EF-hand Calcium-binding Motifs Research Articles

Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins.

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3' end of the MACF cDNA; hence, we refer to it as MACF2. The C-terminal domain of mouse MACF2 was cloned and characterized. The microtubule-binding properties of MACF2 C-terminal domain are similar to that of MACF. The GAR domain was originally found in Gas 2 protein and here we show that it can associate with MTs in transfected cells. Plectin and desmoplakin have GSR-containing domains at their C-termini and we further demonstrate that the GSR-containing domain of plectin, but not desmoplakin, can bind to MTs in vivo.

Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum

A third calcium-dependent protein kinase (CDPK) gene has been isolated from the human malaria parasite Plasmodium falciparum by vectorette technology. The gene consists of five exons and four introns. The open reading frame resulting from removal of the four introns encodes a protein of 562 amino acid residues with a predicted molecular mass of 65.3 kDa. The encoded protein, termed PfCDPK3, consists of four distinct domains characteristic of a member of the CDPK family and displays the highest homology (46% identity and 69% similarity) to PfCDPK2, the second CDPK of P. falciparum. The N-terminal variable domain is rich in serine/threonine and lysine and contains multiple consensus phosphorylation sites for a range of protein kinases. The catalytic domain possesses all conserved motifs of the protein kinase family except for the highly conserved glutamic acid residue in subdomain VIII, which is replaced by a glutamine residue. The sequence of the junction domain comprising 31 amino acid residues is less conserved. The calmodulin-like regulatory domain contains four EF-hand calcium-binding motifs, each consisting of a loop of 12 amino acid residues which is flanked by two α-helices. Southern blotting of genomic DNA digests showed that the Pfcdpk3 gene is present as a single copy per haploid genome. A 2900 nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfCDPK3 is involved in sexual stage-specific events. It is proposed that PfCDPK3 may serve as a link between calcium and gametogenesis of P. falciparum.

Structural basis of the Ca 2+-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I

Background: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. Results: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 Å resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11–annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100—annexin interaction. Conclusions: By solving the structure of a second annexin N terminus–S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca 2+-regulated annexin I–S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends-PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH(2) terminus. However, unlike dystonin, mACF7 does not contain a coiled-coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest-specific protein, Gas2. In this paper, we demonstrate that the NH(2)-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

NADPH Oxidase Genes from Tomato (Lycopersicon esculentum) and Curly‐Leaf Pondweed (Potamogeton crispus)

Abstract: The oxidative burst is an integral component of plant resistance to pathogens. There is accumulating evidence that the oxidative burst is catalyzed by an enzyme with similarities to the phagocyte NADPH oxidase. We have cloned a full length homolog of the gp91 (phox) subunit of the plasma membrane NADPH oxidase complex from tomato named LeRBOM. The predicted protein contains 989 amino acids. The large N‐terminal domain contains two EF hand calcium binding motifs and one conserved glycosylation site. Six putative membrane spans are present in the C‐terminal half of the predicted protein. Extensive homology with the human gp91 (phox) subunit was found including conservation of amino acid residues important for heme coordination and substrate binding. We have also isolated partial genomic clones from tomato and from the aquatic plant Potamogeton crispus. These species serve as models for studies of signal transduction leading to NADPH oxidase activation. In tomato, LeRBOH1 expression was too low to be detected on Northern blots. RT‐PCR indicated that LeRBOH1 was expressed in all tissues tested.

Characterization and immunolocalization of a cytosolic calcium-binding protein from Brassica napus and Arabidopsis pollen.

