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Calmodulin: a the Ca2ϩ signal. Hence, separate intracellular loci or are potentially distinct compartments of prototypical localized Ca2ϩ signalling2 (Fig. 1a). Therefore, Ca2ϩ signals in the nucleus exert different effects from those generated in the or near the plasma calcium sensor membrane of the same cell3. Additionally, the modulation of the amplitude or frequency of Ca2ϩ spikes (AM and FM, respectively) encodes important David Chin and Anthony R. Means signalling information4. This has recently been illustrated for cases in which an optimal frequency of intracellular Ca2ϩ oscillations is important for the Calmodulin is the best studied and prototypical example of the expression of different genes5.

2ϩ E–F-hand family of Ca -sensing . Changes in Calcium-regulated proteins: calmodulin 2ϩ intracellular Ca2ϩ concentration regulate calmodulin in three How do Ca signals produce changes in cell func- tion? The information encoded in transient Ca2ϩ distinct ways. First, at the cellular level, by directing its signals is deciphered by various intracellular Ca2ϩ- binding proteins that convert the signals into a wide subcellular distribution. Second, at the molecular level, by variety of biochemical changes. Some of these 2ϩ promoting different modes of association with many target proteins, such as C, bind to Ca and are directly regulated in a Ca2ϩ-dependent manner. proteins. Third, by directing a variety of conformational states in Other Ca2ϩ-binding proteins, however, are inter- mediaries that couple the Ca2ϩ signals to biochemical calmodulin that result in target-specific activation. The and cellular changes (Fig. 1b). Among this latter calmodulin-dependent regulation of protein illustrates the group are a family of proteins that is distinguished by a structural motif known as the E–F hand. An E–F ϩ potential mechanisms by which Ca2 -sensing proteins can hand consists of an N-terminal helix (the E helix) 2ϩ recognize and generate affinity and specificity for effectors in a immediately followed by a centrally located, Ca - coordinating loop and a C-terminal helix (the Ca2ϩ-dependent manner. F helix). The three-dimensional arrangement of these domains is reminiscent of the thumb, index and middle fingers of a hand, hence the name ‘E–F hand’. These proteins respond to Ca2ϩ in one of two ways Calcium (as Ca2ϩ) is an element that is crucial for (Fig. 1b). One group (e.g. and ) numerous biological functions. In many organisms, do not undergo a significant change in confor- the vast majority of Ca2ϩ is complexed with phos- mation on binding Ca2ϩ and function as Ca2ϩ buffers phates to form exo- or endoskeletons that not only or Ca2ϩ transporters. The second group, the Ca2ϩ serve as structural scaffolds but also buffer the levels sensors, undergo a Ca2ϩ-induced change in confor- ϩ Ϫ of Ca2 within extracellular fluids at ~10 3 M. By mation6. The most prominent examples of sensors contrast, the resting concentrations of intracellular include C (a protein dedicated to regu- ϩ Ϫ free Ca2 (~10 7 M) is 104 times lower than that out- lating striated-), the multifunc- side cells, providing the potential for the ready tional Ca2ϩ transducer calmodulin (CaM), the S100 import of Ca2ϩ into cells, where it can act as a second family of proteins and, most recently, the neuronal messenger. myristoylated proteins such as recoverin7. Various extracellular stimuli promote the move- The molecular and cellular mechanisms under- ment of Ca2ϩ either from outside the cell (via lying the ability of a majority of the Ca2ϩ-sensor plasma-membrane Ca2ϩ channels) or from intracellular proteins to integrate Ca2ϩ signals into specific cellular stores into the intracellular milieu (Fig. 1a). The responses are not clearly understood. Much of what Ca2ϩ is released in elemental aliquots called sparks, we do know about the mechanisms that the sensor puffs or waves depending on the extent of the intra- proteins use to transduce Ca2ϩ signals is based on cellular area covered. This free Ca2ϩ is only briefly information gained from CaM, probably the most The authors are in available to act as a cellular signal, however, because intensively studied member of the E–F-hand family ϩ ϩ the Dept of Ca2 -binding proteins and Ca2 pumps immediately of sensors. In the remainder of this article, CaM will Pharmacology combine to sequester and transport it to intracellular therefore serve as a model or prototype for other ϩ and Cancer storage sites or outside the cell. potential Ca2 transducers. A review of some of the ϩ Biology, Duke The short pulses of Ca2 exert specific changes in mechanisms responsible for regulating CaM at the University Medical cellular function depending on their route of entry subcellular and molecular levels might reveal valuable ϩ ϩ Center, Durham, into the cell, their local sites of action and, finally, by clues as to how Ca2 -sensor proteins convert Ca2 NC 27710, USA. their pattern of modulation. The particular mem- signals into cellular function. E-mail: chin0001@ brane channel or intracellular receptor responsible CaM is expressed in all eukaryotic cells where it ϩ mc.duke.edu; for the release of Ca2 exerts considerable influence participates in signalling pathways that regulate ϩ means001@ on the eventual effects of the Ca2 signal1. The mode many crucial processes such as growth, proliferation mc.duke.edu of cellular entry also influences the site of action of and movement. It is relatively small (vertebrate CaM

