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Keywords:

  • calmodulin;
  • CaM-binding domains;
  • Ca2+-signaling;
  • cellular signaling;
  • CaM-target activation

Abstract

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

Several crystal and NMR structures of calmodulin (CaM) in complex with fragments derived from CaM-regulated proteins 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 Ca2+-pump, the Ca2+-activated K+-channel, the Ca2+/CaM-dependent kinase kinase and the anthrax exotoxin. Unexpected aspects of CaM/target interaction observed in these complexes include: (a) binding of the Ca2+-pump domain to only the C-terminal part of CaM (b) dimer formation with fragments of the K+-channel (c) insertion of CaM between two domains of the anthrax exotoxin (d) binding of Ca2+ 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 IIα (CaMKIIα). In the latter complexes, CaM engulfs the CaM-binding domain peptide with its two Ca2+-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 activation through the displacement of an autoinhibitory or regulatory domain from the target protein upon binding of CaM. The novel structures of CaM-target complexes discussed 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 Ca2+-pump.


Abbreviations
CaM

calmodulin

MLCK

myosin light chain kinase

CaMKIIα

CaM-dependent kinase IIα

ER

endoplasmic reticulum

SR

sarcoplasmic reticulum

SERCA

sarcoplasmic Ca2+ pump

PKC

protein kinase C

PtdIns

phosphatidylinositol

CaMKK

CaM-dependent kinase kinase

InsP3

inositol-1,4,5-trisphosphate

Ca2+ signaling and calmodulin

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

The calcium metal ion (Ca2+) 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 contraction and cell survival [1–4]. The intracellular concentration of free Ca2+ is tightly controlled and usually very low inside the cytosol (0.1 µm) whereas the extracellular concentration of Ca2+ is roughly 10 000-fold higher (1 mm). 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 Ca2+ ions into the cytosol. In addition, several intracellular organelles function as Ca2+ stores, which can release Ca2+ upon stimulation by, for instance, inositol-1,4,5-trisphosphate (InsP3) or cyclic ADP-ribose [5–7]. The sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) are major Ca2+ stores, but mitochondria and the nucleus also participate actively in the release of Ca2+ through the InsP3-receptor or ryanodine receptor [3,4,8–11]. The export of Ca2+ ions from the cytosol into the extracellular space or into intracellular organelles is achieved by ATP-driven Ca2+-pumps and exchangers.

The key proteins involved in the transport of Ca2+ ions, (e.g. voltage gated Ca2+ channels, the InsP3-receptor and ryanodine receptors, sarcoplasmic Ca2+-pump (SERCA), endoplasmic Ca2+-pump, plasma membrane Ca2+-pump and others) are regulated by many different signals including secondary messenger molecules (e.g. InsP3, cADPR, etc.), protein phosphorylation, neurotransmitters, membrane potential and of course by the concentration of Ca2+ ions itself [12–15]. In addition, Ca2+ ions are involved in the regulation of phospholipase C, the enzyme generating InsP3 from PtdIns3,4P2, PtdIns-3-kinase, the enzyme metabolizing InsP3 to InsP4 and in the regulation of protein kinase C (PKC), which can phosphorylate the InsP3 receptor [16,17]. These interdependencies generate an intricate network of feedback loops which can result in oscillations of Ca2+ concentration of defined amplitude, frequency and location [18–25]. The mechanism of how the information contained in Ca2+ 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 Ca2+ oscillations [26–29].

The approximately 100-fold increase in free Ca2+ concentration upon stimulation of a cell allows Ca2+-binding proteins to bind Ca2+ ions. Several hundred Ca2+-binding proteins have been identified and most of them share a common Ca2+ binding motif [30–33]. This motif comprises 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 Ca2+-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 Ca2+-binding motifs are found in pentraxins, annexins and in C2 domains [36–39]. The C2 domains consist of about 130 amino acids forming a compact β-sandwich composed of two four-stranded antiparallel β-sheets and bind up to three Ca2+ ions in the loops connecting the strands [40,41]. Over 100 intracellular proteins have been found to contain C2 domains and several proteins important for signal transduction are regulated by Ca2+ binding to C2 domains. Ca2+ 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 activating protein and many others [42–48]. In synaptic vesicles a group of transmembrane proteins called synaptotagmins are involved in the Ca2+-dependent neurotransmitter release and C2 domains function as Ca2+ effector domains [49–52].

