Protein-protein docking and analysis reveal that two homologous bacterial adenylyl cyclase toxins interact with calmodulin differently

Calmodulin (CaM), a eukaryotic calcium sensor that regulates diverse biological activities, consists of N- and C-terminal globular domains (N-CaM and C-CaM, respectively). CaM serves as the activator of CyaA, a 188-kDa adenylyl cyclase toxin secreted by Bordetella pertussis, which is the etiologic agent for whooping cough. Upon insertion of the N-terminal adenylyl cyclase domain (ACD) of CyaA to its targeted eukaryotic cells, CaM binds to this domain tightly ( approximately 200 pm affinity). This interaction activates the adenylyl cyclase activity of CyaA, leading to a rise in intracellular cAMP levels to disrupt normal cellular signaling. We recently solved the structure of CyaA-ACD in complex with C-CaM to elucidate the mechanism of catalytic activation. However, the structure of the interface between N-CaM and CyaA, the formation of which contributes a 400-fold increase of binding affinity between CyaA and CaM, remains elusive. Here, we used site-directed mutations and molecular dynamic simulations to generate several working models of CaM-bound CyaA-ACD. The validity of these models was evaluated by disulfide bond cross-linking, point mutations, and fluorescence resonance energy transfer experiments. Our study reveals that a beta-hairpin region (amino acids 259-273) of CyaA-ACD likely makes contacts with the second calcium binding motif of the extended CaM. This mode of interaction differs from the interaction of N-CaM with anthrax edema factor, which binds N-CaM via its helical domain. Thus, two structurally conserved, bacterial adenylyl cyclase toxins have evolved to utilize distinct binding surfaces and modes of activation in their interaction with CaM, a highly conserved eukaryotic signaling protein.     

Calcium dependence of the interaction between calmodulin and anthrax edema factor

Edema factor (EF), a toxin from Bacillus anthracis (anthrax), possesses adenylyl cyclase activity and requires the ubiquitous Ca2+-sensor calmodulin (CaM) for activity. CaM can exist in three major structural states: an apo state with no Ca2+ bound, a two Ca2+ state with its C-terminal domain Ca2+-loaded, and a four Ca2+ state in which the lower Ca2+ affinity N-terminal domain is also ligated. Here, the interaction of EF with the three Ca2+ states of CaM has been examined by NMR spectroscopy and changes in the Ca2+ affinity of CaM in the presence of EF have been determined by flow dialysis. Backbone chemical shift perturbations of CaM show that EF interacts weakly with the N-terminal domain of apoCaM. The C-terminal CaM domain only engages in the interaction upon Ca2+ ligation, rendering the overall interaction much tighter. In the presence of EF, the C-terminal domain binds Ca2+ with higher affinity, but loses binding cooperativity, whereas the N-terminal domain exhibits strongly reduced Ca2+ affinity. As judged by chemical shift differences, the N-terminal CaM domain remains bound to EF upon subsequent Ca2+ ligation. This Ca2+ dependence of the EF-CaM interaction differs from that observed for most other CaM targets, which normally interact only with the Ca2+-bound CaM domains and become active following the transition to the four Ca2+ state.     

An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis

The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.     

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.     

Calmodulin is involved in the Ca2+-dependent activation of ceramide kinase as a calcium sensor

We recently demonstrated that the activation of ceramide kinase (CERK) and the formation of its product, ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca(2+) (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca(2+) as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca(2+)-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca(2+). The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM.CERK complexes. An in vitro CaM binding assay also demonstrated Ca(2+)-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca(2+) sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca(2+)-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca(2+)-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein kinase II, which had been assumed to be the main target of CaM in mast cells.     

Troponin C/calmodulin chimeras as erythrocyte plasma membrane Ca2+-ATPase activators

Calmodulin (CaM) and troponin C (TnC) are EF-hand proteins that play fundamentally different roles in animal physiology. TnC has a very low affinity for the plasma membrane Ca2+-ATPase and is a poor substitute for CaM in increasing the enzyme's affinity for Ca2+ and the rate of ATP hydrolysis. We use a series of recombinant TnC (rTnC)/CaM chimeras to clarify the importance of the CaM carboxyl-terminal domain in the activation of the plasma membrane Ca2+-ATPase. The rTnC/CaM chimera, in which the carboxyl-terminal domain of TnC is replaced by that of CaM, has the same ability as CaM to bind and transmit the signal to Ca2+ sites on the enzyme. There is no further functional gain when the amino-terminal domain is modified to make the rTnC/CaM chimera more CaM-like. To identify which regions of the carboxyl-terminal domain of CaM are responsible for these effects, we constructed the chimeras rTnC/3CaM and rTnC/4CaM, where only one-half of the C-terminal domain of CaM (residues 85-112 or residues 113-148) replaces the corresponding region in rTnC. Neither rTnC/3CaM nor rTnC/4CaM can mimic CaM in its affinity for the enzyme. Nevertheless, with respect to the signal transduction process, rTnC/4CaM, but not rTnC/3CaM, shows the same behaviour as CaM. We conclude that the whole C-terminal domain is required for binding to the enzyme while Ca2+-binding site 4 of CaM bears all the requirements to increase Ca2+ binding at PMCA sites. Such mechanism of binding and activation is distinct from that proposed for most other CaM targets. Furthermore, we suggest that Ala128 and Met124 from CaM site 4 may play a crucial role in discriminating CaM from TnC.     

