Structural determinants of calmodulin binding to the intracellular C-terminal domain of the metabotropic glutamate receptor 7A

Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.     

Dissociation of RalA from synaptic membranes by Ca2+/calmodulin.

Ras-related small GTP-binding proteins execute many cellular functions, such as cell growth, differentiation, cytoskeletal reorganization, membrane trafficking, and membrane fusion. RalA belongs to the superfamily of Ras-related small GTP-binding proteins. Synaptic vesicles (SV) contain small GTP-binding proteins, where RalA, Rab3A, and Rab5A are the major GTP-binding proteins. It has been postulated that a cycling of these proteins between membrane-bound and soluble states is required for regulating cellular functions. Calmodulin (CaM) was found to dissociate Rab3A from SV membranes by forming a 1:1 complex with Ca2+/CaM. RalA was also found to be a Ca2+/CaM-binding protein. Therefore, we examined if Ca2+/CaM can also cause the RalA to dissociate from SV membranes. In this study, we identified that Ca2+/CaM dissociates RalA as well as Rab3A from synaptic vesicles.     

A novel Ca2+-induced Ca2+ release mechanism in A7r5 cells regulated by calmodulin-like proteins

Intracellular Ca2+ release is involved in setting up Ca2+ signals in all eukaryotic cells. Here we report that an increase in free Ca2+ concentration triggered the release of up to 41 +/- 3% of the intracellular Ca2+ stores in permeabilized A7r5 (embryonic rat aorta) cells with an EC50 of 700 nm. This type of Ca2+-induced Ca2+ release (CICR) was neither mediated by inositol 1,4,5-trisphosphate receptors nor by ryanodine receptors, because it was not blocked by heparin, 2-aminoethoxydiphenyl borate, xestospongin C, ruthenium red, or ryanodine. ATP dose-dependently stimulated the CICR mechanism, whereas 10 mm MgCl2 abolished it. CICR was not affected by exogenously added calmodulin (CaM), but CaM1234, a Ca2+-insensitive CaM mutant, strongly inhibited the CICR mechanism. Other proteins of the CaM-like neuronal Ca2+-sensor protein family such as Ca2+-binding protein 1 and neuronal Ca2+ sensor-1 were equally potent for inhibiting the CICR. Removal of endogenous CaM, using a CaM-binding peptide derived from the ryanodine receptor type-1 (amino acids 3614-3643) prevented subsequent activation of the CICR mechanism. A similar CICR mechanism was also found in 16HBE14o-(human bronchial mucosa) cells. We conclude that A7r5 and 16HBE14o-cells express a novel type of CICR mechanism that is silent in normal resting conditions due to inhibition by CaM but becomes activated by a Ca2+-dependent dissociation of CaM. This CICR mechanism, which may be regulated by members of the family of neuronal Ca2+-sensor proteins, may provide an additional route for Ca2+ release that could allow amplification of small Ca2+ signals.     

Protein-peptide interaction studies demonstrate the versatility of calmodulin target protein binding

Calmodulin (CaM) is a prototypical Ca2+-sensor protein that can control many important biological functions by binding to hundreds of target proteins. To gain insight into the versatility of CaM-target recognition, we have analyzed the complex structures for many types of CaM-binding peptides and some target proteins. In particular, some recently reported novel complex structures reveal that the versatile target binding of CaM is accommodated by its flexible domain arrangement and the malleability of its interfaces.     

Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.     

A Ca2+/calmodulin-binding peroxidase from Euphorbia latex: novel aspects of calcium-hydrogen peroxide cross-talk in the regulation of plant defenses

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor found in all eukaryotes, where it participates in the regulation of diverse calcium-dependent physiological processes. In response to fluctuations of the intracellular concentration of Ca(2+), CaM binds to a set of unrelated target proteins and modulates their activity. In plants, a growing number of CaM-binding proteins have been identified that apparently do not have a counterpart in animals. Some of these plant-specific Ca(2+)/CaM-activated proteins are known to tune the interaction between calcium and H(2)O(2) in orchestrating plant defenses against biotic and abiotic stresses. We previously characterized a calcium-dependent peroxidase isolated from the latex of the Mediterranean shrub Euphorbia characias (ELP) [Medda et al. (2003) Biochemistry 42, 8909-8918]. Here we report the cDNA nucleotide sequence of Euphorbia latex peroxidase, showing that the derived protein has two distinct amino acid sequences recognized as CaM-binding sites. The cDNA encoding for an E. characias CaM was also found and sequenced, and its protein product was detected in the latex. Results obtained from different CaM-binding assays and the determination of steady-state parameters showed unequivocally that ELP is a CaM-binding protein activated by the Ca(2+)/CaM system. To the best of our knowledge, this is the first example of a peroxidase regulated by this classic signal transduction mechanism. These findings suggest that peroxidase might be another node in the Ca(2+)/H(2)O(2)-mediated plant defense system, having both positive and negative effects in regulating H(2)O(2) homeostasis.     

