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T Vorherr

Publications and source records attributed to T Vorherr.

At least 37 records · Page 2Linked to original sources
PubMed1992

Fluorescence energy transfer analysis of calmodulin-peptide complexes.

The interactions between calmodulin and the tryptophan residues of synthetic peptides corresponding to the calmodulin binding domains of skeletal muscle myosin light-chain kinase and the plasma membrane calcium pump were examined. The single tryptophan residue contained in each peptide became relatively immobilized and inaccessible to iodide ion upon binding to calmodulin, indicating that the indole side chain was inserted into a hydrophobic cleft in the surface of calmodulin. Fluorescence energy transfer from peptidyl tryptophan residues to an AEDANS moiety attached to cysteine-26 of spinach calmodulin was measured. Included in these analyses was a tryptophan-containing peptide analog of the calmodulin binding domain of neuromodulin. These data indicated that the indole ring of each peptide inserted 32-35 A away from cysteine-26 and may therefore interact with the carboxyl-terminal lobe of CaM in its "bent" conformation [Persechini & Kretsinger (1988a) J. Cardiovasc. Pharmacol. 12 (Suppl 5), S1-S12; Ikura et al. (1992) Science 256, 632-638; Vorherr et al. (1992) Eur. J. Biochem. 204, 931-937]. The interchange of tryptophan-3 and phenylalanine-21 of the calcium pump peptide increased the efficiency of energy transfer to the AEDANS-moiety approximately 12-fold, reducing the calculated distance to 20 A. These data suggest that phenylalanine-21 of the calcium pump peptide interacts with the hydrophobic cleft in the amino-terminal lobe of CaM.

Amino Acid Sequence↗
PubMed1992

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.

Amino Acid Sequence↗
PubMed1992

Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).

Affinity Labels↗
PubMed1992

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.

Amino Acid Sequence↗
PubMed1992

Construction and molecular dynamics simulation of calmodulin in the extended and in a bent conformation.

Analysis of sequence similarity and comparison of the three-dimensional (3D) structures of troponin C and calmodulin have revealed a sequence in the central helix of calmodulin with a high probability for bending. The three amino acids known to form a bend in the N-terminal portion of troponin C are also found in the central helix of calmodulin. The modelling of a bent calmodulin structure, using the dihedral angles of the three residues in the bend of troponin C as a 3D template, results in a conformation of calmodulin where the N- and C-terminal domains are able to form contacts. Dynamics simulations starting from the X-ray structure of calmodulin and from the modelled bent calmodulin were carried out to compare flexibility and correlated movements of Ca2+ in the binding loops. Both conformations of calmodulin remained stable during the period of simulation. In the simulation of calmodulin in the extended form, the motions of the Ca2+ atoms in the two domains (Ca2+1 and Ca2+2 in one domain, and Ca2+3 and Ca2+4 in the other) are correlated. In the simulation of the bent form, an additional correlation between the Ca atoms in the two different domains is observed. The results are compatible with the occurrence of a bent conformation of calmodulin in the presence of targets, and with increased Ca2+ affinity and cooperativity of the Ca(2+)-binding loops in the calmodulin-peptide complexes.

Calcium↗
PubMed1992

Identification of two domains which mediate the binding of activating phospholipids to the plasma-membrane Ca2+ pump.

The stimulation of the purified human erythrocyte calcium pump by acidic phospholipids was investigated using synthetic peptides corresponding to a putative phospholipid-responsive domain [Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N. & Carafoli, E. (1990) Biochemistry 29, 8070-8076] and to the calmodulin-binding domain of the pump. The peptides interfered with the activation of the enzyme by phosphatidylserine and phosphatidic acid in competition assays. The peptide corresponding to the calmodulin-binding domain was found to be the most efficient antagonist. Direct binding measurements using fluorescent derivatives of the peptides confirmed the interaction between the acidic phospholipids and the peptides, and fluorescence titrations of dansylated calmodulin with the purified ATPase showed a direct effect of acidic phospholipids on calmodulin binding. A proteolyzed preparation of the Ca(2+)-ATPase lacking the calmodulin-binding domain confirmed that the phospholipid-induced stimulation is mediated by two sites, one located in the C-terminal portion of the previously identified 44-amino-acid phospholipid-responsive domain, the other in the calmodulin-binding domain.

Amino Acid Sequence↗
PubMed1992

A monoclonal antibody recognizes an epitope in the first extracellular loop of the plasma membrane Ca2+ pump.

