The tumor-sensitive calmodulin-like protein is a specific light chain of human unconventional myosin X

Human calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca(2+)-dependent interactions with specific target proteins. Using gel overlays, we identified a approximately 210-kDa protein binding specifically and in a Ca(2+)-dependent manner to CLP, but not to calmodulin. Yeast two-hybrid screening yielded a CLP-interacting clone encoding the three light chain binding IQ motifs of human "unconventional" myosin X. Pull-down experiments showed CLP binding to the IQ domain to be direct and Ca(2+)-dependent. CLP interacted strongly with IQ motif 3 (K(d) approximately 0.5 nm) as determined by surface plasmon resonance. Epitope-tagged myosin X was localized preferentially at the cell periphery in MCF-7 cells, and CLP colocalized with myosin X in these cells. Myosin X was able to coprecipitate CLP and, to a lesser extent, calmodulin from transfected COS-1 cells, indicating that CLP is a specific light chain of myosin X in vivo. Because unconventional myosins participate in cellular processes ranging from membrane trafficking to signaling and cell motility, myosin X is an attractive CLP target. Altered myosin X regulation in (tumor) cells lacking CLP may have as yet unknown consequences for cell growth and differentiation.     

Target selectivity in EF-hand calcium binding proteins

EF-hand calcium binding proteins have remarkable sequence homology and structural similarity, yet their response to binding of calcium is diverse and they function in a wide range of biological processes. Knowledge of the fine-tuning of EF-hand protein sequences to optimize specific biochemical properties has been significantly advanced over the past 10 years by determination of atomic resolution structures. These data lay the foundation for addressing how functional selectivity is generated from a generic ionic signal. This review presents current ideas about the structural mechanisms that provide the selectivity of different EF-hand proteins for specific cellular targets, using S100 and calmodulin family proteins to demonstrate the critical concepts. Three factors contribute significantly to target selectivity: molecular architecture, response to binding of Ca(2+) ions, and the characteristics of target binding surfaces. Comparisons of calmodulin and S100 proteins provide insights into the role these factors play in facilitating the variety of binding configurations necessary for recognizing a diverse set of targets.     

Characterization of a calcium-modulated protein from transformed chicken fibroblasts.

A calcium-modulated protein has been isolated from secondary cultures of virus transformed chicken embryo fibroblasts and has been characterized in terms of its physical, chemical, and functional properties. These properties demonstrate that this protein is a calmodulin and distinguish it from other calcium-modulated proteins found in various muscle and non-muscle tissues. In addition, this transformed cell calmodulin has been shown to be indistinguishable in both structure and function from normal cell calmodulins isolated from chicken gizzard and brain. A novel change in the electrophoretic behavior of these calmodulin preparations that is dependent on sample history has been observed. These alterations may be the basis for previous reports of tissue specific differences in calmodulin and for some of the differences occasionally observed in peptide maps of calmodulins.     

Selective degradation of oxidized calmodulin by the 20 S proteasome

We have investigated the mechanisms that target oxidized calmodulin for degradation by the proteasome. After methionine oxidation within calmodulin, rates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragments released from the proteasome. Oxidized calmodulin is initially degraded into large proteolytic fragments that are released from the proteasome and subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the selective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. There is a linear correlation between decreases in secondary structure and the rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in alpha-helical content and rates of degradation. In contrast, alterations in the surface hydrophobicity of oxidized calmodulin do not alter the rate of degradation by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensitive sites in oxidized calmodulin that are cleaved by the proteasome in a nonprocessive manner.     

Parasite annexins--new molecules with potential for drug and vaccine development

In the last few years, annexins have been discovered in several nematodes and other parasites, and distinct differences between the parasite annexins and those of the hosts make them potentially attractive targets for anti-parasite therapeutics. Annexins are ubiquitous proteins found in almost all organisms across all kingdoms.Here, we present an overview of novel annexins from parasitic organisms, and summarize their phylogenetic and biochemical properties, with a view to using them as drug or vaccine targets. Building on structural and biological information that has been accumulated for mammalian and plant annexins, we describe a predicted additional secondary structure element found in many parasite annexins that may confer unique functional properties, and present a specific antigenic epitope for use as a vaccine.     

