Glycation of lens MIP26 affects the permeability in reconstituted liposomes.

We studied the role of glycation of lens putative gap junctional protein, MIP26, on the permeability as well as on calmodulin mediated gating activity in reconstituted liposomes. Calf lens membranes were incubated with 0-100 mM glucose for 3 days and MIP26 was isolated. There was a glucose concentration dependent increase in the glycation of MIP26 which reached to 2.48 moles/mole of protein with 100 mM glucose. Gel electrophoresis showed that there was no degradation of MIP26 to MIP22 during incubation. Channel permeability was determined by reconstituting MIP26 into asolectin liposomes. There was a MIP26 glycation dependent decrease in the permeability to sucrose. Furthermore, proteoliposomes containing nonglycated MIP26 showed complete uncoupling of the channels with calmodulin whereas the channels containing glycated MIP26 were only partially uncoupled. These results suggest that glycation of MIP26 does interfere with the gating activity in reconstituted liposomes.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Phosphofructokinase is a calmodulin binding protein

A trial to purify myosin light chain kinase from crude myosin led to the isolation of a Mr 85 000 calmodulin binding protein different from this enzyme. Because it showed inherent phosphofructokinase activity we investigated its relation to this enzyme. We demonstrated identity to phosphofructokinase by a close to identical amino acid composition, by antigenic identity and a set of completely identical peptide maps. The calmodulin binding property was also shown for a fraction of the enzyme prepared by standard methods. First experiments show that Ca2+--calmodulin is a potent regulator of phosphofructokinase polymerization.     

Calcium binding to complexes of calmodulin and calmodulin binding proteins

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.     

Ion binding to calmodulin

Summary Over the past few years calcium has emerged as an important bioregulator. Upon external stimulation, the cell generates a transient Ca2+ increase, which is transformed into a cellular event through a molecular cascade. The first step in this cascade is the binding of calcium to proteins present in the cytosol. These proteins capable of binding Ca²⁺ under physiological conditions all belong to the same evolutionary family that evolved from a common ancestor. However, they strongly differ in the properties of their calcium binding sites. Calmodulin, the ubiquitous calcium binding protein present in all eukaryotic cells, is very close to the ancestor protein, presents four calcium binding sites which bind calcium, magnesium and monovalent ions competitively and is involved in the triggering of cellular processes. Parvalbumin, another member of the family, is more specialized and found mostly in fast-twitch skeletal muscle. It binds calcium and magnesium with high affinity and seems to be involved in muscle relaxation. On the other hand, troponin C which confers Ca²⁺ sensitivity to acto-myosin interaction exhibits both triggering and relaxing sites. The study of intracellular Ca^2– binding proteins has shown that calcium binding proteins have evolved from a simple common structure to fulfill different functions.Abbreviations CaBP calcium-binding protein - ICaBP the vitamin D-dependent intestinal Cat+binding protein - S-100 the glial S-100 protein - RLC the phosphorylatable myosin regulatory light chain - CaM calmodulin - Pa parvalbumin - TnC troponin C - TnI troponin I - Hepes N-2-hydroxyethylpipezarine, N-2-ethane-sulfonic acid - W7 N-(6-Aminohexyl)-5-chloro-l-Naphtalene sulfonamide - SDS sodium dodecyl sulfate - NMR nuclear magnetic resonance     

NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein

Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low deltaG(o)20 values of approximately 2.7 (N-domain) and approximately 1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20-25 degrees C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] > or = 0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities. For the intact protein (+/-Ca2+), the bimodal denaturation profiles can be analyzed to give two deltaG(o)20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.     

Effects of calmodulin and calmodulin binding protein BP-10 on phosphorylation of thylakoid membrane protein

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Modulating calmodulin binding specificity through computational protein design

We report the computational redesign of the protein-binding interface of calmodulin (CaM), a small, ubiquitous Ca(2+)-binding protein that is known to bind to and regulate a variety of functionally and structurally diverse proteins. The CaM binding interface was optimized to improve binding specificity towards one of its natural targets, smooth muscle myosin light chain kinase (smMLCK). The optimization was performed using optimization of rotamers by iterative techniques (ORBIT), a protein design program that utilizes a physically based force-field and the Dead-End Elimination theorem to compute sequences that are optimal for a given protein scaffold. Starting from the structure of the CaM-smMLCK complex, the program considered 10(22) amino acid residue sequences to obtain the lowest-energy CaM sequence. The resulting eightfold mutant, CaM_8, was constructed and tested for binding to a set of seven CaM target peptides. CaM_8 displayed high binding affinity to the smMLCK peptide (1.3nM), similar to that of the wild-type protein (1.8nM). The affinity of CaM_8 to six other target peptides was reduced, as intended, by 1.5-fold to 86-fold. Hence, CaM_8 exhibited increased binding specificity, preferring the smMLCK peptide to the other targets. Studies of this type may increase our understanding of the origins of binding specificity in protein-ligand complexes and may provide valuable information that can be used in the design of novel protein receptors and/or ligands.     

