REVIEW ARTICLE Novel aspects of calmodulin target recognition and activation Stefan W. Vetter 1 and Estelle Leclerc 2 1 Department of Molecular Biology and 2 Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA Several crystal and NMR structures of calmodulin (CaM) in complex with fragments derived from CaM-regulated pro- teins have been reported recently and reveal novel ways for CaM to interact with its targets. This review will discuss and compare features of the interaction between CaM and its target domains derived from the plasma membrane Ca 2+ -pump, the Ca 2+ -activated K + -channel, the Ca 2+ / CaM-dependent kinase kinase and the anthrax exotoxin. Unexpected aspects of CaM/target interaction observed in these complexes include: (a) binding of the Ca 2+ -pump domain to only the C-terminal part of CaM (b) dimer for- mation with fragments of the K + -channel (c) insertion of CaM between two domains of the anthrax exotoxin (d) binding of Ca 2+ ions to only one EF-hand pair and (e) binding of CaM in an extended conformation to some of its targets. The mode of interaction between CaM and these targets differs from binding conformations previously observed between CaM and peptides derived from myosin light chain kinase (MLCK) and CaM-dependent kinase IIa (CaMKIIa). In the latter complexes, CaM engulfs the CaM- binding domain peptide with its two Ca 2+ -binding lobes and forms a compact, ellipsoid-like complex. In the early 1990s, a model for the activation of CaM-regulated proteins was developed based on this observation and postulated activa- tion through the displacement of an autoinhibitory or regulatory domain from the target protein upon binding of CaM. The novel structures of CaM-target complexes dis- cussed here demonstrate that this mechanism of activation may be less general than previously believed and seems to be not valid for the anthrax exotoxin, the CaM-regulated K + - channel and possibly also not for the Ca 2+ -pump. Keywords: calmodulin; CaM-binding domains; Ca 2+ - signaling; cellular signaling; CaM-target activation. Ca 2+ signaling and calmodulin The calcium metal ion (Ca 2+ ) plays an uniquely important role in the physiology of higher organisms and is involved in the regulation of many cellular processes ranging from gene transcription and neurotransmitter release to muscle con- traction and cell survival [1–4]. The intracellular concentra- tion of free Ca 2+ is tightly controlled and usually very low inside the cytosol (0.1 l M ) whereas the extracellular con- centration of Ca 2+ is roughly 10 000-fold higher (1 m M ). Various stimuli, such as changes in membrane polarization or small receptor ligands can trigger the opening of calcium channels residing in the plasma membrane, resulting in the influx of Ca 2+ ions into the cytosol. In addition, several intracellular organelles function as Ca 2+ stores, which can release Ca 2+ upon stimulation by, for instance, inositol- 1,4,5-trisphosphate (InsP 3 ) or cyclic ADP-ribose [5–7]. The sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) are major Ca 2+ stores, but mitochondria and the nucleus also participate actively in the release of Ca 2+ through the InsP 3 -receptor or ryanodine receptor [3,4,8–11]. The export of Ca 2+ ions from the cytosol into the extracellular space or into intracellular organelles is achieved by ATP-driven Ca 2+ -pumps and exchangers. The key proteins involved in the transport of Ca 2+ ions, (e.g. voltage gated Ca 2+ channels, the InsP 3 -receptor and ryanodine receptors, sarcoplasmic Ca 2+ -pump (SERCA), endoplasmic Ca 2+ -pump, plasma membrane Ca 2+ -pump and others) are regulated by many different signals inclu- ding secondary messenger molecules (e.g. InsP 3 ,cADPR, etc.), protein phosphorylation, neurotransmitters, mem- brane potential and of course by the concentration of Ca 2+ ions itself [12–15]. In addition, Ca 2+ ions are involved in the regulation of phospholipase C, the enzyme generating InsP 3 from PtdIns3,4P 2 , PtdIns-3-kinase, the enzyme metaboli- zing InsP 3 to InsP 4 and in the regulation of protein kinase C (PKC), which can phosphorylate the InsP 3 receptor [16,17]. These interdependencies generate an intricate network of feedback loops which can result in oscillations of Ca 2+ concentration of defined amplitude, frequency and location [18–25]. The mechanism of how the information contained in Ca 2+ oscillation is decoded