Structural investigation into the differential target enzyme regulation displayed by plant calmodulin isoforms

The conserved calmodulin (CaM) isoform SCaM-1 and the divergent SCaM-4 from soybean bind to many of the same target enzymes, but differentially activate or competitively inhibit them. Class 1 target enzymes are activated by both calcium (Ca(2+))-bound SCaM-1 (Ca(2+)-SCaM-1) and Ca(2+)-bound SCaM-4 (Ca(2+)-SCaM-4), while class 2 enzymes are activated by Ca(2+)-SCaM-1 but competitively inhibited by Ca(2+)-SCaM-4, and class 3 enzymes are activated by Ca(2+)-SCaM-4 but competitively inhibited by Ca(2+)-SCaM-1. To determine whether these differences can be attributed to unique interactions with the CaM-binding domains (CaMBD) of these enzymes, we have studied the binding of each protein to peptides derived from the CaMBD of a representative target enzyme from each of these three classes. Using a combination of NMR spectroscopy and isothermal titration calorimetry, we demonstrate that the N- and C-domains of either Ca(2+)-SCaM bind to each peptide to form structurally compact complexes driven by the burial of hydrophobic surfaces. Interestingly, the interactions with the CaMBD peptides from classes 1 and 2 are similar for the two proteins; however, binding to the peptide from class 3 is structurally and thermodynamically distinct for Ca(2+)-SCaM-1 and -4. We also demonstrate that both calcium-free SCaM-1 (apo-SCaM-1) and calcium-free SCaM-4 (apo-SCaM-4) bind to the CaMBD from cyclic nucleotide phosphodiesterase, and that the interactions are similar to each other and to the interactions with apo-mammalian CaM. Therefore, the apo-SCaMs are also capable of binding to the same target enzymes, which could provide an additional mechanism for CaM-dependent signaling in plants.     

Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).

Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.     

Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue

Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.     

Functional characterization of a Chrysanthemum dichrum stress-related promoter

A 1,474-bp stress-inducible CdDREBa promoter was identified from Chrysanthemum dichrum, revealing several candidate stress-related cis-acting elements (MYC-box, MYB site, GT-1, and W-box) within it. In Arabidopsis leaf tissues transformed with a CdDREBa promoter-β-glucuronidase (GUS) gene fusion, serially 5'-deleted CdDREBa promoters were differentially activated by cold and salinity. Histochemical and quantitative assays of GUS expression allowed us to localize a critical part of the promoter located between upstream 430 and 351 nt. This 80-bp fragment enhanced GUS expression under salinity stress when fused to -90/+8 CaMV 35S minimal promoter. Further promoter internal-deletion assays indicated that a low temperature-responsive element was located between positions -430 and -390, and a salinity inducible one between -385 and -351. Our results showed that there was a novel stress-related critical region except for the known cis-acting element (MYC-box, GT-1) in CdDREBa promoter.     

The GATA-binding protein CGF-1 is closely related to GT-1

Many light-regulated genes contain a conserved GATA motif in their 5-upstream region. We have characterized in detail the GATA-binding factor, CGF-1, which binds within a 73 bp TATA-proximal light/circadian regulatory element in the Arabidopsis cab2 promoter and to two more sites farther upstream. CGF-1 was found to be distinct from other metal-dependent GATA-binding factors, but to have the same sequence requirements for binding and similar physical and chemical properties as GT-1, a factor required for light regulation of the tobacco rbcS-3A gene. CGF-1 was found to be constitutively present in extracts and was shown to be immunologically related to GT-1. The close similarity between CGF-1 and GT-1 suggests that a GT-1-like factor is involved in the phytochrome/circadian regulation of the cab2 gene. CGF-1 and GT-1 were also found to have similar sequence specificities to another constitutively-regulated GATA factor, IBF-2b, which binds the I box region of the tomato nitrate reductase gene. Of three complexes detected using an IBF-2b-specific probe, only one was identical to CGF-1/GT-1. The other two were similar to IBF-2b, demonstrating that CGF-1/GT-1, although very similar, are actually distinct from IBF-2b. These data indicate that more than one factor can bind to the same short sequence and may indicate how constitutively present factors like GT-1 can play a role in light regulation.     

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.     

Structurally homologous binding of plant calmodulin isoforms to the calmodulin-binding domain of vacuolar calcium-ATPase

The discovery that plants contain multiple calmodulin (CaM) isoforms having variable sequence identity to mammalian CaM has sparked a flurry of new questions regarding the intracellular role of Ca(2+) regulation in plants. To date, the majority of research in this field has focused on the differential enzymatic regulation of various mammalian CaM-dependent enzymes by the different plant CaM isoforms. However, there is comparatively little information on the structural recognition of target enzymes found exclusively in plant cells. Here we have used a variety of spectroscopic techniques, including nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, to study the interactions of the most conserved and most divergent CaM isoforms from soybean, SCaM-1, and SCaM-4, respectively, with a synthetic peptide derived from the CaM-binding domain of cauliflower vacuolar calcium-ATPase. Despite their sequence divergence, both SCaM-1 and SCaM-4 interact with the calcium-ATPase peptide in a similar calcium-dependent, stoichiometric manner, adopting an antiparallel binding orientation with an alpha-helical peptide. The single Trp residue is bound in a solvent-inaccessible hydrophobic pocket on the C-terminal domain of either protein. Thermodynamic analysis of these interactions using isothermal titration calorimetry demonstrates that the formation of each calcium-SCaM-calcium-ATPase peptide complex is driven by favorable binding enthalpy and is very similar to the binding of mammalian CaM to the CaM-binding domains of myosin light chain kinases and calmodulin-dependent protein kinase I.     

Molecular dissection of GT-1 from Arabidopsis.

We isolated and characterized an Arabidopsis cDNA encoding the DNA binding protein GT-1. This protein factor, which contains 406 amino acids, is highly homologous to the previously described tobacco DNA binding protein GT-1a/B2F but is 26 amino acids longer. Recombinant Arabidopsis GT-1, which was obtained from in vitro translation, bound to probes consisting of four copies of pea small subunit of ribulose bisphosphate carboxylase rbcS-3A box II and required the same GGTTAA core binding site as the binding activity of an Arabidopsis nuclear protein preparation. However, unlike the truncated tobacco GT-1a prepared from Escherichia coli extracts, the full-length Arabidopsis GT-1 bound to pea rbcS-3A box III and Arabidopsis chlorophyll a/b binding protein CAB2 light-responsive elements, both of which contain GATA motifs. Deletion and mutational analyses suggested that the predicted trihelix region of GT-1 is essential for DNA binding. Moreover, GT-1 binds to target DNA as a dimer, and its C-terminal region contains a putative dimerization domain that enhances the binding activity. Transient expression of the GT-1::beta-glucuronidase fusion protein in onion cells revealed the presence of a nuclear localization signal(s) within the first 215 amino acids of GT-1.     

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.     

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10–20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.     

Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo

Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (–902 to –656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.