Comparison of mRNA-display-based selections using synthetic peptide and natural protein libraries

mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.     

Identification of the calmodulin binding domain of connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Calmodulin-Mediated Cell Cycle Regulation: New Mechanisms for Old Observations

The significance of divalent calcium ions (Ca2+) to cell cycle progression has been a subject of study for several decades, with a regulatory role for Ca2+ suggested in distinct cell types and multiple organisms. Our interest in proliferative vascular diseases led us to focus on mammalian vascular smooth muscle cells (VSMC) in particular, in which we and others had shown that a coordinate elevation in the intracellular free Ca2+ concentration is required for G1 to S phase cell cycle progression. However, the molecular basis for this Ca2+-sensitive cell cycle transition was not known.Our recent discovery of a functional protein-protein interaction between the late G1-active cyclin E1 and the major calcium signal-transducing factor Calmodulin (CaM) sheds new light on the mechanism(s) through which Ca2+ concentrations regulate cell cycle. Having identified a CaM-binding site on cyclin E1, our studies support a direct role for CaM in mediating Ca2+-sensitive cyclin E/cdk2 activity and G1 to S phase transitions in VSMC. The CaM binding site identified on cyclin E1 has a Kd for CaM consistent with that of known CaM-binding proteins, and is composed of a 22 amino acids N-terminal sequence that is highly conserved across several mammalian species. Deletion of this binding site abolished CaM binding and Ca2+-sensitive cyclin E/cdk2 activity.Here we provide our perspectives on the literature supporting a role for Ca2+ in cell cycle regulation, focusing on the evidence implicating CaM in this functionality, and discuss the potential for therapeutic modulation of CaM-dependent cell cycle machinery.     

钙不依赖性钙调素结合蛋白的研究进展

钙调素是普遍存在于真核生物细胞中、发挥多种生物学调控作用的信号组分.钙调素不仅在有Ca2 情况下通过与钙依赖性钙调素结合蛋白作用而传递信号,也能在相对无Ca2 条件下直接结合钙不依赖性钙调素结合蛋白而传递信号.综述了无钙离子结合钙调素及钙不依赖性钙调素结合蛋白的结构特性、钙不依赖性钙调素结合蛋白的种类及其可能的生物学作用,这将有助于我们深入认识钙调素介导信号途径的特异性、复杂性和多样性.     

Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome

Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans.     

A new multigene family encoding calcium-dependent calmodulin-binding membrane proteins of Paramecium tetraurelia

Ca2+/calmodulin (CaM) regulates various physiological processes in a wide variety of organisms, metazoa and protists alike. To better understand Ca2+/CaM-dependent processes, particularly those with membrane-associated components, we studied Ca2+/CaM-binding membrane proteins in Paramecium tetraurelia, a unicellular model system. A CaM-binding protein, PCM1 (Paramecium CaM-binding membrane-bound protein), from a detergent-solubilized ciliary membrane fraction was identified and purified through Ca2+-dependent CaM-affinity chromatography. PCM1 has an apparent molecular mass of approx. 65kDa. It binds radiolabeled CaM in blot overlay assays and binds to CaM-affinity columns, both only in the presence of 10 microM or higher Ca2+. Three peptide sequences from PCM1 were obtained, and polymerase chain reaction (PCR) and Southern hybridization experiments were designed accordingly, leading to a partial cDNA clone for PCM1 and the discovery of three homologs: PCM2, PCM3 and PCM4. Amino acid sequences predicted by the full-length coding sequence for PCM3 and partial genes for PCM1, PCM2 and PCM4 are very similar (approx. 85% amino-acid identities). Their sequences indicate that they are hitherto novel proteins with beta/gamma-crystallin domains, cysteine-rich regions and potential CaM-binding domains. These protein motifs are suggested to mediate protein-protein interaction important for Ca2+/CaM signal transduction event(s) through the PCM family of proteins.     

Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.     

The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels

We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two alpha-helices (A and B). These two helices encompass approximately 85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of approximately 130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels.     

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.     

Endogenously bound calmodulin is essential for the function of the inositol 1,4,5-trisphosphate receptor

Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP3R). Despite many efforts, the exact mechanism by which CaM regulates the IP3R still remains elusive. Here we show, using unidirectional 45Ca2+ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP3R. Removing endogenously bound CaM by titration with a high affinity (pM) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP3-induced Ca2+ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP3. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP3-induced Ca2+ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM1234, reactivated the IP3R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP3R, and this resulted in a complete loss of the IP3R activity. Our data support a new model whereby CaM is constitutively associated with the IP3R and functions as an essential subunit for proper functioning of the IP3R.