Two low-molecular-weight proteins have been purified from Brassica napus pollen and a gene corresponding to one of them has been isolated. The gene encodes an 8.6-kD protein with two EF-hand calcium-binding motifs and is a member of a small gene family in B. napus. The protein is part of a family of pollen allergens recently identified in several evolutionarily distant dicot and monocot plants. Homologs have been detected in Arabidopsis, from which one gene has been cloned in this study, and in snapdragon (Antirrhinum majus), but not in tobacco (Nicotiana tabacum). Expression of the gene in B. napus was limited to male tissues and occurred during the pollen-maturation phase of anther development. Both the B. napus and Arabidopsis proteins interact with calcium, and the potential for a calcium-dependent conformational change was demonstrated. Given this affinity for calcium, the cloned genes were termed BPC1 and APC1 (B. napus and Arabidopsis pollen calcium-binding protein 1, respectively). Immunolocalization studies demonstrated that BPC1 is found in the cytosol of mature pollen. However, upon pollen hydration and germination, there is some apparent leakage of the protein to the pollen wall. BPC1 is also concentrated on or near the surface of the elongating pollen tube. The essential nature of calcium in pollen physiology, combined with the properties of BPC1 and its high evolutionary conservation suggests that this protein plays an important role in pollination by functioning as a calcium-sensitive signal molecule.

The Calcium-binding Sites of Heparinase I fromFlavobacterium heparinum Are Essential for Enzymatic Activity

In the accompanying paper (Shriver, Z., Liu, D., Hu, Y., and Sasisekharan, R. (1999) J. Biol. Chem. 274, 4082-4088), we have shown that calcium binds specifically to heparinase I and have identified two major calcium-binding sites (CB-1 and CB-2) that partly conform to the EF-hand calcium-binding motif. In this study, through systematic site-directed mutagenesis, we have confirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity. More specifically, we identified critical residues (viz. Asp210, Asp212, Gly213, and Thr216 in CB-1 and Asn375, Tyr379, and Glu381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Mutations in CB-1 resulted in a lower kcat, but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the kcat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides. Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I. Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I. These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism. Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.

Involvement of both dockerin subdomains in assembly of the Clostridium thermocellum cellulosome.

Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.

Characterization of Sm20.8, a member of a family of schistosome tegumental antigens

Two cDNA clones each encoding a 20.8-kDa protein (Sm20.8) were identified from the human blood fluke Schistosoma mansoni sporocyst and adult worm cDNA expression libraries by antibodies derived from rabbits vaccinated with irradiated cercariae and purified over an NP-40 extract of 3h schistosomula. Each identified cDNA has an open reading frame encoding a protein of 181 amino acids and shows homology (29–30%) with Sm21.7, Sm22.6, and Sj22.6, previously identified as belonging to a family of soluble schistosome tegumental antigens. An EF-hand calcium-binding motif is found in Sm20.8 protein in two different positions. However, neither motif binds 45calcium ( 45Ca) Recombinant Sm20.8 showed immunoreactivity with sera from infected humans and rabbits vaccinated with irradiated cercariae. Polyclonal rabbit sera against the Sm20.8 recognized the native protein in an extract of infected snail (sporocyst), cercariae, 3 hour schistosomules (3 h NP-40) and an adult worm preparation but not in uninfected snail tissue or eggs. Further demonstration that Sm20.8 was expressed in the different developmental stages of the parasite was by RT-PCR. Confocal microscopy demonstrates that Sm20.8 localizes to the tegument of adult worms and 3 h np-40. The IgG fraction specific to Sm20.8 mediated complement killing of schistosomules in vitro by 34%. Vaccination of mice with naked DNA containing the Sm20.8 gene and subsequently challenged with cercariae showed 30% reduction in worm burden compared to controls.

Upregulation of a New Microglial Gene,mrf-1, in Response to Programmed Neuronal Cell Death and Degeneration

Cerebellar granule neurons isolated from postnatal day 7 (P7) rats and grown in normal K+ medium begin to degenerate at approximately 4 d in vitro (DIV) and die. To search for genes upregulated in the process of neuronal cell death, differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5 DIV. One of the genes isolated was microglial response factor-1 (mrf-1), which encoded a sequence of 177 amino acids with a single EF-hand calcium-binding motif. By Northern blots, the transcript was upregulated in cerebellar culture at 4 DIV, peaked at 6 DIV, and decreased at 7 DIV. Upregulation was also found when the apoptosis of granule cells was induced by replacing high K+ medium with normal K+ medium. However, when non-neuronal cells were thoroughly eliminated with aphidicolin, an antimitotic agent, the upregulation at 4-7 DIV did not occur. By immunocytochemistry, MRF-1 was detected at 5 DIV in OX-42-positive cells (microglia), and it exhibited an increase in response to granule cell death. MRF-1 levels in microglia purified from cerebral cortex also upregulated in the presence of 5 DIV granule cells. In the developing cerebellum in vivo, levels of mrf-1 mRNA transiently increased in the early postnatal stages, reaching a peak at P7 when cerebellar neurons and astrocytes undergo extensive apoptosis. In adult brain sections, MRF-1 was detected in the perikarya and processes of ramified/resting microglia, and peripheral motor nerve dissection prominently increased the expression in activated microglia surrounding injured central motoneurons. Therefore, mrf-1 appears to be one of the microglial genes that respond to neuronal cell death and degeneration.

