important for spec- important distinguishing structural prop- tion of KChIP1 with its target helix (high- ifying their diverse biological functions. erty is the distribution of charged and hy- lighted red in Fig. 1A) is very similar to In summary, the structures of NCS and drophobic side-chains on the protein sur- the interaction of the N-terminal domain S100 proteins both contain EF-hands ar- face. For NCS proteins, the surface of CaM bound to myosin light chain ki- ranged in a compact globular fold, in con- properties of the C-terminal domain are nase (MLCK) (Fig. 2C). trast to the two independent domains quite variable, whereas the N-terminal Although the hydrophobic patch on the found in calmodulin. The highly con- domain is more conserved. N-terminal domain is conserved among served and compact main chain confor- Surface representations of hydrophobic- NCS proteins, the surface properties of mation found in either NCS or S100 ity and the charge density of the various the C-terminal domain appear to be proteins serves as a scaffold that can be NCS structures are shown in Fig. 1A.All unique. For example, frequenin exhibits adapted with amino acid side-chain NCS structures exhibit a similar exposed exposed hydrophobic residues (highlighted groups of different size and͞or charge to hydrophobic surface located on the N- yellowinFig.1A) in the C-terminal do- alter the protein surface and facilitate terminal half of the protein (F35, W31, main that, together with the conserved unique target interactions. This type of F56, F57, Y86, and L90 for recoverin in hydrophobic crevice in the N-terminal surface remodeling appears to be an ef- Fig. 2A). The exposed hydrophobic resi- domain, form one continuous and elon- fective means of modifying target specific-

Ikura and Ames PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1161 Downloaded by guest on September 27, 2021 Now a major question is how CaM binds and regulates all these proteins. Crystallographic and NMR studies on various CaM target proteins have shed light on this unusual property of CaM. The first structure determined for CaM in complex with a target protein showed a remarkable conformational change in CaM’s two EF-hand domains upon bind- ing to a peptide derived from myosin light chain kinase (MLCK) (107, 108). This structure revealed two important proper- ties. First, the central domain linker (resi- dues 78–81) is highly flexible and can be bent dramatically upon binding to the tar- get protein (Fig. 2A; refs. 109–111). The flexibility of the domain linker permits the orientation of the two domains of CaM to change independently to accom- modate the structural nature of the target protein. Secondly, two hydrophobic an- choring residues from the smooth muscle MCLK peptide (Trp-800 and Leu-813) bind simultaneously to the hydrophobic pocket in N- and C-terminal domains, which is extremely rich in methionine resi- dues (four Met residues in each pocket, see Fig. 2B). CaM contains nine methi- onines corresponding to 6% of the entire sequence, which is significantly higher than the average of known proteomes (1%). Conservative mutations of these methionines to leucines at different sites significantly altered CaM’s activity to stimulate cAMP (112). The vital role of methionines in interac- tions with target proteins is evident in a number of new structures reported for CaM in complex with CaMKII (113), CaM kinase kinase (114), NO synthatase (115), glutamate decarboxylase (Fig. 2C; ref. 15), MARCKS (116), Ca2ϩ-activated Kϩ channel (Fig. 2C; ref. 21), and anthrax (Fig. 2C; ref. 14). These structures reinforce the significance of the Fig. 2. Structure and target recognition by calmodulin. (A) Superposition of selected NMR structures of aforementioned structural properties of apo-calmodulin (Protein Data Bank entry 1dmo). (B) Space-filling representation of hydrophobic target CaM by clearly illustrating that CaM can binding sites (yellow) flanked by surrounding methionine residues (orange). (C) Main-chain ribbon adopt largely different, global conforma- diagrams of calmodulin (yellow) bound to the target proteins myosin light chain kinase (MLCK), SK tions depending on the structural entity channel, GAD, and EF (red). Bound Ca2ϩ ions are shown in blue. that CaM binds. In addition to plasticity of the protein fold, the amino acid side ity, thereby increasing the repertoire of the protein calcineurin (13) chains that interact with target proteins, in downstream effector proteins. and nitric oxide synthetase (NOS) (16) particular the Met residues, are remark- were