is not clear, but it has been shown, for example, that the kinase activity of CaM- dependent kinase II is sensitive to Ca 2+ oscillations [26–29]. The approximately 100-fold increase in free Ca 2+ con- centration upon stimulation of a cell allows Ca 2+ -binding proteins to bind Ca 2+ ions. Several hundred Ca 2+ -binding proteins have been identified and most of them share a common Ca 2+ binding motif [30–33]. This motif comprises Correspondence to S. W. Vetter, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Fax: + 1 858 784 2857, Tel.: + 1 858 784 2425, E-mail: svetter@scripps.edu Abbreviations: CaM, calmodulin; MLCK, myosin light chain kinase; CaMKIIa, CaM-dependent kinase IIa; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; SERCA, sarcoplasmic Ca 2+ pump; PKC, protein kinase C; PtdIns, phosphatidylinositol; CaMKK, CaM-dependent kinase kinase; InsP 3 , inositol-1,4,5-trisphosphate. (Received 10 September 2002, revised 2 December 2002, accepted 5 December 2002) Eur. J. Biochem. 270, 404–414 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03414.x about 30 amino acids and consists of a helix-loop-helix where the two helices are arranged similar to the extended thumb and index finger of a hand: it is commonly called the EF-hand motif. In almost all Ca 2+ -binding proteins two EF-hand motifs are in close proximity forming an EF-hand pair. This consists of four helices arranged in the form of a twisted four helix bundle [34,35]. Structurally different Ca 2+ -binding motifs are found in pentraxins, annexins and in C 2 domains [36–39]. The C 2 domains consist of about 130 amino acids forming a compact b-sandwich composed of two four-stranded antiparallel b-sheets and bind up to three Ca 2+ ions in the loops connecting the strands [40,41]. Over 100 intracellular proteins have been found to contain C 2 domains and several proteins important for signal transduc- tion are regulated by Ca 2+ binding to C 2 domains. Ca 2+ binding enables phospholipid binding to the C2 domain and subsequent recruitment of the enzymes to phospholipid membranes. Examples of such proteins are cytoplasmic phospholipase A, phosphoinositide-specific phospholipase C, PtdIns-3-kinase, protein kinase C, RAS-GTPase activa- ting protein and many others [42–48]. In synaptic vesicles a group of transmembrane proteins called synaptotagmins are involved in the Ca 2+ -dependent neurotransmitter release and C2 domains function as Ca 2+ effector domains [49–52]. Calmodulin is a highly conserved, soluble, intracellular, 15 kDa Ca 2+ -binding protein ubiquitously found in ani- mals, plants, fungi and protozoa, and is regarded as a major transducer of Ca 2+ signals in mammalian cells [53,54]. Many proteins involved in Ca 2+ signal transduction alter their activity in response to changes in free Ca 2+ levels, but are themselves not able to bind Ca 2+ ions. Some of these proteins utilize CaM as a sensor and mediator of the initial Ca 2+ signal. CaM relays the Ca 2+ signal by binding free Ca 2+ ions to its C- and N-terminal EF-hand pairs, which causes a conformational change and enables Ca 2+ /CaM to bind to specific CaM-binding domains. The binding of Ca 2+ /CaM to its target proteins alters their activity in a calcium dependent manner. The details of the actual mechanism of activation are only partially understood for most CaM-regulated proteins. A major obstacle in obtaining detailed insights in the struc- tural basis for CaM/target recognition and activation is the fact that many CaM targets are large multimeric or transmembrane proteins which makes it difficult to obtain NMR or crystal structures of CaM in complex with functional target proteins. Structure of calmodulin and structural changes upon Ca 2+ binding The overall structure of Ca 2+ /CaM as determined by X-ray crystallography is shown in Fig. 1 [55,56]. The protein is dominated by two EF-hand pairs forming the C- and N-terminal lobes and a long a-helix connecting the two lobes. The two EF-hand pairs share 48% sequence identity and 75% sequence homology and the peptide backbone of the two lobes can be superimposed with a mean square derivation of  0.7 A ˚ [57]. Yet, reflecting the differences in sequence, the Ca 2+ -binding affinities are different for the N- and C-terminal lobe, with the C-terminal lobe having a 10-fold higher Ca 2+ -binding affinity (K d  0.2 l M ) than the N-terminal lobe (K d  2 l M ) [58,59]. Mg 