Characterization of calcium-binding sites in development-specific protein S of Myxococcus xanthus using site-specific mutagenesis

Protein S, the most abundant protein synthesized during development of the Gram-negative bacterium Myxococcus xanthus, assembles on the surface of the spores. It can be dissociated from the spores using divalent metal chelators and will reassemble on the spores in the presence of calcium. The amino acid sequence of protein S contains regions which have homology to the calcium-binding sites of calmodulin. Protein S was found to bind 2 mol of calcium/mol of protein with Kd values of 27 and 76 microM. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin (Ser40----Arg,Ser129----Arg), and a double mutant was also constructed. Each mutant gene was then transduced into the genome of a M. xanthus strain from which the wild-type genes had been deleted. All three mutants produced protein S normally during development. One of the mutants (Ser129----Arg) had normal amounts of protein S on its spores, whereas the other (Ser40----Arg) bound much less and the double mutant had virtually none. Analysis of the calcium binding affinities of the purified proteins showed that [Arg40]protein S and [Arg40, Arg129]protein S did not bind detectable quantities of calcium, whereas [Arg129]protein S bound less calcium than the wild-type protein and with a reduced affinity.     

Polycation-dependent, Ca2+-antagonized phosphorylation of calmodulin by casein kinase-2 and a spleen tyrosine protein kinase

Ten distinct protein kinases have been tested for their ability to phosphorylate calmodulin. Only casein kinase-2 and a spleen tyrosine protein kinase (TPK-III) proved effective, their phosphorylation efficiency being dramatically enhanced by histones and other polybasic peptides while being depressed by 50 microM Ca2+. Phosphorylation by CK-2 takes place with a Km of 12 microM calmodulin, leading to the incorporation of more than 1.5 mol P/mol substrate. Ser81 and Thr79 are among the residues affected. On the other hand, the two tyrosyl residues of calmodulin are both phosphorylated by TPK-III, Tyr99 being preferred over Tyr138.     

Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin

We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.     

Calmodulin from neurospora crassa. General properties and conformational changes

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.     

The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.     

Aluminum interaction with calmodulin. Evidence for altered structure and function from optical and enzymatic studies

The interaction of aluminum ions with bovine brain calmodulin has been examined by fluorescence spectroscopy, circular dichroic spectrophotometry and equilibrium dialysis, and by the calmodulin-dependent activation of 3',5'-cyclic nucleotide phosphodiesterase. These experiments show that aluminum binds stoichiometrically and cooperatively to calmodulin. Binding of aluminum at a molar ratio of 2:1 to calmodulin suffices to induce a major structural change. Estimates from spectroscopic data indicate that the binding affinity for the first mol of aluminum bound to the protein is about one order of magnitude stronger than that of calcium to its comparable site. These estimates agree with a dissociation constant of 0.4 microM derived from equilibrium dialysis experiments. Interaction of aluminum with calmodulin induces a helix-coil transition and enhances the hydrophobic surface area much more than calcium does. A molar ratio of 4:1 for [aluminum]/[calmodulin] is sufficient to block completely the activity of the calcium-calmodulin-dependent phosphodiesterase. Highly hydrated aluminum ions apparently promote solvent-rich, disordered polypeptide regions in calmodulin which, in turn, profoundly influence the protein's flexibility.     

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.     

Radioiodination of tyrosine residue(s) of ox testis and of wheat germ calmodulins

Radioiodination of the two tyrosine residues (Tyr-99 and Tyr-138) of ox testis calmodulin was performed using several methods, and studied through the specific activity, and the [125I]iodoamino acid analysis of the radiolabeled calmodulins. Hydrolysis by thrombin of 125I-calmodulin labeled by the lactoperoxidase method and subsequent isolation of peptides TM1 and TM2 by gel electrophoresis showed preferential labeling by 125I of Tyr-99 (TM1) over Tyr-138 (TM2). Analysis of [125I]iodoamino acids of radiolabeled TM1, TM2 and calmodulin demonstrated that [125I]monoiodotyrosine was predominant, the remainder being [125I]diiodotyrosine. Radioiodination of wheat germ calmodulin, which contains a single tyrosine residue (Tyr-139), showed that only TM2 was labeled by 125I on the Tyr-139 residue and also on the His-108 residue (radiolabeled monoiodotyrosine, diiodotyrosine and monoiodohistidine being present).     