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(a) Ca2+ (a) (b)

Ca2+

Amplitude Frequency modulated (AM) modulated (FM)

Ca2+ Ca2+

Nucleus

trends in Cell Biology Ca2+ FIGURE 2 2+ (b) Ca buffers and ϩ The Ca2 -regulated conformational change in calmodulin. The transporters ϩ main chain structure of Ca2 -free (apo) CaM (a) and 2ϩ Ca 4–CaM (b) are shown in red with their respective N-terminal domains on top. Methionine side chains are shown Ca2+ 2+ Effectors Ca sensors in purple to denote the location of potential hydrophobic 2ϩ trends in Cell Biology pockets in each of the two domains. Ca binding produces large changes in the helices in both domains, resulting in the FIGURE 1 exposure of several hydrophobic residues. (a) Sources of intracellular Ca2ϩ signals. Ca2ϩ enters cells via extracellular plasma-membrane receptors or from intracellular 2ϩ stores, producing transient local or global changes in its (Fig. 2). In the absence of Ca , the N-terminal distribution. The Ca2ϩ oscillations are modulated in their domain of the apo-CaM molecule adopts a ‘closed’ amplitudes (AM) or frequencies (FM) and are therefore capable conformation in which the helices in both E–F of conveying signalling information in complex ways. (b) E–F- hands are packed together. By contrast, still in the 2ϩ hand Ca2ϩ-binding proteins are classified as buffers/transporters absence of Ca , the C-terminal domain of apo-CaM and sensors. The Ca2ϩ sensors change conformation on binding adopts a ‘semiopen’ conformation in which a par- Ca2ϩ and transduce changes in cell function by regulating tially exposed hydrophobic patch is accessible to downstream effectors. solvent. This might allow the C-terminal domain of CaM to interact with some target proteins at resting levels of intracellular free Ca2ϩ (Ref. 8). has 148 residues), evolutionarily highly conserved In the event of a transient rise in Ca2ϩ, the Ca2ϩ and comprises four E–F hands. The first two E–F ion is coordinated in each Ca2ϩ-binding loop of hands combine to form a globular N-terminal Ca2ϩ–CaM by seven, primarily carboxylate, ligands. domain that is separated by a short flexible linker from The binding of Ca2ϩ leads to substantial alterations a highly homologous C-terminal domain consisting of in the interhelical angles within the E–F hands in E–F hands 3 and 4 (Fig. 2). each domain and dramatically changes the two Ca2ϩ sensors must be able to detect and respond domains of CaM to produce more ‘open’ confor- to a biologically relevant range of intracellular free mations (Fig. 2). These structural rearrangements in Ca2ϩ concentrations. CaM fits this profile as its CaM result in the concerted exposure of hydrophobic 2ϩ ϭ ϫ Ϫ7 ϫ Ϫ6 affinity for Ca (Kd 5 10 M to 5 10 M) falls groups in a methionine-rich crevice of each domain within the range of intracellular Ca2ϩ concentrations that is distinct from the Ca2ϩ-binding loops. The ex- Ϫ Ϫ exhibited by most cells (10 7 M to 10 6 M). However, posure to solvent of these hydrophobic residues is it has additional discrimination for Ca2ϩ, as the C- akin to a Ca2ϩ-controlled unfolding of CaM and un- terminal pair of E–F hands has a three- to fivefold leashes considerable free energy. It is this capacity to higher affinity for Ca2ϩ than the N-terminal pair of convert the Ca2ϩ-binding event into biochemical en- sites. By contrast, many Ca2ϩ-binding proteins with ergy that characterizes the Ca2ϩ-sensor proteins and Ͻ Ϫ7 2ϩ a considerably higher affinity (Kd 10 M) act as is the basis of their ability to transduce Ca signals. buffers by sequestering excess free Ca2ϩ, whereas Ca2ϩ-binding proteins with a considerably lower Calmodulin: location, mobility and translocation Ͼ Ϫ5 affinity (Kd 10 M) could not act as sensors Is CaM regulated at the subcellular level, and how because they are unable to detect the range of is this related to Ca2ϩ signalling? The concentration changes in intracellular free Ca2ϩ concentrations and location of CaM do appear to play an important that normally occur in cells. role in regulating its biological activity. CaM consti- The two domains of CaM adopt different tutes at least 0.1% of the total protein present in ϩ Ϫ Ϫ conformations in the absence or presence of Ca2 cells (10 6 M – 10 5 M) and is expressed at even