Evidence for differing roles for each lobe of the calmodulin-like domain in a calcium-dependent protein kinase

Calcium-dependent protein kinases (CDPKs) are structurally unique Ser/Thr kinases found in plants and certain protozoa. They are distinguished by a calmodulin-like regulatory apparatus (calmodulin-like domain (CaM-LD)) that is joined via a junction (J) region to the C-terminal end of the kinase catalytic domain. Like CaM, the CaM-LD is composed of two globular EF structural domains (N-lobe, C-lobe), each containing a pair of Ca(2+) binding sites. Spectroscopic analysis shows that the CaM-LD is comprised of helical elements, but the isolated CaM-LD does not form a conformationally homogeneous tertiary structure in the absence of Ca(2+). The addition of substoichiometric amounts of Ca(2+) is sufficient to stabilize the C-terminal lobe in a construct containing J and CaM-LD (JC) but not in the CaM-LD alone. Moreover, as J is titrated into Ca(2+)-saturated CaM-LD, interactions are stronger with the C-lobe than the N-lobe of the CaM-LD. Measurements of Ca(2+) affinity for JC reveal two cooperatively interacting high affinity binding sites (K(d)(,mean) = 5.6 nm at 20 mm KCl) in the C-lobe and two weaker sites in the N-lobe (K(d,mean) = 110 nm at 20 mm KCl). The corresponding Ca(2+) binding constants in the isolated CaM-LD are lower by more than 2 orders of magnitude, which indicates that the J region has an essential role in stabilizing the structure of the CDPK regulatory apparatus. The large differential affinity between the two domains together with previous studies on a plasmodium CDPK (Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M., and Kappes, B. (1994) Biochemistry 33, 3714-3721) suggests a model whereby even at normally low cytosolic levels of Ca(2+), the C-lobe interacts with the junction, but the kinase remains in an autoinhibited state. Activation then occurs when Ca(2+) levels rise to fill the two weaker affinity binding sites in the N-lobe, thereby triggering a conformational change that leads to release of the autoinhibitory region.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

Loss of autoinhibition of the plasma membrane Ca(2+) pump by substitution of aspartic 170 by asparagin. A ctivation of plasma membrane calcium ATPase 4 without disruption of the interaction between the catalytic core and the C-terminal regulatory domain

The plasma membrane calcium ATPase (PMCA) actively transports Ca(2+) from the cytosol to the extra cellular space. The C-terminal segment of the PMCA functions as an inhibitory domain by interacting with the catalytic core. Ca(2+)-calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp(170), in the putative "A" domain of human PMCA isoform 4xb, plays a critical role in autoinhibition. In the absence of calmodulin a PMCA containing a site-specific mutation of D170N had 80% of the maximum activity of the calmodulin-activated PMCA and a similar high affinity for Ca(2+). The mutation did not change the activation of the PMCA by ATP. Deletion of the C-terminal segment further downstream of the calmodulin-binding site led to an additional increase in the maximal activity of the mutant, which suggests that the mutation did not affect the inhibition because of this portion of the C-terminal segment. The calmodulin-activated PMCA was more sensitive to vanadate inhibition than the autoinhibited enzyme. In contrast, inhibition of the D170N mutant required higher concentrations of vanadate and was not affected by calmodulin. Despite its higher basal activity, the mutant had an apparent affinity for calmodulin similar to that of the wild type enzyme, and its rate of proteolysis at the C-terminal segment was still calmodulin-dependent. Altogether these results suggest that activation by mutation D170N does not involve the displacement of the calmodulin-binding autoinhibitory domain from the catalytic core and may arise directly from changes in the accessibility to the calcium-binding residues of the pump.     

Regulatory interaction of sodium channel IQ-motif with calmodulin C-terminal lobe

An increasing number of ion channels have been found to be regulated by the direct binding of calmodulin (CaM), but its structural features are mostly unknown. Previously, we identified the Ca(2+)-dependent and -independent interactions of CaM to the voltage-gated sodium channel via an IQ-motif sequence. In this study we used the trypsin-digested CaM fragments (TR(1)C and TR(2)C) to analyze the binding of Ca(2+)-CaM or Ca(2+)-free (apo) CaM with a sodium channel-derived IQ-motif peptide (NaIQ). Circular dichroic spectra showed that NaIQ peptide enhanced alpha-helicity of the CaM C-terminal lobe, but not that of the CaM N-terminal lobe in the absence of Ca(2+), whereas NaIQ enhanced the alpha-helicity of both the N- and C-terminal lobes in the presence of Ca(2+). Furthermore, the competitive binding experiment demonstrated that Ca(2+)-dependent CaM binding of target peptides (MLCKp or melittin) with CaM was markedly suppressed by NaIQ. The results suggest that IQ-motif sequences contribute to prevent target proteins from activation at low Ca(2+) concentrations and may explain a regulatory mechanism why highly Ca(2+)-sensitive target proteins are not activated in the cytoplasm.     