Metal ion binding properties and conformational states of calcium- and integrin-binding protein

Calcium- and integrin-binding protein (CIB) is a novel member of the helix-loop-helix family of regulatory calcium-binding proteins which likely has a specific function in hemostasis through its interaction with platelet integrin alphaIIbbeta(3). The significant amino acid sequence homology between CIB and other regulatory calcium-binding proteins such as calmodulin, calcineurin B, and recoverin suggests that CIB may undergo a calcium-induced conformational change; however, the mechanism of calcium binding and the details of a structural change have not yet been investigated. Consequently, we have performed a variety of spectroscopic and microcalorimetric studies of CIB to determine its calcium binding characteristics, and the subsequent conformational changes that occur. Furthermore, we provide the first evidence for magnesium binding to CIB and determine the structural consequences of this interaction. Our results indicate that in the absence of any bound metal ions, apo-CIB adopts a folded yet highly flexible molten globule-like structure. Both calcium and magnesium binding induce conformational changes which stabilize both the secondary and tertiary structure of CIB, resulting in considerable increases in the thermal stability of the proteins. CIB was found to bind two Ca(2+) ions in a sequential manner with dissociation constants (K(d)) near 0.54 and 1.9 microM for sites EF-4 and EF-3, respectively. In contrast, CIB bound only one Mg(2+) ion to EF-3 with a K(d) near 120 microM. Together, our results suggest that CIB may exist in multiple structural and metal ion-bound states in vivo which may play a role in its regulation of target proteins such as platelet integrin.     

Electric charge balance mechanism of extended soluble proteins

Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function-regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and globular proteins can provide clues useful for identification of extended proteins from complete genomes including orphan sequences. In the present study, we examined the structure and amino acid sequence of extended proteins. We found that extended proteins have a large net electric charge, high charge density, and an even balance of charge between the terminal domains, indicating that electrostatic interaction is a dominant factor in stabilization of extended proteins. Additionally, the central domain exposed to water contained many amphiphilic residues. Extended proteins can be identified from these physical properties of the tertiary structure, which can be deduced from the amino acid sequence. Analysis of physical properties of amino acid sequences can provide clues to the mechanism of protein folding. Also, structural changes in extended proteins may be caused by formation of molecular complexes. Long-range effects of electrostatic interactions also appear to play important roles in structural changes of extended proteins.     

Competitive binding of calmodulin isoforms to calmodulin-binding proteins: implication for the function of calmodulin isoforms in plants.

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32 residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzyme activation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoforms act on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaM isoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although they exhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectively bound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalization experiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distribution patterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressed in most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitive nature of CaM isoforms may allow modulation of Ca(2+)/CaM signaling pathways by virtue of relative abundance and differential target activation potency.     

Characterization of a novel calmodulin from Dictyostelium discoideum

We have purified calmodulin from the eukaryotic microorganism Dictyostelium discoideum (Clarke, M., Bazari, W. L., and Kayman, S. C. (1980) J. Bacteriol. 141, 397-400) and have compared it to calmodulin purified from bovine brain. The two proteins behaved almost identically during fractionation on ion exchange and gel filtration columns and on isoelectric focusing gels. Dictyostelium calmodulin had one-third the specific activity of brain calmodulin in the Ca2+-dependent activation of brain cyclic nucleotide phosphodiesterase; this activation was inhibited for both proteins by 25 microM trifluoperazine. Dictyostelium calmodulin also activated erythrocyte (Ca2+ + Mg2+)-ATPase and interacted with the inhibitory subunit of skeletal muscle troponin. Competition radioimmune assays showed that Dictyostelium calmodulin could compete with brain calmodulin for antibodies to brain calmodulin. These similarities indicate a close relationship between Dictyostelium and brain calmodulin and suggest that the functional capabilities of the protein have been conserved even among evolutionarily distant species. However, substantial differences in primary structure were detected by amino acid analyses and peptide mapping. Most interesting is the lack of trimethyllysine in Dictyostelium calmodulin. This unusual amino acid, which is commonly found in calmodulins, is therefore not essential for interaction between calmodulin and the calmodulin-regulated proteins tested here.     

