Structural dynamics of the complex of calmodulin with a minimal functional construct of eukaryotic elongation factor 2 kinase and the role of Thr348 autophosphorylation

The calmodulin (CaM) activated α‐kinase, eukaryotic elongation factor 2 kinase (eEF‐2K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF‐2K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution‐state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca²⁺‐CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C‐lobe surface, complementary to the one involved in recognizing the calmodulin‐binding domain (CBD) of TR, provides a secondary TR‐interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca²⁺‐CaM•TR complex. Solution NMR data suggest that CaM_H107K, which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaM_H107K to induce TR autophosphorylation compared to wild‐type CaM.     

Both N- and C-lobes of calmodulin are required for Ca-dependent regulations of CaV1.2 Ca channels

We investigated the concentration- and Ca²⁺-dependent effects of CaM mutants, CaM₁₂ and CaM₃₄, in which Ca²⁺-binding to its N- and C-lobes was eliminated, respectively, on the Ca_V1.2 Ca²⁺ channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM₁₂ and CaM₃₄ (0.7-10 μM) applied with 3 mM ATP produced channel activity after “rundown”. Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca²⁺], suggesting that both functional lobes of CaM were necessary for the Ca²⁺-dependent shift. However, channel activity induced by the CaM mutants showed Ca²⁺-dependent decrease, implying a Ca²⁺ sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca²⁺-dependent regulations of Ca_V1.2 Ca²⁺ channels.     

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Trifluoperazine (TFP; Stelazine?) is an antagonist of calmodulin (CaM), an essential regulator of calcium‐dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca²⁺)₄‐CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM‐kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca²⁺)₄‐CaM and explore differential effects on the N‐ and C‐domains of CaM, stoichiometric TFP titrations of CaM were monitored by ¹⁵N‐HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca²⁺)₄‐CaM. In both cases, the preferred site was in the C‐domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP‐binding sites in apo CaM appeared distinct from those in (Ca²⁺)₄‐CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ‐motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N‐domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed,” “semi‐open,” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca²⁺)₄‐CaM, needs to be considered a potential target of drug action. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Altered RyR2 regulation by the calmodulin F90L mutation associated with idiopathic ventricular fibrillation and early sudden cardiac death

Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitation–contraction coupling. Defective CaM–RyR2 interaction is associated with heart failure. A novel CaM mutation (CaM^F90L) was recently identified in a family with idiopathic ventricular fibrillation (IVF) and early onset sudden cardiac death. We report the first biochemical characterization of CaM^F90L. F90L confers a deleterious effect on protein stability. Ca²⁺-binding studies reveal reduced Ca²⁺-binding affinity and a loss of co-operativity. Moreover, CaM^F90L displays reduced RyR2 interaction and defective modulation of [³H]ryanodine binding. Hence, dysregulation of RyR2-mediated Ca²⁺ release via aberrant CaM^F90L–RyR2 interaction is a potential mechanism that underlies familial IVF.     

Functional properties of the Cav1.2 calcium channel activated by calmodulin in the absence of α2δ subunits

Voltage-activated Ca_v1.2 calcium channels require association of the pore-forming α_1C subunit with accessory Ca_vβ and α₂δ subunits. Binding of a single calmodulin (CaM) to α_1C supports Ca²⁺-dependent inactivation (CDI). The human Ca_v1.2 channel is silent in the absence of Ca_vβ and/or α₂δ. Recently, we found that coexpression of exogenous CaM (CaM_ex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of Ca_vβ. Here we discovered that CaM_ex and its Ca²⁺-insensitive mutant (CaM₁₂₃₄) rendered active α_1C/Ca_vβ channel in the absence of α₂δ. Coexpression of CaM_ex with α_1C and β_2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by ≈ +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca²⁺) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaM_ex and CaM₁₂₃₄ accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal Ca_v1.2 channel. The data suggest a previously unknown action of CaM that in the presence of Ca_vβ translates into activation of the α₂δ-deficient calcium channel and alteration of its properties.     

Recognition of β-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca²⁺‐calmodulin‐dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T‐cell activation and cardiac hypertrophy in humans. Calcium binding to CaN_B (the regulatory subunit) triggers conformational change in CaN_A (the catalytic subunit). Two isoforms of CaN_A (α, β) are both abundant in brain and heart and activated by calcium‐saturated calmodulin (CaM). The individual contribution of each domain of CaM to regulation of calcineurin is not known. Hydrodynamic analyses of (Ca²⁺)₄‐CaM_1–148 bound to βCaNp, a peptide representing its CaM‐binding domain, indicated a 1:1 stoichiometry. βCaNp binding to CaM increased the affinity of calcium for the N‐ and C‐domains equally, thus preserving intrinsic domain differences, and the preference of calcium for sites III and IV. The equilibrium constants for individual calcium‐saturated CaM domains dissociating from βCaNp were ～1 μM. A limiting K_d ≤ 1 nM was measured directly for full‐length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. βCaNp binding to ¹⁵N‐(Ca²⁺)₄‐CaM_1–148 monitored by ¹⁵N/¹HN HSQC NMR showed that association perturbed the N‐domain of CaM more than its C‐domain. NMR resonance assignments of CaM and βCaNp, and interpretation of intermolecular NOEs observed in the ¹³C‐edited and ¹²C‐¹⁴N‐filtered 3D NOESY spectrum indicated anti‐parallel binding. The sole aromatic residue (Phe) located near the βCaNp C‐terminus was in close contact with several residues of the N‐domain of CaM outside the hydrophobic cleft. These structural and thermodynamic properties would permit the domains of CaM to have distinct physiological roles in regulating activation of βCaN. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