2+ and K + ions also bind to CaM, but with 10 3 )10 4 lower affinity than Ca 2+ enabling CaM to respond specifically to increases in Ca 2+ concentration [60]. The dissociation constant (K d ) of CaM for Ca 2+ decreases significantly in ternary complexes when Ca 2+ / CaM is bound to target peptides, mainly as the result of a decrease in the dissociation rate k off of the Ca 2+ ion from the EF-hands [61–63]. CaM–peptide complex dissociation has been suggested to be initiated by the loss of Ca 2+ ions from the N-terminal CaM lobe, likely followed by loss of Ca 2+ from the C-terminal lobe and subsequently followed by rapid loss of the peptide [62]. Due to the presence of four EF-hand (helix-loop-helix) motifs, each of which is about 30 residues in length, only a small portion of the 148 residue long CaM molecule does not participate in Ca 2+ binding. This includes 8–10 residues in the central region of the protein sequence (residues 76– 84). In the crystal structure, this central linker region is folded into a long a-helix, connecting the fourth helix of the N-terminal lobe with the first helix of the C-terminal lobe. The length and expected rigidity of the central helix, as suggested from the crystal structure, does not agree well with data from small angle X-ray scattering and fluores- cence energy transfer measurements in solution, which generally indicate a shorter distance between the C- and N-terminal lobes of CaM [64–69]. The conformation and functional significance of this linker region has been subject to intensive debate. Mutations within the central linker with the aim to disrupt or to stabilize the helical conformation, or to change the length of the helix, did not abolish the ability of CaM to recognize and to activate its targets [70–74]. It was ultimately shown beyond doubt by NMR as well as by analyzing X-ray diffraction data that the observed a-helical conformation of the central linker is a consequence of crystal packing [75,76]. In solution, the central linker region is very flexible and unstructured. Consequently, the N- and C-terminal lobes do not adopt a defined orientation relative to each other in solution, but display a tumbling motion, being held together by the central linker. Comparison of the structures of apo-CaM and Ca 2+ / CaM determined by NMR and X-ray crystallography reveals that the overall structure and distribution of secondary structure elements is very similar in both molecules (Fig. 1) [77–79]. The major conformational change induced by the binding of Ca 2+ ions into the EF-hand pairs is a significant alteration of the relative orientation of the helices in each lobe [31,58,80]. As the result of a twist-like motion, the relative angles between the helices change from 121°)144° in apo-CaM to 86°)116° in Ca 2+ /CaM [79]. This rearrangement of the helices leads to the exposure of several hydrophobic residues to the solvent, which form a large hydrophobic, concave patch or channel on the surface of each lobe (Fig. 2). Structure of CaM bound to short peptides Most physiological relevant CaM targets are proteins, but CaM also binds to a number of synthetic peptides corresponding to CaM binding domains, to peptide hormones and toxins, as well as to small drug like molecules [81–84]. Analysis of CaM binding peptides revealed that Ó FEBS 2003 CaM target recognition and activation (Eur. J. Biochem. 270) 405 they share no sequence homology, but fulfill minimal structural characteristics: they all have the potential to fold into a basic, amphiphilic a-helix [85–89]. They display large hydrophobic residues in conserved positions either 1-5-10 or 1-8-14, which point to one face of in a presumed a-helical conformation. These structural characteristics have been used to predict several dozen potential CaM-binding sites from DNA and protein databases, as well as to engineer synthetic CaM-binding peptides [85,86,90]. Biophysical characterization of complexes between CaM and synthetic peptides indicated that the ternary complexes have a more compact shape than Ca 2+ /CaM by itself [64– 66,91]. A collapsed structure of ternary Ca 2+ /CaM/peptide complexes was also concluded from photoaffinity and cross- linking experiments [92,93]. In order to reduce the distance between the C- and N-terminal lobes it is necessary to allow a kink or bend in the central helix. An early model, proposed before the first structure of a