Structure of the N-terminal calcium sensor domain of centrin reveals the biochemical basis for domain-specific function

Centrin is an essential component of microtubule-organizing centers in organisms ranging from algae and yeast to humans. It is an EF-hand calcium-binding protein with homology to calmodulin but distinct calcium binding properties. In a previously proposed model, the C-terminal domain of centrin serves as a constitutive anchor to target proteins, and the N-terminal domain serves as the sensor of calcium signals. The three-dimensional structure of the N-terminal domain of Chlamydomonas rheinhardtii centrin has been determined in the presence of calcium by solution NMR spectroscopy. The domain is found to occupy an open conformation typical of EF-hand calcium sensors. Comparison of the N- and C-terminal domains of centrin reveals a structural and biochemical basis for the domain specificity of interactions with its cellular targets and the distinct nature of centrin relative to other EF-hand proteins. An NMR titration of the centrin N-terminal domain with a fragment of the known centrin target Sfi1 reveals binding of the peptide to a discrete site on the protein, which supports the proposal that the N-terminal domain serves as a calcium sensor in centrin.     

Functional significance of the central helix in calmodulin

The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and II are separated from domains III and IV by a long central helix consisting of residues 65-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (Kact) were 2- and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins.     

The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations

Oncogenic mutant K-Ras promotes cancer cell proliferation, migration, invasion, and survival by assembling signaling complexes. To date, the functional and structural roles of K-Ras mutations within these complexes are incompletely understood despite their mechanistic and therapeutic significance. Here, we review recent advances in understanding specific binding between K-Ras and the calcium sensor calmodulin. This interaction positively and negatively regulates diverse functions of K-Ras in cancer, suggesting flexibility in K-Ras/calmodulin complex formation. Also, structural data suggest that oncogenic K-Ras likely samples several conformational states, influencing its distinct assemblies with calmodulin and with other proteins. Understanding how K-Ras interacts with calmodulin and with other partners is essential to discovering novel inhibitors of K-Ras in cancer.     

Subunit dissociation,unfolding, and inactivation of bothrojaracin,a C-type lectin-like protein from snake venom

Snake venoms contain a large number of hemostatically active proteins that are structurally related to Ca(2+)-dependent animal lectins. These proteins, called C-type lectin-like proteins (CLPs), are generally found as heterodimers composed of two homologous subunits linked by a disulfide bond. Here, bothrojaracin (BJC), a CLP from Bothrops jararaca venom that is also a thrombin inhibitor, has been used as a model to study the subunit dissociation and unfolding of CLPs from snake venom. Dithiothreitol (DTT) up to 10 mM produces minor effects on the tertiary structure and activity of BJC. On the other hand, chromatographic studies and fluorescence polarization measurements indicate that the interchain disulfide bond is disrupted by DTT, although the dimeric association is maintained. Treatment of BJC with urea produces a progressive red shift in the emission spectra of the tryptophan residues, and circular dichroism measurements show that BJC retains significant secondary structure in the presence of 8 M urea, suggesting only partial unfolding. The effects of urea are fully reversible, as there is complete recovery of BJC activity after removal of the denaturing agent. Addition of DTT to a protein sample previously treated with 8 M urea produces a slightly larger spectral shift than that observed with urea alone. Furthermore, in this condition BJC loses its secondary structure, and its subunits are dissociated. After removal of urea and DTT, BJC is inactive toward thrombin, suggesting the irreversibility of their combined action. Altogether, our data show that (i) BJC is highly resistant to urea or DTT effects, requiring the simultaneous action of both agents to fully denature the protein, and (ii) BJC monomers are tightly associated, and the presence of DTT combined with high urea concentrations is necessary to disrupt them. On the basis of these results we propose the first denaturation model for a CLP from snake venom.     

Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins

In the preceding paper (Lukas, Iverson, Schleicher, Watterson 1984 Plant Physiol 75: 788-795), we reported that the amino acid sequence of spinach calmodulin has at least 13 amino acid sequence differences from vertebrate calmodulin. In the present study, we investigated the effect of these amino acid sequence substitutions on the enzyme activator properties of vertebrate and plant calmodulins. Calmodulins from spinach and the green alga Chlamydomonas reinhardtii activate chicken gizzard myosin light chain kinase in a manner similar but not identical to chicken calmodulin. In contrast, these calmodulins have very different NAD kinase activator properties. The concentration required for half-maximal activation of pea seedling NAD kinase by spinach calmodulin (3-4 nanomolar) is lower than the corresponding concentrations of chicken (20 nanomolar) and Chlamydomonas (40 nanomolar) calmodulins. However, the maximum level of activation obtained with Chlamydomonas calmodulin is 4- to 6-fold higher than spinach or chicken calmodulin. These data indicate that the limited structural heterogeneity among calmodulins have differential effects on their biochemical activities.     

