Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.     

Structural basis for simultaneous binding of two carboxy-terminal peptides of plant glutamate decarboxylase to calmodulin

Activation of glutamate decarboxylase (GAD) by calcium-bound calmodulin (CaM) is required for normal plant growth through regulation of gamma-aminobutyrate and glutamate metabolism. The interaction of CaM with the C-terminal domain of GAD is believed to induce dimerization of the enzyme, an event implicated for Ca(2+)-dependent enzyme activation. Here, we present the solution structure of CaM in complex with a dimer of peptides derived from the C-terminus of Petunia hybrida GAD. The 23 kDa ternary complex is pseudo-symmetrical with each domain of CaM bound to one of the two antiparallel GAD peptides, which form an X-shape with an interhelical angle of 60 degrees. To accommodate the dimeric helical GAD target, the two domains of CaM adopt an orientation markedly different from that seen in other CaM-target complexes. Although the dimeric GAD domain is much larger than previously studied CaM-binding peptides, the two CaM domains appear closer together and make a number of interdomain contacts not observed in earlier complexes. The present structure of a single CaM molecule interacting with two target peptides provides new evidence for the conformational flexibility of CaM as well as a structural basis for the ability of CaM to activate two enzyme molecules simultaneously.     

C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells

Glutamate decarboxylase (GAD) converts L-glutamate to gamma-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca(2+)/calmodulin (CaM) to modulate enzyme activity. However, rice possesses two distinct types of GAD, OsGAD1 and OsGAD2. Although they both have a C-terminal extension, the former peptide contains an authentic CaM-binding domain (CaMBD), which is common to dicotyledonous plants, while the latter does not. Therefore, the role of the C-terminal extension in functional expression of OsGAD2 was investigated. An in vitro enzyme assay using recombinant OsGAD2 proteins revealed low activity in the presence or absence of Ca(2+)/CaM. However, a truncated version of GAD2 (OsGAD2DeltaC) had over 40-fold higher activity than wild-type GAD at physiological pH. These two DNA constructs were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Free amino acids were isolated from several lines for each construct to determine GABA content. Calli overexpressing OsGAD2 and OsGAD2DeltaC had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2DeltaC rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility. These data suggest that the C-terminal extension of OsGAD2 plays a role as a strong autoinhibitory domain, and that truncation of this domain causes the enzyme to act constitutively, with higher activity both in vitro and in vivo.     

Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants

Plants accumulate high levels of Gamma amino butyric acid (GABA) in response to different environmental stresses and GABA metabolism has different functions such as osmotic and pH regulation, bypass of tricarboxylic acid cycle, and C:N balance. The cytoplasmic male sterile (CMS) II mutant of Nicotiana sylvestris has a deletion in the mitochondrial gene nad7 which encodes the NAD7 subunit of complex I which causes increased leaf respiration, impaired photosynthesis, slower growth and increased amino acid levels. In this study we aimed to elucidate the role of GABA and GABA metabolism in different genotypes of the same plant system under salt stress (100mM NaCl) in short (24h) and long (7, 14 and 21 days) terms. We have investigated the differences in leaf fresh and dry weights, relative water content, photosynthetic efficiency (F(v)/F(m)), glutamate dehydrogenase (GDH, EC 1.4.1.4) and glutamate decarboxylase (GAD, EC 4.1.1.15) enzyme activities, GABA content and GAD gene expression profiles. GDH activity showed variations in CMSII and wild type (WT) plants in the first 24h. GAD gene expression profiles were in good agreement with the GAD enzyme activity levels in CMSII and WT plants after 24h. In long-term salinity, GAD activities increased in WT but, decreased in CMSII. GABA accumulation in WT and CMSII plants in short and long term was induced by salt stress. Variations in GDH and GAD activities in relation to GABA levels were discussed and GABA metabolism has been proposed to be involved in better performance of CMSII plants under long term salinity.     