A monoclonal antibody against the human erythrocyte Ca2+ pump (1E4) reacted with the enzyme in intact erythrocytes. Using trypsinized preparations of the pump the antibody only reacted with the N-terminal fragments of 33.5 and 35 kDa. The fragments span from the N terminus (35 kDa) or from residue 19 (33.5 kDa) to residue 314 of the hPMCA4 isoform of the pump. Exhaustive degradation with a number of agents produced smaller peptides which reacted with the antibody. Sequencing analysis on two chymotryptic fragments of 8.8 and 13.5 kDa identified the epitope in an approximately 80-residue domain beginning with Gly-81. Two peptides corresponding to the putative extramembrane portions of this region of the pump were synthesized. The antibody reacted with one of them, spanning residues Phe-121 to Gly-152 and containing the first putative external loop of the pump. Peptides corresponding to overlapping portions of this peptide were synthesized, leading to the location of the epitope in a 13-residue sequence (Glu-130 to Glu-142) in the first predicted extracellular loop (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Mathews, S., Strehler-Page, M-A., James, P., Vorherr, T., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159).

Amino Acid Sequence↗
PubMed1992

Regulation of the calcium ion pump of sarcoplasmic reticulum: reversible inhibition by phospholamban and by the calmodulin binding domain of the plasma membrane calcium ion pump.

A 45 amino acid peptide (A45) corresponding to the phospholamban (PLN) binding domain of the sarcoplasmic reticulum (SR) ATPase was synthesized. Circular dichroism experiments have shown that the peptide had a predominantly random-coil conformation but adopted a higher proportion of secondary structure in the presence of a synthetic 32 amino acid peptide corresponding to the hydrophilic portion of PLN. A similar conformational change was induced by the synthetic calmodulin binding domain of the plasma membrane Ca2+ pump (peptide C28W), which acts as an endogenous inhibitor of the pump and is homologous to PLN. Cross-linking experiments have shown that peptide C28W interacted with peptide A45. The Ca(2+)-pumping activity of cardiac SR, which contains endogenous PLN, was stimulated about 30% by peptide A45. The stimulation was maximal at submicromolar Ca2+ levels and tended to disappear at higher Ca2+ concentrations. By contrast, the Ca(2+)-pumping activity of skeletal muscle SR, which lacks endogenous PLN, was unaffected. Peptide C28W strongly inhibited the pumping activity of skeletal muscle SR, and peptide A45 reversed the inhibition. The results suggest that peptide A45 competed with the ATPase for phospholamban or for peptide C28W, removing the inhibition of the pump. Thus, the exogenous inhibitor of the SR Ca(2+)-ATPase, PLN, and the internal inhibitor of the plasma membrane Ca(2+)-ATPase, peptide C28W, are functionally analogous.

Amino Acid Sequence↗
PubMed1992

The calmodulin-binding site of the plasma membrane Ca2+ pump interacts with the transduction domain of the enzyme.

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.

Amino Acid Sequence↗
PubMed1991

Phospholamban is related to the autoinhibitory domain of the plasma membrane Ca(2+)-pumping ATPase.

The Ca2+ pumps of the plasma membrane (PM ATPase) and of sarcoplasmic reticulum (SR ATPase) share a number of structural and functional properties. A major difference is the regulatory mechanism. The PM ATPase contains a C-terminal autoinhibitory domain; calmodulin binds to it, removing the inhibition. The SR ATPase contains a domain that interacts with the inhibitor protein phospholamban when the latter is in the nonphosphorylated state; phosphorylation of phospholamban removes the inhibition. Peptides corresponding to the autoinhibitory domain of the PM ATPase were synthesized and found to inhibit the SR ATPase. A 28-residue peptide (C28W), containing the entire autoinhibitory domain, was the most powerful (IC50 = 15 microM; lmax greater than 90%). The inhibition was Ca2+ dependent and more pronounced at submicromolar Ca2+ concentrations. C28W is about 50% homologous to the cytosolic domain of phospholamban, the hydrophilic portion of which was found to interact directly with calmodulin (Kd = about 700 nM). However, while calmodulin reversed the inhibition of the SR ATPase by C28W, it failed to reverse that induced by nonphosphorylated phospholamban.

Amino Acid Sequence↗
PubMed1991

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.

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.

Amino Acid Sequence↗
PubMed1991

The plasma membrane Ca2+ pump contains a site that interacts with its calmodulin-binding domain.

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.