The phosphorylation of choline acetyltransferase

Human placental Choline Acetyltransferase (ChAT) has been shown to be phosphorylated in vitro by kinases present in rat brain. Phosphorylation occurs at a single site with the exclusive phosphoamino acid being serine. ChAT phosphorylation was shown to be calcium, and not cyclic nucleotide, dependent and was inhibited by inhibitors of calcium/calmodulin protein kinases including anti-calmodulin anti-sera. ChAT phosphorylation was stimulated by calmodulin (9 fold) and, to a lesser extent, by phosphatidylserine (4 fold). These results indicate the involvement of a calcium/calmodulin and possibly also a calcium/phosopholipid kinase. This finding was confirmed by demonstrating ChAT phosphorylation using both purified multifunctional calcium/calmodulin protein kinase (CaMK) and calcium/phospholipid protein kinase C (PKC) from rat brain. A stoichiometric incorporation of 0.9 mol phosphate/mol ChAT was achieved by CaMK. Phosphorylated ChAT could be isolated from freshly prepared rat brain synaptosomes. The results obtained with this model system support the hypothesis that in vivo a fraction of ChAT exists phosphorylated.     

Characteristics of calmodulin binding to purified human lymphocyte plasma membranes

We have explored the role of calmodulin in plasma membrane-related phenomena in lymphocyte activation by measurement of [125I]calmodulin binding to highly purified plasma membrane of human peripheral blood lymphocytes. Calcium-dependent calmodulin binding to lymphocyte membrane was found to reach equilibrium within 5 min of incubation at 37 degrees C and to be saturable and specific. A single class of high affinity-binding sites was identified, with a dissociation constant (Kd) of 1 to 3 X 10(-8) M and a total binding capacity (Bt) of 1 to 2 pmol/mg membrane protein. The free calcium concentration necessary for half-maximal binding was 100 to 300 nM. This was strikingly similar to the cytoplasmic-free calcium activity [Ca2+]i measured by the Quin-2 fluorescence technique, particularly after stimulation with phytomitogens. Calmodulin binding was inhibitable by trifluoperazine (TFP), W-7, and chloropramazine, all of which are calmodulin antagonists. The concentration of TFP that caused 50% inhibition of lymphocyte proliferative responses to phytomitogens was found to be identical to the concentration of TFP which causes 50% inhibition of calmodulin binding to lymphocyte plasma membrane. SDS-polyacrylamide gel electrophoresis followed by gel overlay and autoradiography with iodinated calmodulin revealed five calcium-dependent, TFP-inhibitable, calmodulin-binding polypeptides.     

Calmodulin N-methyltransferase. Partial purification and characterization

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.     

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.     

Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase

In the red cell incubated with ortho-[32P] phosphate, CaCl₂ and calcium ionophore A 23187, phosphorylation of erythrocyte pyruvate kinase was demonstrated using the double antibody technique and autoradiography. Phosphorylation was inhibited by calmodulin inhibitors, trifluoperazine or ZnCl₂. In the presence of purified erythrocyte calmodulin, CaCl₂ and [γ-³²P] ATP, the partially purified erythrocyte pyruvate kinase containing cytozol protein kinases was phosphorylated. This was also inhibited by trifluoperazine or ZnCl₂. From these results, it was concluded that erythrocyte pyruvate kinase is phosphorylated by a calcium-calmodulin dependent process.     

Cooperativity among calmodulin's drug binding sites

The binding of felodipine, a dihydropyridine Ca2+ antagonist, to calmodulin has been studied by equilibrium dialysis and fluorescence techniques. Analysis using the Hill equation gives a Hill coefficient of 2. A plot of bound [felodipine] vs. free [felodipine]2 gives a Bmax of 1.9 mol/mol and a K0.5 of 22 microM. Two calmodulin antagonists, prenylamine and R24571, which have previously been shown to potentiate the fluorescent enhancement observed when felodipine binds to calmodulin [Johnson, J. D. (1983) Biochem. Biophys. Res. Commun. 112, 787], produce a reduction in Hill coefficient to 0.7 and 1.0, respectively, and account for the observed potentiation of felodipine binding. Titrations of felodipine with calmodulin in the absence and presence of prenylamine and R24571 suggest that these drugs decrease the K0.5 of calmodulin for felodipine by 25-fold. Thus, potentiating drugs (prenylamine and R24571) bind to either of the two felodipine binding sites and, through an allosteric mechanism, result in felodipine binding to the remaining site with greatly enhanced affinity. Two types of potentiating drugs are observed. Prenylamine exhibits a Hill coefficient of 0.8 whereas felodipine, R24571, and diltiazem exhibit Hill coefficients of 2 in their potentiation of felodipine binding. Titrations of felodipine and calmodulin with Ca2+ exhibit cooperativity with a Hill coefficient of 4. Half-maximal binding occurs near pCa 6.0. In the presence of R24571, the calcium dependence of felodipine binding is biphasic, now exhibiting a much higher affinity (pCa 7.6) component. A model is presented to explain the relationship of these various allosterically regulated conformers of calmodulin and their interactions and activation with its target proteins.     