CaM/peptide complex had been solved, suggested a 45° bend within the central helix at residue Ser81 [94,95]. Some speculation about the true binding mode of peptides to CaM was finally resolved when the atomic resolution structures of three CaM-peptide complexes from smooth muscle myosin light chain kinase (smMLCK), skeletal myosin light chain kinase (skMLCK) and CaM-dependent kinase IIa (CaMKIIa) were solved by Fig. 1. Ribbon presentations of CaM and CaM in complex with target peptides. CaM is colored blue, the CaM targets are red, Ca 2+ ions are yellow. The N-terminal lobe of CaM is orientated to the top, the C-terminal lobe to the bottom of the figures. Structural data were taken from the Protein Data Bank, accession codes: apo-CaM (1CFD) Ca 2+ /CaM (1CLL); CaM/CaMKIIa (1CM1); CaM/CaMKK (1IQ5); CaM/Ca 2+ -pump (1CFF); CaM/K + -channel (1G4Y), CaM/anthrax exotoxin (1K93). Table 1. Key features of CaM/peptide complexes. CaM target Target size (amino acids) CaM conformation Target conformation Anchor residues Ca 2+ ions bound Method Year smMLCK 20 Collapsed Helix 1-5-8-14 4 NMR 1992 skMLCK 26 Collapsed Helix 1-8-14 4 X-ray 1992 CaMKIIa 25 Collapsed Helix 1-5-10 4 X-ray 1992 CaMKK 27 Collapsed Helix + b-hairpin loop 1-5-16 4 NMR 1999 Ca 2+ -pump 20 Extended Helix, binds to C-terminal lobe only 1-5-8 4 NMR 1999 K + -channel 95 Extended 3 Helices, forms dimer Nonclassical 2 (N-terminal lobe) X-ray 2001 Anthrax exotoxin 510 Extended Complex Nonclassical 2 (C-terminal lobe) X-ray 2002 406 S. W. Vetter and E. Leclerc (Eur. J. Biochem. 270) Ó FEBS 2003 multidimensional NMR and X-ray crystallography in 1992 (Table 1) [96–98]. The structures of all three complexes are quite similar and only the complex between CaM and the peptide derived from CaMKIIa will be discussed here to highlight the main features. The CaM/CaMKIIa complex is of ellipsoidal shape and much more compact (50 · 30 · 30 A ˚ 3 ) than Ca 2+ /CaM without the peptide (maximal length of 65 A ˚ ) (Fig. 1). The central linker region is unwound and allows the C- and N-terminallobestobendby100° and to rotate by 120° relative to their orientation seen in the Ca 2+ /CaM crystal structure. The peptide is bound in an a-helical conformation and is engulfed by Ca 2+ /CaM into a hydrophobic channel formed by bringing the C- and N-terminal lobes close together (Fig. 2). The binding of CaM-binding peptides is largely driven by hydrophobic interactions between hydrophobic anchor residues of the peptide with the hydrophobic surface cavities of CaM. Methionine residues, unusually abundant in CaM, play a particularly important role in the binding of target peptides. The methionine side chains are very flexible and the sulfur atom has a larger polarizability than carbon, resulting in stronger van der Waals interactions. The hydrophobic patches of each lobe are surrounded by several charged residues, creating charged binding channel outlets. The C-terminal end of the peptide-binding channel has a negatively charged rim, whereas the N-terminal hydrophobic patch has clusters of negatively and positively charged residues. This charge distribution on the molecular surface contributes to peptide binding via electrostatic inter- actions and determines the relative binding orientation of CaM-binding domain peptides. Basic residues at the N-ter- minus of the peptide form salt bridges with acidic residues surrounding the peptide-binding channel of the C-terminal lobe of CaM (Fig. 2). The peptide-binding orientation is also discussed for the complex of CaM with the peptide derived from CaM-dependent kinase kinase (CaMKK). The binding mode of CaM to target peptides observed in these early structures was in agreement with a proposed model for the regulation of CaM-targets by auto-inhibitory domains. Limited proteolysis or mutation of the plasma membrane Ca 2+ -pump, the cAMP phosphodiesterase, myosin light chain kinase, calcineurin or neuronal nitric oxide synthase led to constitutively activated enzymes [99– 103]. In the early 1990s, a model was developed in which an auto-inhibitory domain becomes displaced from the active site upon binding of CaM, leading to the activation of the enzymes. Plasma membrane Ca 2+ -pump However, it has always been clear that the binding mode, where CaM engulfs the target domain in a collapsed conformation, could not generally apply to all CaM- regulated enzymes. The CaM-binding domain of the Ca 2+ -pump has hydrophobic anchor residues in positions Fig. 2. The CaM/CaMKIIa complex. The N-terminal lobe of CaM is rendered as a ribbon, the C-terminal lobe is represented by its electrostatic surface, the CaMKIIa peptide is shown as a blue ribbon. Negative electrostatic potential is indicated in red. The concave shape and hydrophobic lining of the peptide-binding surface is visible. Ó FEBS 2003 CaM target recognition and activation (Eur. J. Biochem. 