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(a) (b) (c) cytokinesis9. Other fluorescently labelled CaM molecules have provided information on its cellular mobility and location. Experiments with serum- deprived Swiss 3T3 fibroblasts first indicated that the majority of CaM was freely diffusible, but the CaM was then immobilized in response to stimulation by serum10. However, other studies on unstimulated smooth-muscle cells showed that most CaM is bound, possibly to Ca2ϩ-independent binding proteins, at resting concentrations of free Ca2ϩ. In response to a rise in Ca2ϩ, CaM exhibits a com- plex pattern of cellular localization, including a significant redistribution from the to the nu- cleus11. This stimulus-dependent movement of CaM trends in Cell Biology to the nucleus and its activation has also been de- FIGURE 3 tected in neurons12. CaM has also been seen to Distribution of calmodulin and tubulin in the mitotic spindle. accumulate slowly in the nucleus of - (a) A spindle visualized with an anticalmodulin antibody. treated pancreatic acinar cells13. The mechanism of (b) A Nomarski image of a mitotic spindle formed by incubation translocation in smooth-muscle cells apparently in a Xenopus extract. (c) A spindle visualized with involves the passive diffusion of CaM into the an antitubulin antibody. nucleus, where it might associate with targets in a Ca2ϩ-dependent manner14. higher levels in rapidly growing cells, especially The synchronization between CaM and Ca2ϩ signals those undergoing cell division and differentiation. is also being explored. A direct relationship between The local intracellular availability of CaM is likely to a rise in the levels of intracellular free Ca2ϩ and the be biologically significant because various CaM- Ca2ϩ-dependent activation of CaM was first observed dependent effectors are regulated over a wide range during a response to wound healing in fibroblasts15. Ϫ Ϫ ϩ of free CaM concentrations (10 12 M – 10 6 M). Ca2 oscillations in the secretory granules of pan- Recent studies of CaM tagged with green-fluor- creatic acinar cells have also been correlated with escent protein (GFP) show that CaM is found through- oscillations in the local concentration of CaM13. out the cytosol and nucleus in HeLa cells, although Recent studies on sea-urchin eggs undergoing mito- it is concentrated around the mitotic apparatus in sis, however, indicate that the spatial patterns of cells undergoing mitosis (Fig. 3), especially around Ca2ϩ are different from those of Ca2ϩ-activated the centrioles and the cytoplasmic furrow during CaM16. Interestingly, the Ca2ϩ-dependent activation of CaM exhibits a heterogenous distri- bution pattern in the cells that have TABLE I – SOME RECENT EXAMPLES OF CALMODULIN-REGULATED PROTEINS been studied, indicating the presence of discrete populations of CaM. These Protein Comments Ref. studies emphasize the importance of temporal and spatial relationships be- Cabin1 Thymocyte transcriptional regulator 45 tween Ca2ϩ signals and CaM function. NAP-22 Neuronal substrate of C 46 Calmodulin: regulation of effectors Striatin Neuronal, associates with 2A 47 An important obstacle to studies on 2ϩ CAP-19 Neuronal, IQ calmodulin-binding motif 48 many Ca -sensor proteins is the prob- lem of identifying downstream targets. EGF-receptor Human, CaM binds at juxtamembrane 49 Biochemical and genetic approaches MLC phosphatase (targeting subunit) Participant in muscle contraction/relaxation 50 have recently started to identify targets for some of the S100 class of proteins17 Connexin 32 Located at gap junctions 51 and also for members of the myristoyl- 2ϩ 18 ChURP Located in the nucleus 52 ated Ca sensors, such as frequenin . By contrast, CaM has been known for High MW protein phosphoprotein 53 some time to regulate several classes of 2ϩ Beta-2-glycoprotein Membrane-associated protein in kidney 54 proteins and in a Ca -depend- ent manner. The binding of target pro- Retinal proteins Involved in neuronal synaptic transmission 55 teins by CaM raises the affinity of CaM 2ϩ 19 Extracellular proteins Located in animal body fluids 56 for Ca by approximately tenfold and sensitizes the CaM–effector complex to Sperm proteins Spermatocyte, acrosome reaction 57 changes in Ca2ϩ concentrations. Interest- Plant proteins Plasma membrane transporter 58 ingly, many of the most highly charac- terized effectors (e.g. the CaM- Yeast proteins Involved in cell growth and division 59 dependent adenylyl cyclases, phospho- Phosphatidylinositol 3-kinase Component in receptor signalling 60 diesterases, protein kinases and the protein phosphatase ) are directly or