A charge‐sensitive loop in the FKBP38 catalytic domain modulates Bcl‐2 binding

The Bcl‐2 inhibitor FKBP38 is regulated by the Ca²⁺‐sensor calmodulin (CaM). Here we show a hitherto unknown low‐affinity cation‐binding site in the FKBP domain of FKBP38, which may afford an additional level of regulation based on electrostatic interactions. Fluorescence titration experiments indicate that in particular the physiologically relevant Ca²⁺ ion binds to this site. NMR‐based chemical shift perturbation data locate this cation‐interaction site within the β5–α1 loop (Leu90–Ile96) of the FKBP domain, which contains the acidic Asp92 and Asp94 side‐chains. Binding constants were subsequently determined for K⁺, Mg²⁺, Ca²⁺, and La³⁺, indicating that the net charge and the radius of the ion influences the binding interaction. X‐ray diffraction data furthermore show that the conformation of the β5–α1 loop is influenced by the presence of a positively charged guanidinium group belonging to a neighboring FKBP38 molecule in the crystal lattice. The position of the cation‐binding site has been further elucidated based on pseudocontact shift data obtained by NMR via titration with Tb³⁺. Elimination of the Ca²⁺‐binding capacity by substitution of the respective aspartate residues in a D92N/D94N double‐substituted variant reduces the Bcl‐2 affinity of the FKBP38^35–153/CaM complex to the same degree as the presence of Ca²⁺ in the wild‐type protein. Hence, this charge‐sensitive site in the FKBP domain participates in the regulation of FKBP38 function by enabling electrostatic interactions with ligand proteins and/or salt ions such as Ca²⁺. Copyright © 2010 John Wiley & Sons, Ltd.     

Ca2+/calmodulin binding to PSD‐95 mediates homeostatic synaptic scaling down

Postsynaptic density protein‐95 (PSD‐95) localizes AMPA‐type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca²⁺ influx, Ca²⁺/calmodulin (Ca²⁺/CaM) binding to the N‐terminus of PSD‐95 mediates postsynaptic loss of PSD‐95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD‐95 N‐terminus as important for binding to Ca²⁺/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaM^R126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca²⁺/CaM to PSD‐95 induced by a chronic increase in Ca²⁺ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.     

Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket

Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca²⁺) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaM^N98I, CaM^D132E, CaM^D134H and CaM^Q136P) and we used a CaM-RyR2 co-immunoprecipitation and a [³H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [³H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca²⁺, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.     

Structural Dynamics of the Activation of Elongation Factor 2 Kinase by Ca2 +-Calmodulin

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca^2 + ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.     

The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cells

Soybean calmodulin isoform 4 (sCaM4) is a plant calcium‐binding protein, regulating cellular responses to the second messenger Ca²⁺. We have found that the metal ion free (apo‐) form of sCaM4 possesses a half unfolded structure, with the N‐terminal domain unfolded and the C‐terminal domain folded. This result was unexpected as the apo‐forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg²⁺ ions are always present at high concentrations in cells (0.5–2 mM), we suggest that Mg²⁺ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg²⁺ the initially unfolded N‐terminal domain of sCaM4 folds into an α‐helix‐rich structure, similar to the Ca²⁺ form. We have used the NMR backbone residual dipolar coupling restraints ¹D_NH, ¹D_CαHα, and ¹D_C′Cα to determine the solution structure of the N‐terminal domain of Mg²⁺‐sCaM4 (Mg²⁺‐sCaM4‐NT). Compared with the known structure of Ca²⁺‐sCaM4, the structure of the Mg²⁺‐sCaM4‐NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg²⁺‐sCaM4 and CaM‐binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg²⁺‐sCaM4 does not bind to Ca²⁺‐CaM target peptides and therefore is functionally similar to apo‐mCaM. The Mg²⁺‐ and apo‐structures of the sCaM4‐NT provide unique insights into the structure and function of some plant calmodulins in resting cells.     

Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity

Changes in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) are an important signal for various physiological activities. The Na⁺/Ca²⁺ exchangers (NCX) at the plasma membrane transport Ca²⁺ into or out of the cell according to the electrochemical gradients of Na⁺ and Ca²⁺ to modulate [Ca²⁺]_i homeostasis. Calmodulin (CaM) senses [Ca²⁺]_i changes and relays Ca²⁺ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca²⁺ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM₁₂₃₄ (a Ca²⁺-binding deficient mutant) in HEK293T cells and measured the increase in [Ca²⁺]_i contributed by the influx of Ca²⁺ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM₁₂₃₄. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3^rd or 4^th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2^nd and 3^rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca²⁺]_i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Eukaryotic elongation factor-2 kinase (eEF2K): a potential therapeutic target in cancer

Eukaryotic elongation factor-2 kinase (eEF2K), encoded by the EEF2K gene, is well-known to be a Ca²⁺/calmodulin (CaM)-dependent kinase which can negatively modulate protein synthesis. It is highly conserved among eukaryotes from mammals to invertebrates, of which human and mouse may have 99 % overall amino acid identity. This kinase can phosphorylate eukaryotic elongation factor-2 (eEF2) or undergo the process of autophosphorylation at multiple sites to inhibit its function in translation elongation. Due to the fact that regulation of eEF2 by eEF2K is an evolutionarily conserved mechanism, eEF2K activity may confer tumor cell adaption to metabolic stress under acute nutrient depletion, and the high expressed level of eEF2K has been found in several types of malignancies. eEF2K may modulate the expression of some apoptotic proteins such as XIAP, c-FLIP_L, Bcl-X_L, PI3KCI and p70^S6K to inhibit apoptotic process in cancer. On the other hand, it plays a regulatory role in autophagy involved in mTORC1, AMPK and Atg8, thereby promoting cancer cell survival. Additionally, eEF2K may play a crucial role in the crosstalk between apoptosis and autophagy in cancer. Collectively, these findings have led to the conclusions that eEF2K may contribute to carcinogenesis, and thus being utilized as a potential target for future cancer therapy.     

Cloning,Recombinant Expression and Characterization of Wild Type-105-Trp-Calmodulin of the Green Alga Mougeotia scalaris

The single calmodulin gene (CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the CDNA inserted into the prokaryotic expression vector pGEX-2T. The recombinant calmodulin protein (CAM) was expressed as a fusion product together with glutathione S-transferase and isolated on glutathione sepharose. After cleavage and purification, the CaM was characterized by Ca²⁺-dependent shift in SDS-PAGE, by activation of cyclic 3′,5′nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca²⁺ buffers in the PDE test, affinity to Ca²⁺ of Mougeotia CaM was found to be diminished fivefold compared to maize or bovine brain CaMs. There was also a 20-fold increase of half maximal activation (K_act) in the PDE test for Mougeotia CaM relative to maize CaM, while the K_act of maize CaM to that of bovine brain CaM was almost the same. The derived amino acid sequences of CaM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105-Trp (Mougeotia) → Leu (maize). In Mougeotia CaM the 105-Trp, including the neighbouring side chains of 92-Phe and 141-Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

Energetics of calmodulin domain interactions with the calmodulin binding domain of CaMKII

Calmodulin (CaM) is an essential eukaryotic calcium receptor that regulates many kinases, including CaMKII. Calcium‐depleted CaM does not bind to CaMKII under physiological conditions. However, binding of (Ca²⁺)₄‐CaM to a basic amphipathic helix in CaMKII releases auto‐inhibition of the kinase. The crystal structure of CaM bound to CaMKIIp, a peptide representing the CaM‐binding domain (CaMBD) of CaMKII, shows an antiparallel interface: the C‐domain of CaM primarily contacts the N‐terminal half of the CaMBD. The two domains of calcium‐saturated CaM are believed to play distinct roles in releasing auto‐inhibition. To investigate the underlying mechanism of activation, calcium‐dependent titrations of isolated domains of CaM binding to CaMKIIp were monitored using fluorescence anisotropy. The binding affinity of CaMKIIp for the domains of CaM increased upon saturation with calcium, with the C‐domain having a 35‐fold greater affinity than the N‐domain. Because the interdomain linker of CaM regulates calcium‐binding affinity and contribute to conformational change, the role of each CaM domain was explored further by investigating effects of CaMKIIp on site‐knockout mutants affecting the calcium‐binding sites of a single domain. Investigation of the thermodynamic linkage between saturation of individual calcium‐binding sites and CaM‐domain binding to CaMKIIp showed that calcium binding to Sites III and IV was sufficient to recapitulate the behavior of (Ca²⁺)₄‐CaM. The magnitude of favorable interdomain cooperativity varied depending on which of the four calcium‐binding sites were mutated, emphasizing differential regulatory roles for the domains of CaM, despite the high degree of homology among the four EF‐hands of CaM. Proteins 2009. © 2008 Wiley‐Liss, Inc.