also found to be targets of Ca2ϩ͞ ably flexible. Indeed, the hydrophobic Conformational Plasticity in Calmodulin CaM-dependent stimulation. The list of pockets of CaM are so flexible that they In contrast to the highly specialized multi- known CaM-dependent proteins exceeds can accommodate a variety of amino acid side chains, such as those of Trp, Phe, Ile, ple isoforms of S100 and NCS families, 300 in number (105) and now includes Leu, Val, and Lys. This structural plastic- the single protein, CaM, regulates numer- many ion transporters, such as inositol ity at both the level of the individual side ous target proteins that are functionally 1,4,5-trisphosphate receptor (23), ryano- dine receptor (24), plasma membrane chains and the orientation of entire do- and structurally diverse (Table 1). In 1970, ϩ ϩ Ca2 pump (19), and L-type Ca2 channel mains is crucial for CaM to the recog- CaM was first discovered to activate cyclic (22), all negatively or positively regulated nition of numerous target proteins for nucleotide phosphodiesterase (17, 18). by Ca2ϩ͞CaM. Amazingly, some infec- regulation by CaM. Subsequently, many proteins such as ϩ tious bacteria enzymes, such as adenylyl Ca2 -transporting ATPase (102, 103), cyclase from Bacillus anthracis and Borde- Cellular Level of Control myosin light chain kinase (8) and phos- tella pertussis, require host CaM for its The exact and precise recruitment of the phorylase kinase (10) were all found to be toxic enzymatic activity of converting Ca2ϩ signaling toolkit proteins under a regulated by CaM. Protein kinases (104), ATP to cyclic-ADP (106). specific cellular condition depends on

1162 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508640103 Ikura and Ames Downloaded by guest on September 27, 2021 differential gene transcription of its com- tissue-specific expression. The NCS pro- teins that function as Ca2ϩ sensors in ponents. In the case of CaM alone, the teins, recoverin and guanylate cyclase- eukaryotic cells. The NCS and S100 temporal and spatial expression of its activating proteins, are found almost protein families has undergone extensive gene is tightly regulated in most, if not all, exclusively in retinal rod and cone cells rounds of evolutionary mutation to cell types. In many vertebrates, three bona (125, 126), whereas many other NCS pro- equip each isoform with a specific func- fide CaM genes (CaM I, CaM II, and teins, such as neurocalcin and frequenin, tion at a specific location in the cell. In CaM III), which are collectively tran- are present only in the brain and spinal contrast, the evolutionary history of the scribed into at least eight mRNAs, eventu- cord. Mammalian frequenin (NCS-1) is single multifunctional Ca2ϩ sensor pro- ally translated into an identical CaM expressed throughout the central nervous tein, CaM, is strikingly different. Pre- protein consisting of 148 amino acids. system (127). By contrast, hippocalcin is sumably through a These proteins are highly expressed in the localized in hippocampal pyramidal neu- event, CaM has become equipped with brain and the testis (117). Interestingly, rons, and VILIP-3 is found only in cere- two EF-hand domains that are con- recent data using highly polarized cells, bellar Purkinje and granule cells. In ␦ nected by a magical flexible linker that such as , (118) indicate that addition, hippocalcin, neurocalcin- , and enables this multifunctional protein to CaM mRNAs are targeted to different recoverin each possess a calcium-myristoyl change its conformation as needed. This switch that controls subcellular membrane intracellular compartments and that the event presumably happened at a rela- localization and capacity to interact with translocation of mRNA(s), instead of tively early stage of , and the membrane-bound targets (68, 69). In the protein, provides a means to enrich molecular architecture has been kept the CaM protein in a specific site(s) in the contrast, the KChIP proteins lack myris- toylation and generally do not possess a since then as the amino acid sequence cell. Overall, CaM seems to be a limiting of CaM is remarkably conserved from factor in cell function (119). Furthermore, functional myristoyl switch. Instead, splice variants of KChIPs are differentially ex- yeast to human. It is interesting to note the CaM protein has been shown to un- that all known NCS and S100 family dergo posttranslational modifications pressed in various sensory neurons and cardiac cells. The DREAM protein proteins adopt a single globular domain including acetylation, trimethylation, car- architecture, unlike