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Low [Ca2+] High [Ca2+] apo + A indirectly involved in protein . CaM-A CaM-A+ CaM also regulates the activities of the plasma- CaM 2ϩ − B membrane Ca pump, various ion channels, the apo + B CaM-B and isoforms of the inositol CaM CaM + B (1,4,5)-trisphosphate receptor. The list of CaM tar- + C gets is extensive and constantly growing (Table 1). CaM CaM-C CaM-C+ Are there differences in the mechanisms by which − C CaM and other Ca2ϩ transducers regulate their + D targets? CaM performs a variety of roles, and CaM- CaM CaM-D- − binding proteins can be categorized into at least six D classes based on their modes of regulation in the CaM-E++ presence and absence of Ca2ϩ (Fig. 4). One group of effectors, which we designate class A, binds essen- ϩ tially irreversibly to CaM irrespective of Ca2 . CaM CaM-E+ is thus more appropriately considered a subunit of + E + F these proteins. One example is phosphorylase CaM kinase, an that requires denaturing condi- − E − F CaM-F+ tions to dissociate CaM but is activated in the trends in Cell Biology presence of Ca2ϩ. Members of a second group of ef- fectors (class B) bind to CaM in the absence of Ca2ϩ FIGURE 4 (i.e. to the apo-CaM form) but dissociate reversibly Ca2ϩ-dependent functions of various classes of calmodulin-binding (CaM-binding) 2ϩ in the presence of Ca (Ref. 20). Examples include proteins. CaM and various classes of targets exist in free or bound states. Target proteins such as neuromodulin and neurogranin, classes A, B and C are associated with CaM or Ca2ϩ-free CaM (apo-CaM) at low which might serve as intracellular reservoirs for (resting) intracellular free Ca2ϩ concentrations (red). When Ca2ϩ concentrations are 2ϩ CaM at resting concentrations of Ca but liberate high (green), class B dissociates from CaM, classes D, E and F associate with ϩ ϩ Ca2 -activated CaM in response to a transient Ca2 CaM, classes A, C, E and F are activated by CaM (ϩ), and class D is inactivated signal. by CaM (Ϫ). A third group of effectors (class C) includes smooth-muscle -light-chain kinase (MLCK) and calcineurin. These class-C effectors form low- various classes of effectors. As CaM, like many Ca2ϩ affinity, inactive complexes with CaM at low con- sensors, is a relatively small protein, it must there- centrations of Ca2ϩ, when CaM is unoccupied or fore use multiple interaction surfaces to accomplish partially occupied by Ca2ϩ [Ͻ2 (mole Ca2ϩ) (mole these ends. These interaction sites enable CaM to CaM)Ϫ1]. At high concentrations of Ca2ϩ, these tar- convert the energy provided by Ca2ϩ binding into gets engage in a high-affinity complex and are acti- effector regulation. vated by CaM21,22. A fourth class of proteins (class D) binds to CaM in the presence of Ca2ϩ, but, in this Calmodulin–effector coupling: binding and case, CaM inhibits their function. This group in- activation cludes enzymes such as select members of the G- The Ca2ϩ-controlled exposure of hydrophobic protein-receptor kinases23, as well as the inositol groups in the two domains of CaM releases a con- (1,4,5)-trisphosphate receptor type 124. siderable amount of biochemical energy, which is A fifth group of effectors (class E), such as the transduced into two separable effects: a change in CaM-dependent protein kinases I, II and IV, exhibit the affinity of CaM for the effector and/or an al- more conventional behaviour and are activated by teration in the effector’s function. Studies focusing on Ca2ϩ–CaM. The class-E targets also exhibit an access- one group of CaM-regulated enzymes in particular, ory form of regulation in which CaM binding pro- the CaM-dependent protein kinases, have provided motes their regulation (specifically via phosphoryl- important insights into some of the mechanisms ation) by another CaM-regulated kinase (i.e. a underlying these phenomena. A short peptide of CaM-kinase kinase ), which we designate class F. In ~20 residues that is responsible for binding the specific case of the multimeric CaM kinase II, Ca2ϩ–CaM, designated a CaM-binding domain, has both the substrate and the catalytic subunits require been identified in many CaM-regulated proteins CaM binding to promote intermolecular autophos- (Fig. 5a) and in other types of CaM-binding pro- phorylation25. This novel case, in which one CaM- teins26. The crystal structure of CaM kinase I reveals dependent protein (class E) is directly regulated by that the CaM-binding domain directly interacts another CaM-dependent protein (class F), demon- with and sterically obstructs the putative substrate- strates the convergence of different CaM-regulated binding sites of the inactive enzyme27. Furthermore, pathways and is indicative of CaM-signalling the N-terminal part of the CaM-binding sequence cascades. loops away from the enzyme, exposing the The observation that CaM regulates a specific set hydrophobic side chain of Trp303 to solvent and of proteins yet engages in different types of Ca2ϩ- providing potential access for Ca2ϩ–CaM to bind dependent interactions implies that CaM and its (Fig. 5b). This proposal is supported by experiments targets both exhibit certain complementary features showing that the mutation of Trp303 to Ser in CaM that enable CaM recognition but possess other as- kinase I significantly lowered the apparent affinity pects that still allow CaM to discriminate between of CaM kinase I for Ca2ϩ–CaM28.