Amino Acid Sequence↗
PubMed1991

Identification and primary structure of a calbindin 9K binding domain in the plasma membrane Ca2+ pump.

Bovine calbindin 9K has been conjugated to a bifunctional, photoactivatable, cleavable and radioactive cross-linker. It has been photolyzed in the presence of preparations of the purified erythrocyte Ca2+ pump, and shown to interact with it in the presence of Ca2+. The affinity of the interaction has been studied using the fluorescence enhancement of dansylated calbindin 9K incubated with the synthetic calmodulin binding domain of the pump. Two versions of the domain have been used, one corresponding to its full length (28 residues), one to about 2/3 of it (20 residues). The affinity of the interaction was between 5 and 10 times lower than in the case of calmodulin.

Amino Acid Sequence↗
PubMed1991

The calmodulin-binding domain mediates the self-association of the plasma membrane Ca2+ pump.

Dimerization (oligomerization) of the plasma membrane Ca2+ pump increases its activity (Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). Fluorescence titration on preparations of the purified eosin-labeled human erythrocyte ATPase has been used to monitor the oligomerization process. Calmodulin inhibits oligomerization, although it can bind to the oligomerized enzyme. Synthetic peptides corresponding to the calmodulin-binding domain of the pump stimulate its ATPase activity, indicating the formation of heterooligomers of the peptides with the pump. The oligomerization is prevented by the preincubation of the ATPase with calmodulin. Polyclonal antibodies against the synthetic calmodulin-binding domain inhibit its basal and its calmodulin-stimulated ATPase activity and prevent the formation of the oligomers. ATPase preparations truncated at the COOH terminus with calpain to a fragment of 124 kDa which does not contain the calmodulin-binding domain fail to oligomerize with the intact ATPase. The results show that the calmodulin-binding domain mediates the oligomerization of the Ca2+ pump.

Amino Acid Sequence↗
PubMed1990

Mapping of functional domains in the plasma membrane Ca2+ pump using trypsin proteolysis.

The purified erythrocyte Ca2+ pump has been exposed to trypsin under conditions designed to enrich the fragments of molecular mass 90, 85, 81, and 76 kDa, respectively. In SDS-polyacrylamide gels, these fragments are accompanied by a product of molecular mass about 33 kDa. N- and C-terminal sequencing of the fragments blotted on PVDF membranes has located the four high molecular mass fragments and the 33-kDa fragment within the pump structure. The work has extended previous work on the organization of the calmodulin-interacting domain of the pump (Zurini et al., 1984; Benaim et al., 1984) and has tentatively placed the domain of the pump which interacts with acidic phospholipids between transmembrane helices 2 and 3.

Amino Acid Sequence↗
PubMed1990

Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane.

The sequence of more than 1,000 amino acid residues, derived from two different isoforms, has been determined from peptides generated from purified human erythrocyte membrane Ca2(+)-ATPase (hPMCA). Several of these peptide sequences correspond to the previously reported, cDNA deduced sequence of the "teratoma" Ca2+ pump isoform hPMCA1 (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Matthews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159). The complete primary structure of a novel isoform (hPMCA3) has been determined by molecular cloning and nucleotide sequencing of its corresponding cDNA. This new member of the plasma membrane Ca2+ pump family consists of 1,205 amino acid residues with a calculated Mr of 133,930, and it shows 88% similarity (75% identity) with the previously sequenced pump isoform. Specific probes detect major mRNA species of 5.6 kilobases for hPMCA1, and of 7.5 kilobases for hPMCA3, on Northern blots of human K562 erythroleukemic cell RNA. A large number of peptide sequences match perfectly with only one or the other of these isoforms and all peptides (with 6 exceptions corresponding to a contaminant protein or to a third minor Ca2+ pump isoform) are found in either only one or in both of the isoforms. The two erythrocyte Ca2+ pumps display high sequence divergence in a few localized regions that may determine isoform-specific functional specializations; for example, the putative extracellular loop separating transmembrane domains 1 and 2, the highly negatively charged region previously suggested to be involved in Ca2+ binding, and the site of cAMP-dependent protein kinase phosphorylation.

Amino Acid Sequence↗
PubMed1990

Interaction of calmodulin with the calmodulin binding domain of the plasma membrane Ca2+ pump.

Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump (James et al., 1988) were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with the calmodulin binding domain, supporting the suggestion (Benaim et al., 1984) that the latter, or a portion of it, may act as a natural inhibitor of the pump.

Animals↗