Structural and dynamic characterization of a neuron-specific protein kinase C substrate,neurogranin

Neurogranin/RC3 is a neuron-specific, Ca(2+)-sensitive calmodulin binding protein and a specific protein kinase C substrate. Neurogranin may function to regulate calmodulin levels at specific sites in neurons through phosphorylation at serine residue within its IQ motif, oxidation outside the IQ motif, or changes in local cellular Ca(2+) concentration. To gain insight into the functional role of neurogranin in the regulation of calmodulin-dependent activities, we investigated the structure and dynamics of a full-length rat neurogranin protein with 78 amino acids using triple resonance NMR techniques. In the absence of calmodulin or PKC, neurogranin exists in an unfolded form as evidenced by high backbone mobility and the absence of long-range nuclear Overhauser effect (NOE). Analyses of the chemical shifts (13)C(alpha), (13)C(beta), and (1)H(alpha) reveal the presence of a local alpha-helical structure for the region between residues G25-A42. Three-bond (1)H(N)-(1)H(alpha) coupling constants support the finding that the sequence between residues G25 and A42 populates a non-native helical structure in the unfolded neurogranin. Homonuclear NOE results are consistent with the conclusions drawn from chemical shifts and coupling constants. (15)N relaxation data indicate motional restrictions on a nanosecond time scale in the region from D15 to S48. Spectral densities and order parameters data further confirm that the unfolded neurogranin exists in conformation with residual secondary structures. The medium mobility of the nascent helical region may help to reduce the entropy loss when neurogranin binds to its targets, but the complex between neurogranin and calmodulin is not stable enough for structural determination by NMR. Calmodulin titration of neurogranin indicates that residues D15-G52 of neurogranin undergo significant structural changes upon binding to calmodulin.     

Solution structure of betacellulin,a new member of EGF-family ligands

The solution structure of the EGF-like domain of betacellulin (BTCe), a newly discovered member of the epidermal growth factor (EGF) family, has been determined using two-dimensional nuclear magnetic resonance spectroscopy. This is the first report to identify the solution structure of the EGF-family ligand monomers that interact with both ErbB-1 and ErbB-4. The solution structure of BTCe was calculated using 538 NMR-derived restraints. The overall structure of BTCe was stabilized by three disulfide bonds, a hydrophobic core, and 23 hydrogen bonds. It appears that BTCe is comprised of five beta-strands and one short 3(10) helical turn. The secondary structural elements of BTCe are basically similar to those of the other EGF-family proteins, except that several significant variations of the structural properties were found. It is suggested that the structural variations between BTCe and the other EGF-family ligands may affect the specific receptor-recognition properties of EGF-family ligands.     

Aluminum-induced conformational changes in calmodulin alter the dynamics of interaction with melittin

Studies were undertaken to examine the impact of aluminum-induced structural changes in bovine brain calmodulin on the protein's interface region with melittin, a model for calmodulin's target enzymes. Both steady-state and time-dependent fluorescence characteristics of the single tryptophanyl residue of melittin were employed to derive information on aluminum-related changes in the fluorophore's microenvironment. In the presence of stoichiometric amounts of aluminum ions, calmodulin's target region with melittin appears to be more polar than that with aluminum absent. As a result, upon association of melittin with aluminum-calmodulin, the enhancement of helical arrays is less pronounced. The fluorophore's average microenvironment also is modified such that its apparent lifetime is shortened when aluminum is present. In the presence of aluminum ions, the solvation structure of calmodulin is possibly changed, which may be unfavorable for a proper fit between calmodulin and target proteins.     

Intracellular regulators of neuronal sprouting: calmodulin-binding proteins of nerve growth cones

The focus of this study is a quantitative biochemical analysis of the calcium-dependent interactions of calmodulin with a nerve growth cone preparation from fetal rat brain (Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Freidman, and S. Somlo, 1983, Cell 35:573-584). The presence of calmodulin as an integral component of this preparation is demonstrated, and quantitative binding studies are presented. The binding of 125I-calmodulin to nerve growth cone material is shown to be highly specific, calcium dependent, and saturable at nanomolar calmodulin concentrations. Additionally, the growth cones' binding components appear to be membrane proteins. The individual molecular mass species of growth cone proteins displaying calcium-dependent calmodulin binding are also detailed and presented in comparison with those of synaptosomes. This analysis reveals differences between the calmodulin binding proteins of the growth cone preparation and the synaptosome fraction, suggesting the presence in growth cones of a specialized set of components which may be involved in regulatory mechanisms controlling neuritic sprouting.