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(a) Interaction site on calmodulin FIGURE 5 CaM C-domain CaM N-domain (a) Alignment of amino acid sequences from selected calmodulin-binding (CaM-binding) domains. The enzymes are CaM kinase I (CaM KI), CaM kinase II (CaM KII), smooth- and skeletal-muscle myosin-light-chain kinases (smMLCK and CaM KI S K W K Q A F N A T A V V R H M R K skMLCK), the plasma-membrane Ca2ϩ-pump–ATPase (Ca2ϩ CaM KII R K L K G A I L T T M L A T R N F S ATPase) and CaM-kinase kinase (CaM KK). In most cases, a hydrophobic residue (red) from the corresponding peptides R K W Q K T G H A V R A I G R L S S ϩ smMLCK interacts with the C-terminal domain of Ca2 –CaM. The boxed skMLCK R R W K K N F I A V S A A N R F K K residue in CaM KI is Trp303. The N-terminal domain of Ca2ϩ–CaM interacts primarily with the C-terminal half of the Ca2+ATPase I L W F R G L N R I Q T Q I R V V N peptides. (For additional information on CaM binding domains, CaM KK P S W T T V I L V K S M L R K R S F see http://calcium.oci.utoronto.ca/) (b) Two crystal 2ϩ structures showing the main chain of Ca 4–CaM on the left and CaM kinase I on the right. The N-terminal domain of CaM and the ATP-binding lobe of CaM kinase I are both positioned on top, with helices red and sheets green. The Trp303 side chain from the CaM-binding domain of CaM kinase I (black) extends away (b) from the enzyme in the direction of CaM. (c) Crystal structure 2ϩ showing the main chain of Ca 4–CaM (white) in complex with the helical CaM-binding domain of smooth-muscle myosin-light- chain kinase (green). CaM wraps around the helix so that the conserved Trp of the peptide makes contact with Met124 (red) in the C-terminal domain of CaM.

