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Ernesto Carafoli - One of the best experts on this subject based on the ideXlab platform.
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the plasma membrane calcium pump in health and disease
FEBS Journal, 2013Co-Authors: Marisa Brini, Tito Cali, Denis Ottolini, Ernesto CarafoliAbstract:The Ca2+ ATPases of the plasma membrane (PMCA pumps) export Ca2+ from all eukaryotic cells. In mammals they are the products of four separate genes. PMCA types 1 and 4 are distributed ubiquitously; PMCA types 2 and 3 are restricted to some tissues, the most important being the nervous system. Alternative splicing at two sites greatly increases the number of pump isoforms. The two ubiquitous isoforms are no longer considered as only housekeeping pumps as they also perform tissue-specific functions. The PMCAs are classical P-type pumps, their reaction cycle repeating that of all other pumps of the family. Their 3D structure has not been solved, but molecular modeling on SERCA pump templates shows the essential structural pattern of the latter. PMCAs are regulated by Calmodulin, which interacts with high affinity with their cytosolic C-terminal tail. A second Calmodulin-Binding Domain with lower affinity is present in some splicing variants of the pump. The PMCAs are essential to the regulation of cellular Ca2+, but the all-important Ca2+ signal is ambivalent: defects in its control generate various pathologies, the most thoroughly studied being those of genetic origin. Genetic defects of PMCA function produce disease phenotypes: the best characterized is a form of deafness in mice and in humans linked to PMCA2 mutations. A cerebellar X-linked human ataxia has recently been found to be caused by a mutation in the Calmodulin-Binding Domain of PMCA3.
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biogenesis plasma membrane calcium atpase 15 years of work on the purified enzyme
The FASEB Journal, 1994Co-Authors: Ernesto CarafoliAbstract:The plasma membrane calcium pump is the system that ejects Ca2+ out of eukaryotic cells: this is documented in all animal and plant cells, although knowledge on the latter is only now beginning to be established. Information on lower eukaryotic cells, e.g., yeast, is still scarce, but it also is beginning to develop. The pump shares the catalytic properties of ion-motive ATPases of the P-type family, but has distinctive regulation properties: it is modulated by Calmodulin, acidic phospholipids, a number of protein kinases, possibly by the interaction of calcium with its COOH-terminal region, and by aggregation (dimerization) through the Calmodulin Binding Domain. The latter acts as an endogenous inhibitor of pump activity, much as phospholamban does for the sarcoplasmic reticulum pump. The analogy of the regulation mechanisms of the two pumps is heightened by the finding that phosphorylation of the Calmodulin Binding Domain by protein kinase C removes its autoinhibiting function, as other kinases do in th...
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phosphorylation of the Calmodulin Binding Domain of the plasma membrane ca2 pump by protein kinase c reduces its interaction with Calmodulin and with its pump receptor site
Journal of Biological Chemistry, 1994Co-Authors: F Hofmann, Ernesto Carafoli, John Anagli, Thomas VorherrAbstract:Two versions of the Calmodulin Binding Domain of the plasma membrane Ca2+ ATPase, a 24-amino acid peptide, C24W (Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-R-V-V-N-A-F-R-S-S-NH2), and the corresponding phosphothreonine containing peptide, C24W-P (Q-I-L-W-F-R-G-L-N-R-I-Q-T(phospho)-Q-I-R-V-V-N-A-F-R-S-S-NH2), were synthesized. They were used to investigate the effect of threonine phosphorylation by protein kinase C on the Binding of Calmodulin by the Calmodulin Binding Domain and on the inhibitory role of the Domain on the activity of the Ca2+ pump. The phosphopeptide C24W-P was obtained after global phosphorylation of the free Thr side chain on the protected resin bound peptide. The phosphorylated Calmodulin Binding Domain failed to bind Calmodulin; this was shown by gel shift experiments, by fluorescence energy transfer studies and by competition experiments against Calmodulin stimulation of the pump. The inhibition of the Ca2+ pump activity by the Calmodulin Binding Domain in the absence of Calmodulin was also affected by the phosphorylation of the threonine; the inhibition of the fully active calpain-truncated pump by the phosphothreonine containing peptide was lower than that by the unphosphorylated synthetic Domain.