270) 407 1-8-14. However, a splice site within the CaM binding domain gives rise to splice isoforms, which can lack the last anchor residue in position 14 [104]. Interestingly, the Ca 2+ - pump of the plasma membrane can be activated not only by full length Ca 2+ /CaM, but also with just the C-terminal lobe of CaM alone [105]. To address this observation the NMR structure of CaM in complex with the peptide C20W has been solved recently [106]. The peptide C20W corres- ponds to the N-terminal part of the CaM-binding domain whichiscommontoallCa 2+ -pump isoforms, but lacks the third hydrophobic anchor residue in position 14 (Table 1). The structure of the C20W/CaM is shown in Fig. 1 and reveals some unexpected features. Most significantly, the complex does not collapse into an ellipsoid shaped complex, but remains in an extended conformation. The central linker region appears flexible between Arg74 and Glu84 and medium range NOEs, which would indicate an a-helical conformation, are not observed in the NMR experiment. The two lobes do not contact each other and show large variations in their relative orientation. Secondly, the peptide C20W is bound only to the C-terminal lobe. It is bound in an a-helical conformation and is anchored to the peptide binding channel of the C-terminal lobe through hydrophobic interactions involving three hydrophobic residues (Trp1, Leu5 and Ile8). All four methionine residues (Met109, Met124, Met144 and Met145) of the C-terminal lobe of CaM interact with the peptide, underlining the significance of methionine residues for target binding. The hydrophilic face of the amphiphilic peptide helix is exposed to the solvent. Overall, the binding of the hydrophobic face of the C20W peptide to the C-terminal lobe of CaM is equivalent to the binding mode seen in complexes with other peptides (MLCK or CaMKII), except that the C20W peptide does not induce the transition of CaM into a collapsed conformation. It can be speculated that the missing hydrophobic anchor residue in position 14 is responsible for the observed open conformation of the CaM/C20W complex. This is supported by small-angle X-ray scattering experi- ments on the complexes of CaM with the peptides C20W and C24W. The peptide C24W is slightly longer than C20W and contains the third hydrophobic anchor residues of the full length CaM-binding domains. The scattering data indicate an extended conformation of the CaM/C20W complex and a collapsed structure of the CaM/C24W complex [67]. The binding of the C20W peptide of the Ca 2+ -pump CaM-binding domain to only the C-terminal lobe of CaM demonstrates that Ôwrapping aroundÕ a CaM binding domain, as seen in the complexes with MLCK and CaMKIIa, is not necessary to activate certain CaM- regulated proteins. CaM in complex with CaMKK Synthetic CaM-binding peptides have been successfully designed based on the motif of a Ôbasic amphiphilic a-helixÕ and the peptides derived from MLCK, CaMKIIa and the Ca 2+ -pump do bind to CaM in a helical conformation. It was therefore surprising to see a variation of this binding mode for the CaM binding domain peptide derived from Ca 2+ /CaM-dependent kinase kinase [107]. CaMKK is a Ca 2+ /CaM dependent serine/threonine kinase which phos- phorylates CaM-dependent kinases I and IV, modulating a signal transduction cascade leading to Ca 2+ -regulated gene transcription [108]. The CaM-binding domain of CaMKK shows some differences compared to other CaM binding peptides. The hydrophobic anchor residues are further apart, separated by 14 residues, rather then the usual 8 or 12 spacing residues (Table 1). A cluster of basic residues is located near the C-terminal anchor residue, whereas other CaM-binding domains (MLCK, CaMKIIa and the Ca 2+ -pump) possess basic residues near their N-terminal anchor residue. The structure of the complex has been determined by NMR and is shown in Fig. 1 [107]. The complex adopts a collapsed conformation similar