Calcium-dependent and -independent binding of soybean calmodulin isoforms to the calmodulin binding domain of tobacco MAPK phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.     

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.     

The W105G and W99G sorcin mutants demonstrate the role of the D helix in the Ca(2+)-dependent interaction with annexin VII and the cardiac ryanodine receptor

Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca(2+) binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N-terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca(2+) concentration as that of the wild type, whereas W105G requires a significantly higher Ca(2+) concentration. Ca(2+) spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca(2+) concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca(2+) binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation.     

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.     

Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes

This report describes Ca2+-dependent binding of 125I-labeled calmodulin (125I-CaM) to erythrocyte membranes and identification of two new CaM-binding proteins. Erythrocyte CaM labeled with 125I-Bolton Hunter reagent fully activated erythrocyte (Ca2+ + Mg2+)-ATPase. 125I-CaM bound to CaM depleted membranes in a Ca2+-dependent manner with a Ka of 6 x 10(-8) M Ca2+ and maximum binding at 4 x 10(-7) M Ca2+. Only the cytoplasmic surface of the membrane bound 125I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca2+ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar 125I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a KD of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca2+ transporter since: (a) Ka of activation of (Ca2+ + Mg2+)-ATPase and KD for binding were nearly identical, and (b) partial digestion of IOVs with alpha-chymotrypsin produced activation of the (Ca2+ + Mg2+)-ATPase with loss of the high affinity sites. 125I-CaM bound in solution to a class of binding proteins (KD approximately 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to 125I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 Mr (Mr of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 Mr proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca2+-induced effects in the erythrocyte.     

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.     

The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca(2+)/calmodulin-dependent electron transfer.

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca(2+) concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca(2+), whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca(2+) dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta40 and Delta42) retained maximal NO synthesis activity at lower concentrations of free Ca(2+) than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca(2+)/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca(2+) concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca(2+)/CaM. However, reduction of heme in the Delta40 mutant occurred spontaneously on addition of NADPH in the absence of Ca(2+)/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca(2+)/CaM and by destabilizing CaM binding at low Ca(2+) concentrations, consistent with its role as an autoinhibitory domain.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity?

The voltage-dependent sodium channel (VDSC) interacts with intracellular molecules to modulate channel properties and localizations in neuronal cells. To study protein interactions, we applied yeast two-hybrid screening to the cytoplasmic C-terminal domain of the main pore-forming alpha-subunit. We found a novel interaction between the C-terminal domain and calmodulin (CaM). By two-hybrid interaction assays, we specified the interaction site of VDSC in a C-terminal region, which is composed of 38 amino acid residues and contains both IQ-like and Baa motifs. Using a fusion protein of the C-terminal domain, we showed that interaction with CaM occurred in the presence and absence of Ca(2+). Two synthetic peptides, each covering the IQ-like (NaIQ) or the Baa motifs (NaBaa), were used to examine the binding property by a gel mobility shift assay. Although the NaIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca(2+)-bound Ca(2+)CaM, whereas NaIQ binds to both Ca(2+)CaM and Ca(2+)-free apoCaM. Fluorescence spectroscopy of dansylated CaM showed Ca(2+)-dependent spectral changes not only for NaBaa.CaM but also for NaIQ.CaM. The results, taken together with other results, indicate that whereas the NaBaa.CaM complex is formed in a Ca(2+)-dependent manner, the NaIQ.CaM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca(2+) concentration. These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca(2+)-dependent conformational transition of the complex.     

The solution structure of a plant calmodulin and the CaM-binding domain of the vacuolar calcium-ATPase BCA1 reveals a new binding and activation mechanism

The type IIb class of plant Ca(2+)-ATPases contains a unique N-terminal extension that encompasses a calmodulin (CaM) binding domain and an auto-inhibitory domain. Binding of Ca(2+)-CaM to this region can release auto-inhibition and activates the calcium pump. Using multidimensional NMR spectroscopy, we have determined the solution structure of the complex of a plant CaM isoform with the CaM-binding domain of the well characterized Ca(2+)-ATPase BCA1 from cauliflower. The complex has a rather elongated structure in which the two lobes of CaM do not contact each other. The anchor residues Trp-23 and Ile-40 form a 1-8-18 interaction motif. Binding of Ca(2+)-CaM gives rise to the induction of two helical parts in this unique target peptide. The two helical portions are connected by a highly positively charged bend region, which represents a relatively fixed angle and positions the two lobes of CaM in an orientation that has not been seen before in any complex structure of calmodulin. The behavior of the complex was further characterized by heteronuclear NMR dynamics measurements of the isotope-labeled protein and peptide. These data suggest a unique calcium-driven activation mechanism for BCA1 and other plant Ca(2+)-ATPases that may also explain the action of calcium-CaM on some other target enzymes. Moreover, CaM activation of plant Ca(2+)-ATPases seems to occur in an organelle-specific manner.