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the Calmodulin Binding Domain of nitric oxide synthase and adenylyl cyclase
Biochemistry, 1993Co-Authors: Thomas Vorherr, F Hofmann, L Knopfel, S Mollner, T Pfeuffer, Ernesto CarafoliAbstract:Peptides corresponding to regions of the Calmodulin-activated NO-synthase and of the Calmodulin dependent adenylyl cyclase, which could represent the Calmodulin Binding Domains of the two proteins, have been synthesized and tested for Calmodulin Binding. The chosen peptides were those in the sequence of the two proteins which most closely corresponded to the accepted general properties of the Calmodulin Binding Domains, i.e., a hydrophobic sequence containing basic amino acids. In the case of the NO-synthase, the putative high-affinity Calmodulin Binding Domain was identified by urea gel electrophoresis and fluorescence spectroscopy with dansylCalmodulin as peptide NO-30 (amino acids 725-754). The highest affinity Calmodulin Binding site of the Calmodulin-dependent adenylyl cyclase was assigned to peptide AC-28 (amino acids 495-522) by titration with dansylCalmodulin and by the ability to inhibit the Calmodulin-stimulated activity of purified Calmodulin-stimulated adenylyl cyclase. The sequence 495-522 is located in the unit protruding into the cytosol from the sixth putative transmembrane Domain of the molecule. It has the typical hydrophobic/basic composition of canonical Calmodulin Binding Domains, and also contains, like most Calmodulin Binding Domains, an aromatic amino acid in its N-terminal portion. It also contains two Cys residues in the central portion, which is an unusual feature of the Calmodulin Binding Domain of this enzyme.
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the Calmodulin Binding site of the plasma membrane ca2 pump interacts with the transduction Domain of the enzyme
Protein Science, 1992Co-Authors: Rocco Falchetto, Thomas Vorherr, Ernesto CarafoliAbstract:Calpain proteolysis of the plasma membrane Ca2+ pump removes a C-terminal 14-kDa portion which includes the Calmodulin-Binding Domain. This produces a fully activated 124-kDa fragment, which can be inhibited by synthetic versions of the Calmodulin-Binding Domain. The inhibition is strongest when Trp-8 in the latter Domain is replaced by a Tyr residue (Falchetto, R., Vorherr, T., Brunner, J., & Carafoli, E., 1991, J. Biol. Chem. 266, 2930-2936). In the present study, the N-terminus of the 28-residue synthetic Calmodulin-Binding Domain was acetylated with 3H-acetic anhydride, and Phe in position 25 was replaced by a phenylalanine derivatized with a diazirine-based, photoactivatable carbene precursor. This peptide (C28WC*) inhibited the fully active 124-kDa fragment of the pump and became cross-linked to it upon photolysis. After proteolysis of the fragment with Asp-N or Staphylococcus aureus V8 (Glu-C) protease, labeled peptides were isolated by reversed-phase high-performance liquid chromatography and subjected to Edman sequence analysis. The peptides originated from a region of the pump located within the unit protruding into the cytoplasm between transmembrane Domain two and three. This unit has been proposed to be the site of the energy transduction Domain, which would couple the ATP hydrolysis to Ca2+ translocation.
Thomas Vorherr - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of the Calmodulin Binding Domain of the plasma membrane ca2 pump by protein kinase c reduces its interaction with Calmodulin and with its pump receptor site
Journal of Biological Chemistry, 1994Co-Authors: F Hofmann, Ernesto Carafoli, John Anagli, Thomas VorherrAbstract:Two versions of the Calmodulin Binding Domain of the plasma membrane Ca2+ ATPase, a 24-amino acid peptide, C24W (Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-R-V-V-N-A-F-R-S-S-NH2), and the corresponding phosphothreonine containing peptide, C24W-P (Q-I-L-W-F-R-G-L-N-R-I-Q-T(phospho)-Q-I-R-V-V-N-A-F-R-S-S-NH2), were synthesized. They were used to investigate the effect of threonine phosphorylation by protein kinase C on the Binding of Calmodulin by the Calmodulin Binding Domain and on the inhibitory role of the Domain on the activity of the Ca2+ pump. The phosphopeptide C24W-P was obtained after global phosphorylation of the free Thr side chain on the protected resin bound peptide. The phosphorylated Calmodulin Binding Domain failed to bind Calmodulin; this was shown by gel shift experiments, by fluorescence energy transfer studies and by competition experiments against Calmodulin stimulation of the pump. The inhibition of the Ca2+ pump activity by the Calmodulin Binding Domain in the absence of Calmodulin was also affected by the phosphorylation of the threonine; the inhibition of the fully active calpain-truncated pump by the phosphothreonine containing peptide was lower than that by the unphosphorylated synthetic Domain.