to the CaM/CaMKIIa complex and the peptide is engulfed between the N- and C-terminal lobes of CaM. Two features of the complex are distinct from other complexes: the peptide is bound in an ÔinvertedÕ orientation, i.e. binding with its C-terminus to the C-terminal lobe of CaM. In contrast, the MLCK, CaMKKIIa and Ca 2+ -pumppeptidesbindwiththeir N-termini to the C-terminal lobe of CaM. This suggests that the position of basic residues at either the C- or N-terminal end of the binding domain peptides and charge comple- mentarity of binding surfaces determine the binding orien- tation of the peptide in complex with CaM. The second interesting feature is the conformation of the CaMKK binding domain peptide in complex with CaM. The CaMKIIa and MLCK peptides are bound in a largely a-helical conformation, and some of the terminal residues of the peptides are not unexpectedly flexible and structurally not defined. The CaMKK binding domain peptide in contrast is only partially a-helical and contains an addi- tional well-defined b-hairpin like loop. The helical part of the peptide contains a hydrophobic anchor residue Trp444 which interacts with the hydrophobic patch of the N-terminal lobe. The b-hairpin loop contains a hydropho- bic anchor residue Phe459 which interacts with hydrophobic residues of the C-terminal lobe of CaM (Table 1). The spacing between the two anchor residues is 14 residues, which would possibly prevent binding of the anchor residues to the hydrophobic surface cavities of CaM if the peptide would fold into a straight helix. However, the loop allows the peptide to fold backwards and to position the second hydrophobic residues in such a way that it can bind into the C-terminal hydrophobic recognition pocket of CaM. The binding mode of the CaMKK peptide is a significant variation from the ÔclassicalÕ binding mode of a-helical peptides to CaM and has to be added to the growing collection of the structural motifs recognizable by CaM. Ca 2+ activated K + -channel A radically novel target binding mode and possibly a novel mode of target protein activation through dimerization has been revealed with the crystal structure of CaM in complex with a fragment of the Ca 2+ -activated, small conductance K + -channel [109]. The ion-guiding pore of the K + -channel is formed by four a-subunits, each spanning the membrane several times and is gated by the intracellular Ca 2+ 408 S. W. Vetter and E. Leclerc (Eur. J. Biochem. 270) Ó FEBS 2003 concentration [110,111]. The C-terminal cytosolic portion of each a-subunit has one CaM molecule constitutively bound via a domain that shares no similarity to other CaM binding domains and is not Ca 2+ dependent. The structure of the complex between CaM and a 96 residue fragment corresponding to the C-terminal cytosolic portion of the K + -channel provides interesting insights into the mechanism of Ca 2+ activated gating of the channel and presents a new way of CaM binding domain recognition. The structure of the complex is shown in Fig. 1. Most strikingly, CaM remains in an extended conformation and forms a dimeric complex: two K + -channel fragments are clamped together by two molecules of CaM. The K + -channel fragment consists of a long a-helix and a shorter a-helix folded back antiparallel against the longer helix. In the complex, the K + -channel fragment is oriented almost perpendicularly to the long axis of the extended CaM molecule. The portion of the K + -channel fragment containing the two helices is bound to the C-terminal lobe of CaM. The EF-hand pair of the C-terminal lobe remains Ca 2+ free and the binding of this portion of the K + -channel peptide is Ca 2+ -independent. The C-terminal end of the fragment K + -channel peptide protrudes sideways and binds to the N-terminal lobe of a second CaM molecule. The N-terminal lobe of CaM binds two Ca 2+ ions and exposes subsequently the hydrophobic patch typical for Ca 2+ loaded CaM. The hydrophobic patch of CaM interacts with the C-terminus of the long helix of the second K + -channel fragment. This results effectively in the binding of three helices from two different peptide chains by each CaM molecule, compared to the binding of just one helix as seen in all other CaM structures. To accommodate the three helices CaM has to stretch, which is rendered possible by partial unfolding of the central linker. The dimeric CaM/K + -channel peptide complex