Calmodulin is a highly conserved, soluble, intracellular, 15 kDa Ca2+-binding protein ubiquitously found in animals, plants, fungi and protozoa, and is regarded as a major transducer of Ca2+ signals in mammalian cells [53,54]. Many proteins involved in Ca2+ signal transduction alter their activity in response to changes in free Ca2+ levels, but are themselves not able to bind Ca2+ ions. Some of these proteins utilize CaM as a sensor and mediator of the initial Ca2+ signal. CaM relays the Ca2+ signal by binding free Ca2+ ions to its C- and N-terminal EF-hand pairs, which causes a conformational change and enables Ca2+/CaM to bind to specific CaM-binding domains. The binding of Ca2+/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 structural 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 Ca2+ binding

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

The overall structure of Ca2+/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 α-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 Å[57]. Yet, reflecting the differences in sequence, the Ca2+-binding affinities are different for the N- and C-terminal lobe, with the C-terminal lobe having a 10-fold higher Ca2+-binding affinity (Kd ≈ 0.2 µm) than the N-terminal lobe (Kd ≈ 2 µm) [58,59]. Mg2+ and K+ ions also bind to CaM, but with 103−104 lower affinity than Ca2+ enabling CaM to respond specifically to increases in Ca2+ concentration [60].

image

Figure 1. Ribbon presentations of CaM and CaM in complex with target peptides. CaM is colored blue, the CaM targets are red, Ca2+ 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) Ca2+/CaM (1CLL); CaM/CaMKIIα (1CM1); CaM/CaMKK (1IQ5); CaM/Ca2+-pump (1CFF); CaM/K+-channel (1G4Y), CaM/anthrax exotoxin (1K93).

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The dissociation constant (Kd) of CaM for Ca2+ decreases significantly in ternary complexes when Ca2+/CaM is bound to target peptides, mainly as the result of a decrease in the dissociation rate koff of the Ca2+ ion from the EF-hands [61–63]. CaM–peptide complex dissociation has been suggested to be initiated by the loss of Ca2+ ions from the N-terminal CaM lobe, likely followed by loss of Ca2+ 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 Ca2+ 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 α-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 fluorescence 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 α-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 Ca2+/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 Ca2+ 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 Ca2+/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).

image

Figure 2. The CaM/CaMKIIαcomplex. The N-terminal lobe of CaM is rendered as a ribbon, the C-terminal lobe is represented by its electrostatic surface, the CaMKIIα 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.

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Structure of CaM bound to short peptides

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

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 they share no sequence homology, but fulfill minimal structural characteristics: they all have the potential to fold into a basic, amphiphilic α-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 α-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 Ca2+/CaM by itself [64–66,91]. A collapsed structure of ternary Ca2+/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 IIα (CaMKIIα) were solved by 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 CaMKIIα will be discussed here to highlight the main features.

Table 1. Key features of CaM/peptide complexes.
CaM targetTarget size (amino acids)CaM conformationTarget conformationAnchor residuesCa2+ ions boundMethodYear
smMLCK 20CollapsedHelix1-5-8-144NMR1992
skMLCK 26CollapsedHelix1-8-144X-ray1992
CaMKIIα 25CollapsedHelix1-5-104X-ray1992
CaMKK 27CollapsedHelix + β-hairpin loop1-5-164NMR1999
Ca2+-pump 20ExtendedHelix, binds to C-terminal lobe only1-5-84NMR1999
K+-channel 95Extended3 Helices, forms dimerNonclassical2 (N-terminal lobe)X-ray2001
Anthrax exotoxin510ExtendedComplexNonclassical2 (C-terminal lobe)X-ray2002

The CaM/CaMKIIα complex is of ellipsoidal shape and much more compact (50 × 30 × 30 Å3) than Ca2+/CaM without the peptide (maximal length of 65 Å) (Fig. 1). The central linker region is unwound and allows the C- and N-terminal lobes to bend by 100° and to rotate by 120° relative to their orientation seen in the Ca2+/CaM crystal structure. The peptide is bound in an α-helical conformation and is engulfed by Ca2+/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 interactions and determines the relative binding orientation of CaM-binding domain peptides. Basic residues at the N-terminus 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 Ca2+-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 Ca2+-pump

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

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 Ca2+-pump has hydrophobic anchor residues in positions 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 Ca2+-pump of the plasma membrane can be activated not only by full length Ca2+/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 corresponds to the N-terminal part of the CaM-binding domain which is common to all Ca2+-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 α-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 α-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 experiments 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 Ca2+-pump CaM-binding domain to only the C-terminal lobe of CaM demonstrates that ‘wrapping around’ a CaM binding domain, as seen in the complexes with MLCK and CaMKIIα, is not necessary to activate certain CaM-regulated proteins.