Biologically active fluorescent derivatives of spinach calmodulin that report calmodulin target protein binding

Spinach calmodulin (CaM) has been labeled at cysteine-26 with the sulfhydryl-selective probe 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) to produce MIANS-CaM. The interaction of MIANS-CaM with CaM binding proteins was studied by fluorescence enhancement accompanying the protein-protein interactions. MIANS-CaM bound to smooth muscle myosin light-chain kinase with a Kd of 9 nM, causing a 4.6-fold fluorescence enhancement. Caldesmon bound with a Kd of 250 nM, causing a 2-fold fluorescence enhancement. Calcineurin (CaN) bound to MIANS-CaM with a Kd less than 5 nM, causing an 80% increase in fluorescence. On the other hand, binding of the CaM antagonist drugs prenylamine and calmidazolium or the potent peptide antagonist melittin did not alter MIANS fluorescence. MIANS-CaM activated brain cGMP phosphodiesterase and CaN as effectively as unlabeled CaM. Spinach CaM was also labeled with three other sulfhydryl reagents, 6-acryloyl-2-(dimethylamino)naphthalene, (2,5-dimethoxy-4-stilbenyl)maleimide, and rhodamine X maleimide. CaN bound to the highly fluorescent rhodamine X maleimidyl-CaM with a Kd of 1.4 nM, causing a 25% increase in polarization. Both MIANS-CaM and rhodamine X-CaM were used to monitor the Ca2+ dependence of the interaction between CaM and CaN. Half-maximal binding occurred at pCa 6.7-6.8 in the absence of Mg2+, or at pCa 6.3 in the presence of 3 mM Mg2+. In both cases, the dependence of the interaction was cooperative with respect to Ca2+ (Hill coefficients of 1.7-2.0). Use of these fluorescent CaMs should allow accurate monitoring of CaM interactions with its target proteins and perhaps their localization within the cell.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

Acidic calmodulin binding protein, ACAMP-81, is MARCKS protein interacting with synapsin I.

ACAMP-81 is an acidic calmodulin binding protein with molecular mass of 81 kDa. We report partial amino acid analysis of ACAMP-81 and its interaction with synapsin I. 123 amino acids of ACAMP-81 were determined and the sequence was completely identical with that of MARCKS protein which was thought to be a substrate for calcium/phospholipid dependent protein kinase (PKC). We found ACAMP-81 bound to synapsin I with 125I-labeled ACAMP-81 overlay method. ACAMP-81 bound to the cysteine specific cleaved 51 kDa fragment derived from middle/tail region of synapsin I.     

Kinetics of calmodulin binding to calcineurin

Calcineurin (CaN) binds Ca(2+)-saturated calmodulin (CaM) with relatively high affinity; however, an accurate steady-state K(d) value has not been determined. In this report, we describe, using steady-state and stopped-flow fluorescence techniques, the rates of association and dissociation of Ca(2+)-saturated CaM from CaN heterodimer (CaNA/CaNB) and CaNA only. The rate of Ca(2+)/CaM association was determined to be 4.6 x 10(7) M(-1)s(-1). The rate of Ca(2+)/CaM dissociation from CaN was slower than previously reported and was approximately 0.0012 s(-1). In preparations of CaNA alone (no regulatory CaNB subunit), the dissociation rate was slowed further to 0.00026 s(-1). From these data we calculate a K(d) for binding of Ca(2+)-saturated CaM to CaN of 28 pM. This K(d) is significantly lower than previously reported estimates of approximately 1 nM and indicates that CaN is one of the highest affinity CaM-binding proteins identified to date.     

Latent TGF-beta binding protein LTBP-2 decreases fibroblast adhesion to fibronectin

We have analyzed the effects of latent TGF-beta binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.     

Ca2+/calmodulin stimulates GTP binding to the ras-related protein ral-A.