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the Calmodulin Binding Domain of nitric oxide synthase and adenylyl cyclase
Biochemistry, 1993Co-Authors: Thomas Vorherr, F Hofmann, L Knopfel, S Mollner, T Pfeuffer, Ernesto CarafoliAbstract:Peptides corresponding to regions of the Calmodulin-activated NO-synthase and of the Calmodulin dependent adenylyl cyclase, which could represent the Calmodulin Binding Domains of the two proteins, have been synthesized and tested for Calmodulin Binding. The chosen peptides were those in the sequence of the two proteins which most closely corresponded to the accepted general properties of the Calmodulin Binding Domains, i.e., a hydrophobic sequence containing basic amino acids. In the case of the NO-synthase, the putative high-affinity Calmodulin Binding Domain was identified by urea gel electrophoresis and fluorescence spectroscopy with dansylCalmodulin as peptide NO-30 (amino acids 725-754). The highest affinity Calmodulin Binding site of the Calmodulin-dependent adenylyl cyclase was assigned to peptide AC-28 (amino acids 495-522) by titration with dansylCalmodulin and by the ability to inhibit the Calmodulin-stimulated activity of purified Calmodulin-stimulated adenylyl cyclase. The sequence 495-522 is located in the unit protruding into the cytosol from the sixth putative transmembrane Domain of the molecule. It has the typical hydrophobic/basic composition of canonical Calmodulin Binding Domains, and also contains, like most Calmodulin Binding Domains, an aromatic amino acid in its N-terminal portion. It also contains two Cys residues in the central portion, which is an unusual feature of the Calmodulin Binding Domain of this enzyme.
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the Calmodulin Binding site of the plasma membrane ca2 pump interacts with the transduction Domain of the enzyme
Protein Science, 1992Co-Authors: Rocco Falchetto, Thomas Vorherr, Ernesto CarafoliAbstract:Calpain proteolysis of the plasma membrane Ca2+ pump removes a C-terminal 14-kDa portion which includes the Calmodulin-Binding Domain. This produces a fully activated 124-kDa fragment, which can be inhibited by synthetic versions of the Calmodulin-Binding Domain. The inhibition is strongest when Trp-8 in the latter Domain is replaced by a Tyr residue (Falchetto, R., Vorherr, T., Brunner, J., & Carafoli, E., 1991, J. Biol. Chem. 266, 2930-2936). In the present study, the N-terminus of the 28-residue synthetic Calmodulin-Binding Domain was acetylated with 3H-acetic anhydride, and Phe in position 25 was replaced by a phenylalanine derivatized with a diazirine-based, photoactivatable carbene precursor. This peptide (C28WC*) inhibited the fully active 124-kDa fragment of the pump and became cross-linked to it upon photolysis. After proteolysis of the fragment with Asp-N or Staphylococcus aureus V8 (Glu-C) protease, labeled peptides were isolated by reversed-phase high-performance liquid chromatography and subjected to Edman sequence analysis. The peptides originated from a region of the pump located within the unit protruding into the cytoplasm between transmembrane Domain two and three. This unit has been proposed to be the site of the energy transduction Domain, which would couple the ATP hydrolysis to Ca2+ translocation.
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small angle x ray scattering study of Calmodulin bound to two peptides corresponding to parts of the Calmodulin Binding Domain of the plasma membrane ca2 pump
Biochemistry, 1991Co-Authors: Mikio Kataoka, Thomas Vorherr, Joachim Krebs, James F Head, Ernesto CarafoliAbstract:The interaction between Calmodulin (CaM) and two synthetic peptides, C20W and C24W, corresponding to parts of the Calmodulin-Binding Domain of the Ca2+ pump of human erythrocytes, has been studied by using small-angle X-ray scattering (SAXS). The total length of the CaM-Binding Domain of the enzyme is estimated to be 28 amino acids. C20W contains the 20 N-terminal amino acids of this Domain, C24W the 24 C-terminal amino acids. The experiments have shown that the Binding of either peptide results in a complex with a radius of gyration (Rg) smaller than that of CaM. The complex between CaM and C20W revealed an interatomic length distribution function, P(r), similar to that of Calmodulin alone, indicating that the complex retains an extended, dumbbell-shaped structure. By contrast, the Binding of C24W resulted in the formation of a globular structure similar to those observed with many other CaM-Binding peptides.