Calmodulin binds to maize lipid transfer protein and modulates its lipids binding ability

Although plant non-specific lipid transfer proteins (ns-LTPs) are characterized by their ability to bind and transfer a broad range of hydrophobic ligands in vitro, their biological functions in vivo remain unclear. Recently, it has been proposed that ns-LTPs may play a key role in plant defense mechanisms, particularly during the induction of systemic acquired resistance, however, very little is known about the regulation in this process. We report that the binding of maize non-specific lipid transfer protein (Zm-LTP) to calmodulin (CaM) is in a calcium-independent manner. To better understand the interaction mechanism between Zm-LTP and CaM, the CaM-binding site of Zm-LTP was mapped to the region of amino acids 46-60. Point mutations indicate that four amino acid residues, R46, R47, K54 and R58, in this region are crucial for binding. Furthermore, we tested the effects of CaM on the lipid-binding activity of Zm-LTP in the presence of Ca(2+), EGTA, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide and trifluoperazine respectively. We also investigated the structural features of CaM-binding motifs in LTPs from different species and strong differences were observed. Taken together, our results suggest that the interaction with CaM could be a common feature of plant LTPs. The identification and characterization of CaM-binding domain of LTPs should provide new insights into the mechanism by which the physiological functions of LTPs are regulated.     

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca²⁺/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.     

Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution

The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [³⁵S] calmodulin (CaM) in the presence of calcium, and a region of 30–32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca²⁺/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca²⁺/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.     

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.     

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.     

Conformation of the calmodulin-binding domain of metabotropic glutamate receptor subtype 7 and its interaction with calmodulin

Calmodulin (CaM), a Ca(2+)-binding protein, is a well-known regulator of various cellular functions. One of the targets of CaM is metabotropic glutamate receptor 7 (mGluR7), which serves as a low-pass filter for glutamate in the pre-synaptic terminal to regulate neurotransmission. Surface plasmon resonance (SPR), circular dichroism (CD) spectroscopy and nuclear magnetic spectroscopy (NMR) were performed to study the structure of the peptides corresponding to the CaM-binding domain of mGluR7 and their interaction with CaM. Unlike well-known CaM-binding peptides, mGluR7 has a random coil structure even in the presence of trifluoroethanol. Moreover, NMR data suggested that the complex between Ca(2+)/CaM and the mGluR7 peptide has multiple conformations. The mGluR7 peptide has been found to interact with CaM even in the absence of Ca(2+), and the binding is directed toward the C-domain of apo-CaM rather than the N-domain. We propose a possible mechanism for the activation of mGluR7 by CaM. A pre-binding occurs between apo-CaM and mGluR7 in the resting state of cells. Then, the Ca(2+)/CaM-mGluR7 complex is formed once Ca(2+) influx occurs. The weak interaction at lower Ca(2+) concentrations is likely to bind CaM to mGluR7 for the fast complex formation in response to the elevation of Ca(2+) concentration.     

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 μmol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM-binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM-binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca²⁺ sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca²⁺ sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca²⁺ leak, and then lethal arrhythmia.     

Regulation of MAPK phosphatase 1 (AtMKP1) by calmodulin in Arabidopsis

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.     

Different conformational switches underlie the calmodulin-dependent modulation of calcium pumps and channels

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM) bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure. We find that CaM binds with high affinity in a collapsed structure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formation that is indicative of contact interactions between the N- and the C-domains of CaM in complex with these CaM-binding peptides. There is a 4-fold larger amount of excimer formation for CaM bound to the CaM-binding sequence of the Ca-ATPase in comparison to RyR1, indicating a closer structural coupling between CaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPase and RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of the Ca-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remains conformationally disordered irrespective of CaM binding. These results suggest an important role for interdomain contact interactions between the opposing domains of CaM in stabilizing the structure of the peptide complex. The substantially different structural responses associated with CaM binding to Ca-ATPase and RyR1 indicates a plasticity in their respective binding mechanisms that accomplishes different physical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatory domain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1 channel activity.     

C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin

Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.     

Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae

The action of gamma-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H(2)O(2) and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the cross-reaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H(2)O(2) exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.     

A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7.

Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades.     

The plant plasma membrane Ca2+ pump ACA8 contains overlapping as well as physically separated autoinhibitory and calmodulin-binding domains

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.     

Extracellular calmodulin-binding proteins in plants: purification of a 21-kDa calmodulin-binding protein

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl₂ extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca²⁺. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca²⁺ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca²⁺. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.