CaM in complex with CaMKK

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

Synthetic CaM-binding peptides have been successfully designed based on the motif of a ‘basic amphiphilic α-helix’ and the peptides derived from MLCK, CaMKIIα and the Ca2+-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 Ca2+/CaM-dependent kinase kinase [107]. CaMKK is a Ca2+/CaM dependent serine/threonine kinase which phosphorylates CaM-dependent kinases I and IV, modulating a signal transduction cascade leading to Ca2+-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, CaMKIIα and the Ca2+-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/CaMKIIα 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 ‘inverted’ orientation, i.e. binding with its C-terminus to the C-terminal lobe of CaM. In contrast, the MLCK, CaMKKIIα and Ca2+-pump peptides bind with their 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 complementarity of binding surfaces determine the binding orientation 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 CaMKIIα and MLCK peptides are bound in a largely α-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 α-helical and contains an additional well-defined β-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 β-hairpin loop contains a hydrophobic 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 ‘classical’ binding mode of α-helical peptides to CaM and has to be added to the growing collection of the structural motifs recognizable by CaM.

Ca2+ activated K+-channel

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

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 Ca2+-activated, small conductance K+-channel [109]. The ion-guiding pore of the K+-channel is formed by four α-subunits, each spanning the membrane several times and is gated by the intracellular Ca2+ concentration [110,111]. The C-terminal cytosolic portion of each α-subunit has one CaM molecule constitutively bound via a domain that shares no similarity to other CaM binding domains and is not Ca2+ 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 Ca2+ 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 α-helix and a shorter α-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 Ca2+ free and the binding of this portion of the K+-channel peptide is Ca2+-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 Ca2+ ions and exposes subsequently the hydrophobic patch typical for Ca2+ 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) connected by the short N-terminal helix resembles a molecular wrench. Movement of the C-terminal helix as the result of Ca2+ binding to CaM and subsequent dimerization of the α-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

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

The crystal structures of the C-terminal fragment (residues 291–800) of the exotoxin from Bacillus anthracis, with and 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 calmodulin-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 Å) by comparison of three structures: (a) exotoxin without CaM (b) exotoxin with CaM and (c) exotoxin with bound CaM and the pseudosubstrate 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 Ca2+.

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 residues, leading to a very large buried surface area of 5.900 Å2. 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 Å and rotated by 30° upon binding of CaM. In addition, several short sheet structures, helices and loops of exotoxin rearrange and change their conformation.

image

Figure 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.

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The C-terminal lobe of CaM contains two Ca2+ ions and its conformation is very similar to other structures of Ca2+-loaded CaM. The hydrophobic patch typical for Ca2+/CaM is exposed and forms a peptide-binding channel, which interacts with an α-helix of exotoxin. This α-helix shows many properties of a potential CaM-binding site: it forms a positively charged amphiphilic α-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 Ca2+-dependent manner. NMR experiments indicate further that the peptide is bound in an α-helical conformation 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 CaMKIIα. 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 conformation similar to the complexes with the MLCK or CaMKIIα 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 Ca2+-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 Ca2+ from the N-terminal lobe is remarkable, because the complex was crystallized in the presence of Ca2+, making it necessary for CaM to ‘lose’ two Ca2+ ions during its binding to exotoxin.

The binding of CaM to exotoxin increases the catalytic activity of the enzyme approximately 1000-fold. Surprisingly, 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

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References

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 (CaMKIIα, CaMKK, Ca2+-pump, K+-channel, anthrax exotoxin) represent several structurally different CaM-complex architectures. With short, amphiphilic, α-helical peptides (CaMKIIα) a collapsed complex conformation is observed, but also β-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 (Ca2+-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 CaMKIIα, 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 Ca2+ 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 Ca2+ concentration. Also, the structure of CaM in complex with the K+-channel fragment suggests that some CaM targets can be activated through dimerization.

For several Ca2+/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 CaMKIIα and MLCK peptides 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 extended conformation to anthrax exotoxin and activates the enzyme through subtle changes in the surrounding of the active site.

There is no doubt that structures of CaM in complex with large target fragments yield much more information about the mechanisms of CaM target recognition and activation than structures of complexes with short peptides. With this in mind we can look forward to up-coming structures of CaM in complex with full length targets and we are certain that CaM has many more ways to activate its targets than we can imagine today.

References

  1. Top of page
  2. Abstract
  3. Ca2+ signaling and calmodulin
  4. Structure of calmodulin and structural changes upon Ca2+ binding
  5. Structure of CaM bound to short peptides
  6. Plasma membrane Ca2+-pump
  7. CaM in complex with CaMKK
  8. Ca2+ activated K+-channel
  9. Anthrax adenylate cyclase exotoxin
  10. Conclusion
  11. References
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