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the plasma membrane ca2 pump contains a site that interacts with its Calmodulin Binding Domain
Journal of Biological Chemistry, 1991Co-Authors: Rocco Falchetto, Thomas Vorherr, Josef Brunner, Ernesto CarafoliAbstract:A synthetic, 28-residue peptide derived from the Calmodulin-Binding sequence of the plasma membrane Ca2+ pump (C28W) inhibits the ATPase activity of a calpain-produced, truncated fragment of the enzyme. The fragment, which has lost the Calmodulin-Binding Domain, has a molecular mass of 124 kDa and is fully active in the absence of Calmodulin. Replacement of Trp-8 in the peptide by an Ala decreases the overall inhibitory activity, while replacement with a Tyr increases it. However, at very low peptide concentrations the effect of Tyr replacement disappears. The synthetic peptide has been made photoactivatable by replacing Phe in position 9 with a synthetic phenylalanine analogue containing a diazirine group and was radioactively labeled by coupling a [3H]acetyl function to its N terminus. After cross-linking with the derivatized peptide, the 124-kDa fragment has been proteolyzed with either Lys-C, Asp-N, or V8 proteases, and the fragment(s) have been separated. Partial sequencing of the cross-linked, radioactive peptides has identified a site of the pump located C terminally to the phosphoenzyme-forming aspartic acid, spanning residues 537-544 of the hPMCA4 isoform of the enzyme. It is concluded that this sequence is part of a site which binds the Calmodulin-Binding Domain of the pump.
John T Penniston - One of the best experts on this subject based on the ideXlab platform.
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alternative pathways for association and dissociation of the Calmodulin Binding Domain of plasma membrane ca2 atpase isoform 4b pmca4b
Journal of Biological Chemistry, 2012Co-Authors: John T Penniston, Ariel J Caride, Emanuel E StrehlerAbstract:The Calmodulin (CaM)-Binding Domain of isoform 4b of the plasma membrane Ca2+ -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe Binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015–4024). This is a relatively rapid reaction, with an apparent half-time of ∼1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ∼10 min. Using targeted molecular dynamics, we now show that dissociation of Ca2+-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-Binding Domain is quite different from that of Binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.
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intramolecular interactions of the regulatory region with the catalytic core in the plasma membrane calcium pump
Journal of Biological Chemistry, 2003Co-Authors: Rita Padanyi, Adelaida G Filoteo, John T Penniston, Katalin Paszty, Alan R Penheiter, Agnes EnyediAbstract:Abstract The access of three proteases to their sites of cleavage was used as a measure of regulatory interactions in the plasma membrane Ca2+ pump isoform 4b (PMCA4b). When the proteases could not cut at their sites in the C-terminal regulatory region, the interaction was judged to be tight. This was the case in the absence of Ca2+, when chymotrypsin and caspase cut PMCA only very slowly. Ca2+ accelerated the fragmentation, but the digestion remained incomplete. In the presence of Ca2+ plus Calmodulin, the digestion became nearly complete in all cases, indicating a more flexible conformation of the carboxyl terminus in the fully activated state. The acceleration of proteolysis by Ca2+ or Ca2+ plus Calmodulin occurred equally at the caspase site upstream of the Calmodulin-Binding Domain and the chymotrypsin and calpain sites downstream of that Domain. Replacing Trp1093 (a key residue within the Calmodulin-Binding Domain) with alanine had a much more specific effect, because it exposed only proteolytic sites within the Calmodulin-Binding Domain that had previously been shielded in the native protein. At these sites, both calpain and chymotrypsin cut the Trp1093 → Ala mutant in the absence of Calmodulin. These data indicate that, in the auto-inhibited conformation, the Calmodulin-Binding/auto-inhibitory sequence and the regions both upstream and downstream are in close contact with the catalytic core. Trp1093 plays an essential role not only in stabilizing the Ca2+-Calmodulin/Calmodulin-Binding Domain complex but also in the formation or stability of the inhibitory conformation of that Domain when it interacts with the catalytic core of PMCA4b.
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protein kinase c activates the plasma membrane ca2 pump isoform 4b by phosphorylation of an inhibitory region downstream of the Calmodulin Binding Domain
Journal of Biological Chemistry, 1996Co-Authors: Agnes Enyedi, Anil K Verma, Adelaida G Filoteo, John T PennistonAbstract:Abstract The carboxyl-terminal region of the plasma membrane Ca2+ pump isoform 4b contains two autoinhibitory regions which keep the pump inactive in the absence of activators such as Calmodulin. One of these regions is approximately coterminous with the Calmodulin-Binding Domain, while the second region is downstream (Verma, A. K., Enyedi, A., Filoteo, A. G., and Penniston, J. T. (1994) J. Biol. Chem. 269, 1687-1691). The carboxyl-terminal region has also been identified as the site for phosphorylation of this isoform by protein kinase C (Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085). Using constructs lacking various numbers of residues at the carboxyl terminus, we studied the degree of phosphorylation by protein kinase C and the resultant activation of Ca2+ transport. The results showed that the most specific and easy phosphorylation occurred in a region of about 20 residues which is downstream of the Calmodulin-Binding Domain, and that the downstream inhibitory Domain had also about the same size and location. Phosphorylation partially activated the pump by removing only the inhibition due to this region. Binding of Calmodulin to the Calmodulin-Binding Domain activated the pump more fully by removing the inhibition due to both regions, regardless of the state of phosphorylation at the downstream inhibitory region.