shows the two long C-terminal helices of the K + -channel fragments in an anti-parallel, side-by-side orientation. The N-termini of the peptides are facing each other and pointing away from the plane formed by the two C-terminal helices. When viewed from the side, the two K + -channel peptides resemble an isosceles triangle. The N-termini of the K + -channel peptides connect to a transmembrane helix, which was not present in the structure of the CaM/ K + -channel peptide complex, but could be perpendicular to the C-terminal helices. The geometry of two perpendicular helices (C-terminal helix and transmembrane helix) con- nected by the short N-terminal helix resembles a molecular wrench. Movement of the C-terminal helix as the result of Ca 2+ binding to CaM and subsequent dimerization of the a-subunits of the K + -channel could translate into a rotation of the transmembrane helices and thus could allow K + ions to flow through the pore. Anthrax adenylate cyclase exotoxin The crystal structures of the C-terminal fragment (residues 291–800) of the exotoxin from Bacillus anthracis,withand without its activator calmodulin, is the first structure of a CaM-complex with a catalytically functional CaM-target [112–114]. Anthrax exotoxin (edema factor) is a calmo- dulin-dependent adenylate cyclase, but shares no overall structural homology with mammalian adenylate cyclases and uses a slightly different catalytic mechanism. The exotoxin enters host cells through a transporter (protective antigen) produced by the pathogen. Inside the cell, it acquires CaM, becomes activated and then converts large amounts of ATP to cAMP [115]. The structure of this large CaM complex is particularly interesting because it allows to compare the details of target activation by CaM at atomic resolution (about 3 A ˚ ) by comparison of three structures: (a) exotoxin without CaM (b) exotoxin with CaM and (c) exotoxin with bound CaM and the pseudo- substrate 3¢-dATP. The overall structure of CaM/exotoxin complex is shown in Fig. 1. Striking features of the complex are that CaM is inserted deeply between two domains of exotoxin and not merely bound onto exposed helix or surface loop. CaM is bound in an extended conformation, the central section (residues 79–81) is partially unwound and only the C-terminal EF-hand pair is loaded with Ca 2+ . CaM is tightly squeezed between two domains of exotoxin and this results in a large number of interacting residues: 53 CaM residues interact with 63 exotoxin resi- dues, leading to a very large buried surface area of 5.900 A ˚ 2 . The insertion of CaM between the two domains of exotoxin causes large conformational changes in the relative domain orientation (Figs 1 and 3). The 15 kDa large C-terminal portion of the protein is displaced by 15 A ˚ and rotated by 30° upon binding of CaM. In addition, several short sheet structures, helices and loops of exotoxin rearrange and change their conformation. The C-terminal lobe of CaM contains two Ca 2+ ions and its conformation is very similar to other structures of Ca 2+ -loaded CaM. The hydrophobic patch typical for Ca 2+ /CaM is exposed and forms a peptide-binding channel, which interacts with an a-helix of exotoxin. This a-helix shows many properties of a potential CaM-binding site: it forms a positively charged amphiphilic a-helix and contains hydrophobic anchor residues in positions 1-5-10. Fragments of exotoxin and synthetic peptides corresponding to this helical region have been demonstrated to bind CaM in vitro in a Ca 2+ -dependent manner. NMR experiments indicate further that the peptide is bound in an a-helical conforma- tion and fluorescence energy transfer experiments showed a reduced interdomain distance in the CaM/peptide complex, pointing towards a collapsed conformation of the CaM/ peptide complex [112,116]. These experimental observations are very similar to results obtained for synthetic peptides derived from other CaM-binding domains, such as MCLK and CaMKIIa. While there is no NMR or crystal structure of the CaM/exotoxin peptide complex available, it is reasonable to assume that the complex adopts a conforma- tion similar to the complexes with the MLCK or CaMKIIa peptides discussed above. However, the binding of CaM to the functional exotoxin fragment shows that CaM does not collapse around this helix, but instead binds to exotoxin in an extended conformation. The EF-hand pair of the N-terminal lobe of CaM is Ca 2+ -free in the CaM/exotoxin complex and shows a conformation typical for apo-CaM. The EF-hand helices are packed against several helices of the C-terminal domain of exotoxin (Fig. 3). The absence of Ca 2+ from the N-terminal lobe is remarkable, because the complex was crystallized in the presence of Ca 2+ ,makingit Ó FEBS 2003 CaM target recognition and activation (Eur. J. Biochem. 