A homologous hydrophobic residue is conserved in other CaM kinases (Fig. 5a). In the absence of detailed information on complexes between CaM and its intact effectors, spectroscopic and crystallo- graphic studies of Ca2ϩ–CaM complexed with pep- tides corresponding to the CaM-binding domains of four CaM kinases including CaM kinase I show in each case that this conserved hydrophobic residue interacts exclusively with the methionine-rich hydrophobic pocket in the C-terminal domain of Ca2ϩ–CaM29–32 (Fig. 5c). Recently determined three- dimensional structures of Ca2ϩ–CaM bound to peptides from the plasma membrane Ca2ϩ–ATPase pump and a CaM-kinase kinase also reveal additional modes of interaction between CaM and these other CaM-binding peptides33,34. These peptide studies indicate that the C-terminal domain of Ca2ϩ–CaM (c) might confer binding energy on the intact enzymes. Indeed, this appears to be the case because comple- mentary mutagenesis experiments on the Met residues of CaM showed that an evolutionarily in- variant Met124 in the C-terminal domain of Ca2ϩ–CaM that contacts the conserved hydrophobic residues in several CaM-binding peptides (Fig. 5c) is Main chain of necessary for high-affinity binding and activation of 2+− CaM kinase I as well as for three other CaM-dependent Ca 4 CAM protein kinases35,36. In contrast to the C-terminal domain of CAM-binding domain Ca2ϩ–CaM, residues in the hydrophobic pocket of of smMLCK the N-terminal domain of Ca2ϩ–CaM perform vary- ing functions with different CaM-dependent kinases. Conserved tryptophan The results from the crystallographic studies show that hydrophobic residues in the N-terminal do- ϩ Met124 residue main of Ca2 –CaM mainly interact with the C- terminal part of the CaM-binding peptides of smooth- muscle MLCK and CaM kinase II, respectively (Fig. 5a). Progressive C-terminal deletions and chimeric substitutions in the CaM-binding domain of smooth-muscle MLCK showed that the C-terminal trends in Cell Biology half of the CaM-binding domains of these enzymes