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plasma membrane calcium pump isoform 4a has a longer Calmodulin Binding Domain than 4b
Journal of Biological Chemistry, 1996Co-Authors: Anil K Verma, Agnes Enyedi, Adelaida G Filoteo, Emanuel E Strehler, John T PennistonAbstract:Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for Calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the Calmodulin-Binding and autoinhibitory Domains of the two variants. The Calmodulin-Binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the Binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+-Calmodulin) is divided between the 28-residue Calmodulin-Binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49-residue Calmodulin-Binding region.
A J Wand - One of the best experts on this subject based on the ideXlab platform.
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characterization of the secondary structure of Calmodulin in complex with a Calmodulin Binding Domain peptide
Biochemistry, 1992Co-Authors: S M Roth, D M Schneider, Laura A Strobel, Anthony R Means, M F A Van Berkum, A J WandAbstract:The interaction between calcium-saturated chicken Calmodulin and a peptide corresponding to the Calmodulin-Binding Domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive 1H and 15N resonance assignments of Calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of Calmodulin in the complex was facilitated by the use of selective labeling of the protein with alpha-15N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly 15N-labeled Calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C alpha H, and C beta H hydrogens indicates that the secondary structure of the globular Domains of Calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon Binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular Domains of Calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal Domains of Calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of Calmodulin-peptide complexes.
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structure of the smooth muscle myosin light chain kinase Calmodulin Binding Domain peptide bound to Calmodulin
Biochemistry, 1991Co-Authors: S M Roth, Arianna Rubin Means, D M Schneider, Laura A Strobel, Mark F A Vanberkum, A J WandAbstract:The interaction between the peptide corresponding to the Calmodulin-Binding Domain of smooth muscle myosin light-chain kinase and (Ca{sup 2+}){sub 4}-Calmodulin has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of {sup 15}N-labeled peptide in conjunction with {sup 15}N-edited and {sup 15}N-correlated {sup 1}H spectroscopy. The peptide forms a 1:1 complex with calcium-saturated Calmodulin which is in slow exchange with free peptide. The {sup 1}H and {sup 15}N resonances of the bound have been assigned. An extensive set of structural constraints for the bound peptide has been assembled from the analysis of nuclear Overhauser effects and three-bond coupling constants. The backbone conformation of the bound peptide has been determined using these constraints by use of distance geometry and related computational methods. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure. Nonhelical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated Calmodulin.
Hans J Vogel - One of the best experts on this subject based on the ideXlab platform.
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structural studies of soybean Calmodulin isoform 4 bound to the Calmodulin Binding Domain of tobacco mitogen activated protein kinase phosphatase 1 provide insights into a sequential target Binding mode
Journal of Biological Chemistry, 2009Co-Authors: Hiroaki Ishida, Mario Rainaldi, Hans J VogelAbstract:The calcium regulatory protein Calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The Calmodulin-Binding Domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca2+-CaM-Binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca2+-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong Binding through a novel “double anchor” motif. Moreover, the orientation of the helical peptide on the surface of Ca2+-SCaM4 is distinct from other known complexes. The N-lobe of Ca2+-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca2+ are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex.
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calcium dependent and independent Binding of soybean Calmodulin isoforms to the Calmodulin Binding Domain of tobacco mapk phosphatase 1
Journal of Biological Chemistry, 2007Co-Authors: Mario Rainaldi, Aaron P Yamniuk, Tomohiko Murase, Hans J VogelAbstract:Abstract The recent finding of an interaction between Calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca2+ 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 Ca2+ with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca2+, 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 Ca2+-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.
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a peptide analog of the Calmodulin Binding Domain of myosin light chain kinase adopts an alpha helical structure in aqueous trifluoroethanol
Protein Science, 1993Co-Authors: Mingjie Zhang, Tao Yuan, Hans J VogelAbstract:A 22-residue synthetic peptide encompassing the Calmodulin (CaM)-Binding Domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon Binding of the peptide to CaM.