Ral-A is a Ras-related GTP-binding protein that has been suggested to be the downstream target of Ras proteins and is involved in the tyrosine kinase-mediated, Ras-dependent activation of phospholipase D. We reported recently that Ral-A purified from human erythrocyte membrane binds to calmodulin in a Ca2+-dependent manner at a calmodulin binding domain identified near its C-terminal region (Wang, K. L., Khan, M. T., and Roufogalis, B. D. (1997) J. Biol. Chem. 272, 16002-16009). In this study we show the enhancement of GTP binding to Ral-A by Ca2+/calmodulin. The stimulation up to 3-fold by calmodulin was Ca2+-dependent, with half-maximum activation occurring at 180 nM calmodulin and 80 nM free Ca2+ concentration. The present work supports a regulatory role of Ca2+/calmodulin for the activation of Ral-A and suggests a possible direct link between signal transduction pathways of Ca2+/calmodulin and Ral-A proteins.     

Physicochemical and hydrodynamic characterization of P-57, a neurospecific calmodulin binding protein

P-57 is a neurospecific calmodulin binding protein that was discovered by virtue of its unusual interactions with calmodulin-Sepharose [Andreasen, T. J., Luetje, C. W., Heideman, W., & Storm, D. R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T. J., Andreasen, K. I., & Storm, D. R. (1985) J. Biol. Chem. 260, 10784-10788]. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin-Sepharose in the absence of calcium compared to that in the presence of calcium. In this study, we report the chemical and physical properties of P-57 purified from detergent-solubilized bovine brain membranes. The amino acid composition of P-57 is distinctive in that it contains a single phenylalanine residue with no other aromatic amino acids and a relatively high percentage of proline and alanine. In the presence of 0.05% Lubrol PX, its predicted secondary structure from circular dichroism spectroscopy is 1% alpha-helix, 21% beta-sheet, and 78% random coil. The hydrodynamic characteristics of the protein-detergent complex and the molecular weight of the protein were determined by gel filtration and sucrose density gradient sedimentation in the presence and absence of calmodulin. The P-57-detergent complex has an apparent Stokes radius (Rs) of 4.58 nm and a sedimentation coefficient (S20,w) of 1.44 S while the Stokes radius and S20,w for the P-57-calmodulin-detergent complex are 5.33 nm and 2.32 S, respectively. Perrin analysis of a 5-[[[(iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic acid (AEDANS) derivative of P-57 confirmed the Stokes radius determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium binding to tryptic fragments of calmodulin

Fragments of scallop testis calmodulin were prepared by tryptic digestion. One peptide consisted of 75 amino acid residues from N-acetylalanine to lysine at position 75 (F12) and the other of 71 residues from aspartic acid at position 78 to C-terminal lysine (F34). Flow dialysis and equilibrium dialysis experiments revealed the existence of two Ca2+ binding sites in each fragment. Half-saturating concentrations of the Ca2+ titration curves were 11 microM for F12 and 3.2 microM for F34, and Hill coefficients were obtained as 1.14 and 1.84, respectively. The results indicate that the high-affinity sites for Ca2+ are located on the C-terminal region of the calmodulin. The sum of the two Ca2+ titration curves of F12 and F34 fits well to the curves of Ca2+ binding to intact calmodulin. This shows that the characteristic of Ca2+ bindings in intact calmodulin did not change after separation of the whole molecule into two domains, F12 and F34. The domains corresponding to F12 and F34 may exist independently from each other in the intact calmodulin molecule.     

Divalent cation binding to wheat germ calmodulin

The Ca2+ binding to plant (wheat germ) calmodulin was measured in 0.1 M NaCl by a flow-dialysis method. The four macroscopic binding constants best fitted to the data were 0.20, 0.25, 0.025, and 0.0024 microM-1. The cysteine residue of this calmodulin is located at the 27th position from the NH2-terminal (Yazawa, M. et al. (1982) Abstr. 33th Conf. Protein Structure pp. 9-12, Osaka). According to the quantitative analysis of the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with Cys 27, the calmodulin which binds 3 Ca2+ showed the minimum reactivity with DTNB. This suggests that the site for the third Ca2+ binding is located close to Cys 27. Cys 27 was spin-labeled with N-(2,2,6,6-tetramethyl-4-piperidine-1-oxyl)maleimide, and its ESR spectrum was measured in the presence of Mn2+ and/or Ca2+. The rotational relaxation time of the label (1.2 ns) was increased by about one-tenth with 1 to 2 mol of bound Ca2+, but was unchanged with Mn2+. On the other hand, Mn2+ induced a remarkable quenching of the spectrum. From the decrease in the peak heights of the ESR spectrum, the distance between the label and the first bound Mn2+ was estimated to be 0.8 nm. It is concluded that the first Mn2+ binds to a domain near the NH2-terminal. The difference UV absorption spectrum induced by Mn2+ was similar to that induced by Ca2+. However, the amount of Mn2+ needed to saturate the difference spectrum was 1 mol more than the amount of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)