Isolation of two Human cDNAs, HLP3 and HLP4, Homologous to the Neuron-Specific Calcium-Binding Protein Genes

We have cloned and sequenced two types of human cDNA, HLP3 and HLP4, encoding a protein of 191 amino acid residues with four EF-hand calcium-binding motifs. The HLP3 and HLP4 proteins are homologous to the neuron-specific calcium-binding proteins and are likely human counterparts of the neural visinin-like protein(NVP) 1 and NVP2 identified in the rat brain (Kajimoto et al. 1993), displaying 98% and 99% amino acid identities with these sequences, respectively. The human HLP3 and 4 mRNAs are detected only in the brain and found at high amount in the cerebral cortex and cerebellum by Northern blot analysis.

CDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases

Using degenerate oligonucleotide primers corresponding to conserved regions of the calcium-dependent protein kinase (CDPK) family, we carried out a polymerase chain reaction and obtained four distinct partial-length cDNAs from a maize leaf library. We then used these clones as probes for conventional screening and isolated 19 longer clones from another cDNA library of maize seedlings. These clones were classified into four groups based on their DNA cross-hybridization. Two full-length cDNAs, designated as ZmCDPK9 and ZmCDPK7, were sequenced and characterized. The predicted protein of each clone was a typical CDPK with eleven canonical subdomains of protein kinases, and four EF-hand calcium-binding motifs in its N-terminal and C-terminal halves, respectively. The catalytic and regulatory domains were linked by a well-conserved junction domain. The N-terminus of the protein also contained a consensus sequence for an N-myristoylation signal. Northern blot analysis showed that the transcription level of each gene was higher in roots and etiolated leaves than in green leaves. To confirm the calcium dependency of the maize enzymes, the entire coding region of ZmCDPK9 was subcloned into an expression vector so that it was in frame with the vector-encoded peptide tags. A cell-free extract of Escherichia coli transformed with the recombinant plasmid exhibited calcium-dependent phosphorylation activity, using casein as a substrate.

Sequence of a human cDNA encoding Cab45, a Ca2+-binding protein with six EF-hand motifs.

We report the sequence of a human cDNA encoding a deduced 362 amino acid protein with six EF-hand calcium-binding motifs. The protein is a likely human counterpart of the Cab45 protein recently identified in the 3T3-L1 mouse adipocyte cell line [Scherer et al. (1996), J. Cell Biol. 133, 257-268], displaying 87% aa and 83% nt identity with this sequence. The mRNA for human Cab45 is detected ubiquitously in tissue Northern blots.

Kinetic tuning of the EF-hand calcium binding motif: the gateway residue independently adjusts (i) barrier height and (ii) equilibrium.