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are required for Ca2ϩ–CaM-dependent acti- conformation of the intact, folded enzymes restricts vation37,38. Furthermore, deletion studies of CaM their ability to adapt similarly to structural changes kinase I show that the C-terminal portion of its within their CaM-binding domains. It is helpful to CaM-binding sequence confers high affinity for bear these caveats in mind when peptides are used Ca2ϩ–CaM, as well as CaM-dependent activity39. to model effector function. These results complement those from experiments on CaM showing that the hydrophobic pocket in its Perspective and conclusion N-terminal domain generates a high-affinity com- The extensive characterization of CaM provides a plex with CaM kinase II, activates smooth-muscle useful precedent for less-well-understood Ca2ϩ sen- MLCK and combines the functions of high-affinity sors. At the subcellular level, the spatial and temporal binding and activation of CaM kinase I35,40. coordination between Ca2ϩ, CaM and its effectors In comparison with unbound Ca2ϩ–CaM, the are important for channelling all three components Ca2ϩ–CaM–peptide complexes exhibit a dramatic into a productive signalling pathway. The ability of contraction enabling CaM to wrap around and se- CaM to integrate Ca2ϩ signals into different cellular quester the helical CaM-binding peptides (Fig. 5c). contexts by migrating between various compart- Experiments on hydrophilic residues of CaM show ments further underscores this point. At the inter- that charged and polar residues are also required to molecular level, CaM uses different modes of Ca2ϩ- activate the smooth-muscle MLCK by promoting dependent interactions, which are responsible the accessibility of substrate to this particular en- for generating high affinity as well as specificity for zyme. Surprisingly, the hydrophilic residues on targets. At the submolecular level, the Ca2ϩ-triggered CaM responsible for this effect are originally exposure of energy-donating groups on CaM is coupled separated from each other on both domains of free to energy-accepting groups on its targets, leading to Ca2ϩ–CaM by more than 50 Å but then form a changes in Ca2ϩ binding by CaM as well as in the stable ‘latch’ less than 5 Å apart in the Ca2ϩ– function of its effectors. CaM–peptide complex41. These polar groups do not Finally, how are these levels of regulating CaM contact the CaM-binding peptide directly, so they related to each other? It is likely that the mobility of might exert their effects on the intact protein kinase separate pools of CaM derives from the different by interacting with an area distinct from its CaM- interactions between CaM and its targets. Therefore, binding domain. Indeed neutron- and X-ray- some classes of proteins might anchor CaM to spe- scattering studies on the kinase domain of skeletal- cific cellular locations, depending on the stability of muscle MLCK indicate that the CaM-binding a particular CaM–effector complex in the absence or domain is displaced to one side of the enzyme by presence of a Ca2ϩ signal. The affinity of such com- the binding of Ca2ϩ–CaM42. This event could expose plexes is likely to be due to complementary interac- the substrate-binding site of the enzyme and indicates tions between sites on the target proteins and sites how CaM might remove an inhibitory CaM-binding on CaM that change conformation in response to domain away from the kinase domain, thus leading Ca2ϩ. The Ca2ϩ-dependent interactions not only af- to enzyme activation. fect the affinity of the complex but also regulate the The preceding mechanistic studies show that the activity of effectors. This apparent ability of a regulation of enzymes by Ca2ϩ–CaM is a highly or- CaM–effector complex to decode Ca2ϩ signals has dered, cooperative and complementary process that been exemplified in a recent study showing that contributes to both the affinity and specificity for CaM participates in converting Ca2ϩ oscillations into targets. Another surprising outcome is the discovery changes in the autonomous enzymatic activity of at that the structures of Ca2ϩ–CaM–peptide complexes least one target, CaM kinase II44. Additional studies are relevant to their corresponding enzymes. However, on CaM will lead to a more-complete integration of in addition to the obvious limitation in the use of its levels of regulation. Meanwhile, it will be inter- peptides to study mechanisms of enzyme acti- esting to see whether any of the mechanisms exhib- vation, there are mounting indications that peptides ited by CaM will be relevant to other Ca2ϩ sensors. might not be entirely suitable for studying other functions of the full-length target protein. For References example, Ca2ϩ has a considerably higher affinity for 1 Ghosh, A. and Greenberg, M.E. (1995) in neurons: the CaM–MLCK-peptide complex than for the cor- molecular mechanisms and cellular consequences. Science 268, 239–246 19 responding CaM–MLCK-enzyme complex . Also, 2 Allbritton, N.L. and Meyer, T. (1993) Localized calcium spikes and mutations in the CaM-binding domain of smooth- propagating calcium waves. Cell Calcium 14, 691–697 muscle MLCK have a significantly greater effect on 3 Hardingham, G.E. and Bading, H. (1998) Nuclear calcium: a key CaM binding and activation than the same changes regulator of . Biometals 11, 345–358 4 Thomas, A.P. et al. (1996) Spatial and temporal aspects of cellular within the context of the corresponding CaM-binding calcium signaling. FASEB J. 10, 1505–1517 ϩ peptide40. Differences in the Ca2 -dependent inter- 5 Dolmetsch, R.E. et al. (1998) Calcium oscillations increase the efficiency action of CaM with either the CaM-binding peptide and specificity of gene expression. Nature 392, 933–936 of skeletal-muscle MLCK or the intact enzyme have 6 Ikura, M. (1996) Calcium binding and conformational response in 43 E–F hand proteins. Trends Biochem. Sci. 21, 14–17 also been observed by small-angle scattering . One 7 Braunewell, K-H. and Gundelfinger, E.D. (1999) Intracellular neuronal explanation for the adaptability and the higher calcium sensor proteins: a family of E–F hand calcium-binding proteins affinity exhibited by the shorter CaM-binding do- in search of a function. Cell Tissue Res. 295, 1–12 mains is the conformational flexibility inherent in 8 Swindells, M.B. and Ikura, M. (1996) Pre-formation of the semi-open conformation by the apo-calmodulin C-terminal domain and isolated peptides. By contrast, the relatively fixed implications for binding I–Q motifs. Nat. Struct. Biol. 3, 501–504

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328 trends in CELL BIOLOGY (Vol. 10) August 2000