the two independent boxylmethylation, proteolytic cleavage, (KChIP3) is found mostly in the cerebel- lar granular cortex (128), whereas KChIP2 domains of CaM. Protein conforma- and phosphorylation (120–122). These tional plasticity of CaM thus emerged as chemical modifications may modulate and related splice variants are found only ϩ in cardiac myocytes (129). Hence, the tis- a means of achieving functional diversity CaM’s biological activity as a Ca2 sensor sue-specific expression of NCS proteins rather than employing the more tradi- protein; however, the physiological roles and their membrane localization con- tional approach of genetic polymorphism. of such posttranslational events remain to trolled by N-terminal myristoylation help In human and other eukaryotic pro- be fully explored. confer the distinctive physiological roles of teomes, signaling module domains such as Similarly, the CaM target proteins NCS proteins generally. SH2, SH3, and PDZ domains can be identified thus far are all transcription- S100 proteins also exhibit tissue-specific linked with a kinase or a receptor module ally regulated. For instance, the inositol expression patterns and different subcellu- to introduce regulatory mechanisms (130, 1,4,5-trisphosphate receptor (IP R) pos- 3 lar localization. S100 proteins in the cy- 131). Generally, the insertion of protein sesses three isoforms in mammals: Type I tosol act as intracellular calcium sensors domains enables the protein to acquire IP R is highly expressed in cerebellar Pur- 3 and͞or calcium buffers. S100A1 is ex- additional functions one at a time. The kinje cells of the central nervous system, pressed exclusively in cardiac cells, where uniqueness of CaM’s modularity is that by whereas type II is predominantly found it has been implicated in a number of car- simply connecting two EF-hand modules in cardiac ventricular myocytes (123). diomyopathies (81). S100A2, S100A6, and via a flexible linker, an exponential in- These isoforms not only have different S100B are all present in the nucleus, crease in the number of target interac- subcellular distribution, which is dynami- where they control gene expression and cally regulated dependent on physiological tions was achieved. Is this conformational are implicated in various forms of cancer. plasticity unique to CaM, or is there any conditions, but also generate different S100B is expressed also throughout the splice variants with different biophysical other protein(s) that might use the same central nervous system, where it is linked principle for its multifunctionality? Finally, properties in terms of ligand binding to a number of neurodegenerative disor- (124). Moreover, these CaM targets are with regard to NCS and S100 proteins, ders. Many S100 proteins (such as S100B, how could each of the protein isoforms often posttranslationally modified, which S100A4, S100A7, S100A8, and ) coevolve with their respective target pro- may affect their ability to interact with are also extracellularly targeted, where teins to achieve such specificity and exclu- CaM. For example, phosphorylation of they interact with the extracellular domain sivity? Further biochemical and structural the catalytic subunit of CaM kinase mod- of the receptor for advanced glycation end ulates its CaM sensitivity (104). Clearly, products. Extracellular S100 proteins also studies will be needed to address those the CaM–target interaction is modulated display an unusually high affinity for Zn2ϩ key questions. by a number of cellular mechanisms in- or Cu2ϩ, which may serve a unique regu- volving temporal and spatial subcellular latory role in the extracellular space. In We thank several of our colleagues for their helpful discussions and Kyoko Yap, Jane localization of CaM itself and the target summary, the cellular localization and Gooding, Peter Stathopulos, and Chris protein as well as structural modifica- tissue-specific expression of S100 proteins Marshall for their helpful comments on the tions of the CaM-binding site within the help explain their functional diversity. manuscript. This work was supported by the target protein. Canadian Institutes of Health Research Like CaM, the various NCS proteins Summary and Perspectives (M.I.) and the National Institutes of Health are variably expressed in many different In this review, we summarized our cur- (J.B.A.). M.I. holds the Canada Research cell types, and NCS splice variants exhibit rent knowledge on the diversity of pro- Chair of Cancer Structural Biology.

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