270) 409 Fig. 3. Structural changes occurring in the anthrax exotoxin upon binding of CaM. The N-terminal part of the protein (residues 280–450) was used to superimpose the structures of the exotoxin without CaM (blue) and exotoxin in complex with CaM (yellow) and the pseudosubstrate 3¢dATP (electrostatic surface potential shown) bound into the active site. Calmodulin has been omitted in the upper panel for clarity. The lower panel shows calmodulin (electrostatic surface rendering) inserted between the domains of the exotoxin and the relative distance from the substrate bound into the active site. 410 S. W. Vetter and E. Leclerc (Eur. J. Biochem. 270) Ó FEBS 2003 necessary for CaM to ÔloseÕ two Ca 2+ ions during its binding to exotoxin. The binding of CaM to exotoxin increases the catalytic activity of the enzyme approximately 1000-fold. Surpris- ingly, the activation of the enzyme is not achieved by dramatically changing the architecture of the active site, nor by removing an auto-inhibitory domain as in other CaM activated enzymes. Indeed, the binding of CaM does not even occur close to the active site. The N-terminal part of the exotoxin alone and in the presence of CaM can be superimposed and show no dramatic conformational changes as the result of CaM binding (Fig. 3). Instead, several loops close to the active site become stabilized. This relatively minor change around the active site enhances the catalytic activity through better substrate binding and positioning in the active site. Conclusion Target recognition and activation by CaM has been studied intensively over the last two decades and today we are further away from a general model than ever before. The CaM-target complexes discussed here (CaMKIIa,CaM- KK, Ca 2+ -pump, K + -channel, anthrax exotoxin) represent several structurally different CaM-complex architectures. With short, amphiphilic, a-helical peptides (CaMKIIa)a collapsed complex conformation is observed, but also b-hairpin-like loops can be accommodated in such a collapsed complex (CaMKK). Clustering of basic residues at the C- or N-terminus of CaM-binding peptides defines their relative orientation (parallel or antiparallel) in the peptide-binding channel of CaM. Binding of a target peptide to only the C-terminal lobe is observed for a truncated CaM-binding domain (Ca 2+ -pump) and can be attributed to a missing hydrophobic anchor residue. Binding of CaM to its targets in an extended, open conformation was not expected based on the structures of CaM complexed to peptides derived from smMLCK, skMLCK and CaMKIIa, but was found in three out of the four recent structures. The structure of the CaM/K + -channel complex shows three helices bound to CaM, which prevents CaM from adopting a collapsed conformation. In addition, two of these three helices interact with the Ca 2+ free N-terminal EF-hand pair, allowing CaM to be constitutively bound to the K + -channel and utilizing only the C-terminal lobe for detecting changes in Ca 2+ concentration. Also, the structure of CaM in complex with the K + -channel fragment suggests that some CaM targets can be activated through dimeriza- tion. For several Ca 2+ /CaM-regulated proteins a mechanism of activation through replacement of auto-inhibitory domains upon CaM binding has been demonstrated through biochemical studies. A model for the binding of CaM to its targets was based on the X-ray and NMR structures which showed the CaMKIIa and MLCK pep- tides engulfed by CaM. However, this binding seems less general than previously assumed as demonstrated by the structures discussed here. Particularly informative is the structure of CaM in complex with the anthrax exotoxin. Studies using short peptides derived from exotoxin had indicated a collapsed CaM/peptide complex. However, CaM binds in an exten- ded conformation to anthrax exotoxin and activates the enzyme through subtle changes in the surrounding of the active site. 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