Is Ca2+ -Calmodulin-Dependent Protein Phosphorylation in Rat Brain Modulated by Carboxylmethylation?

Calmodulin stimulation of protein kinase activity in calmodulin-depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein-O-carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+-calmodulin-dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate-gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/micrograms protein or 0.15-1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8-azido-ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+-calmodulin protein kinase may modulate the activity of this enzyme in rat brain.     

Volume changes upon addition of Ca2+ to calmodulin: Ca2+-calmodulin conformational states

Measurement of the volume change by a rapid density method upon sequential addition of calcium ion to calmodulin showed relatively large, nonuniform increases for the first 4 moles Ca2+ per mole calmodulin. Substantially larger volume increases (approximately 15 ml/mol protein) were observed upon addition of the second and fourth moles Ca2+ relative to the first and third moles added per mole calmodulin. A total volume increase of approximately 170 ml/mol protein attended the addition of 4 moles Ca2+, as expected for multidentate carboxylate coordination to metal ion. Marginal changes in volume were observed upon further additions, the data showing a remarkably sharp transition after [Ca2+]/[calmodulin] = 4. The results are consistent with an ordered binding of Ca2+ in which pair-wise additions produce similar volume changes; the volume change behavior, however, does not indicate an absence of distinct conformational states for a Ca2+(1)-calmodulin and a Ca2+(3)-calmodulin complex as has been proposed on the basis of 1H-NMR evidences.     

Synthesis of biologically active porcine secretin and [ITyr10] porcine secretin.

Porcine secretin, [Tyr10] secretin, and [Tyr13] secretin were synthesized by solid phase methodology and purified by stepwise gradient elution from a short reversed-phase column with ethanol and acetic acid as organic modifiers. [Tyr10] secretin and [Tyr13] secretin were iodinated by the chloramine-T method and nonmono-, and di-iodinated products separated and isolated by reversed-phase HPLC. Batch incubation analysis is isolated mouse pancreatic islets revealed that secretin and the [Tyr10] analogue were indistinguishable in their effect on the glucose-induced insulin release and cAMP accumulation. [Tyr13] secretin in contrast was significantly less potent in its effect on the glucose-induced insulin release.     

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.     

Calmodulin and ATP dependence of the high and low calcium affinity p-nitrophenylphosphatase from human erythrocyte membranes

In calmodulin depleted membranes from human erythrocytes, the Ca2+-dependent phosphatase showed different sensitivity to calmodulin and ATP with variable affinity towards free calcium concentrations: a calmodulin-dependent activity with high calcium affinity, K1/2 = 1.2 X 10(-7) mol/l calcium, that was fully activated at submicromolar calcium concentrations, higher concentrations being rather inhibitory; an ATP-dependent activity with lower calcium affinity, K1/2 = 10(-6) mol/l calcium, that was fully activated at 10(-5) mol/l calcium in the presence of 50-200 mumol/l ATP and was insensitive to calmodulin, and a calcium dependent phosphatase that was active at a wider ranger of free calcium, 10(-8)-10(-5) mol/l, and required the presence of both calmodulin and ATP.     

Phosphorylation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase regulates myosin light chain kinase

Smooth muscle myosin light chain kinase, purified to homogeneity, has a molecular weight of 130,000 +/- 5,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme has a specific activity under maximal conditions of 30 mumol Pi transferred to myosin light chain/mg kinase/min at 24 C and is totally dependent on calmodulin and calcium for activity. Incubation of myosin kinase with the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase results in the covalent incorporation of up to one mol of phosphate per mol of myosin kinase in the absence of bound calmodulin. Limited tryptic digestion of the radioactively labeled kinase indicates that all of the label has been incorporated into a single tryptic peptide (mol wt approximately 22,000), suggesting that a single site is being phosphorylated. Phosphorylation of myosin kinase lowers the rate at which the kinase phosphorylates myosin light chain. The lower rate of light chain phosphorylation is due to a weaker binding of calmodulin to the phosphorylated kinase than to the unphosphorylated kinase. Cyclic adenosine 3':5'-monophosphate-dependent phosphorylation of the kinase actin-myosin interaction represents a possible link between hormonal binding to smooth muscle receptors and muscle relaxation. A scheme for this sequence of events is presented.