In EF-hand calcium binding sites of known structure, the side chain provided by the ninth EF-loop position lies at the entrance of the shortest pathway connecting the metal binding cavity to solvent. The location of this residue suggests that it could serve as a "gateway", providing steric and electrostatic control over the kinetics of Ca2+ binding and dissociation. To test this hypothesis, the present study has engineered the putative gateway side chain of a model EF-hand site and determined the resulting effects on metal ion affinity and dissociation kinetics. The model site chosen was that of the Escherichia coli galactose binding protein (GBP), in which the putative gateway is a Gln side chain. Nine engineered GBP molecules were generated and isolated, each exhibiting native-like activity and global conformation. Two control substitutions at the fourth EF-loop position, a noncoordinating surface residue, had no significant effect on either the equilibrium or the kinetics of the model site. The remaining seven proteins, which possessed unique substitutions at the ninth EF-loop position (Glu, Asn, Asp, Thr, Ser, Gly, Ala), in each case significantly altered the affinity or dissociation kinetics of the site for Tb3+, used as a probe metal ion. Neutral side chains at the gateway position yielded a 590-fold range of Tb3+ dissociation kinetics but only a 3-fold range of Tb3+ affinities, indicating that the size or polarity of these substitutions alters the transition state barrier for metal binding and release without substantially shifting the equilibrium. In contrast, acidic side chains yielded as much as a 34-fold decrease in the Tb3+ off-rate and a 33-fold increase in Tb3+ affinity, suggesting that a negative charge at the gateway position increases the equilibrium stability of the bound metal ion and thereby slows metal release. Thus, kinetic tuning by the gateway side chain exhibits both transition state and ground state tuning mechanisms. In natural EF-hand sequences, different gateway side chains are used by slow buffering sites and rapid signaling sites, providing evidence that the gateway position is an important physiological determinant of metal binding kinetics.

A carrot cDNA encoding an atypical protein kinase homologous to plant calcium-dependent protein kinases.

Calcium-dependent protein kinases (CDPKs) in plants typically contain a C-terminal calmodulin-like domain with four EF-hand calcium-binding motifs. We have isolated a carrot somatic embryo cDNA clone which encodes a new, divergent isoform of this family, designated CRK (CDPK-related kinase). The catalytic domain of CRK shares a high degree of homology with the catalytic domains of plant CDPKs (53.5% average identity with its two closest phylogenetic relatives, CDPK431 (carrot) and AK1 (Arabidopsis). However, the C-terminal domain of CRK bears significantly less homology to calmodulin (22.0% identity to barley calmodulin) than other plant CDPKs (38.0% average identity between barley calmodulin and the C-terminal domains of CDPK431 and AK1). This degeneracy also involves the EF-hand motifs of CRK, which have diverged to varying extents. The predicted structure of CRK also contains an extended N-terminal domain 145 amino acids in length possessing a consensus N-myristoylation signal. CRK transcripts are most abundant in somatic embryos, with lesser accumulations in flowers and leaves and lowest levels in roots. Homologous genomic DNA sequences that hybridize with CRK cDNA but not with a carrot CDPK probe have been detected in a variety of higher plant taxa, including monocotyledonous species, suggesting that this CDPK-related kinase is widely conserved among angiosperms.

The C-terminal domain of alpha-spectrin is structurally related to calmodulin.

An alignment of amino acid sequences suggests that the spectrin domain, which contains two EF-hand calcium-binding motifs, is structurally related to calmodulin. It is possible to align approximately 160 residues at the C-terminus of alpha-spectrin with the entire calmodulin sequence. We have expressed this domain in Escherichia coli and purified it. Circular dichroic and nuclear magnetic resonance spectroscopy show that the protein is folded and mostly helical. The conformation of the protein, as monitored spectroscopically, is sensitive to calcium at 0.1-1.0 mM. Equilibrium dialysis shows that there are two binding sites within this domain, with affinities in the 0.5 mM range. The domain can be split into N-terminal and C-terminal halves which fold independently. Only the N-terminal subdomain binds calcium. These data suggest that the C-terminus of alpha-spectrin has a domain with a calmodulin fold and two calcium-binding sites. Sequence alignments suggest that the related domains in alpha-actinin, and possibly in dystrophin, may share the same calmodulin-like structure. However, only non-muscle alpha-actinins appear to have one or two EF-hand(s) with the calcium-binding consensus sequence, and a strict consensus is not found in the muscle alpha-actinins or dystrophins.

The gene family of EF-hand calcium-binding proteins from the flagellum of Trypanosoma brucei

The flagellum of Trypanosoma brucei contains calmodulin, and a separate family of antigenically related EF-hand calcium-binding proteins which we call calflagins. The following study evaluates the structure and genomic organization of the calflagin family. Genomic Southern blots indicated that multiple copies of calflagin genes occurred in T. brucei, and that all of these copies were contained in a single 23 kb XhoI-XhoI fragment on chromosomes 15 and 16 mRNAs of 1.2 and 1.6 kb were identified in bloodstream and procyclic life-cycle stages. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. The calflagin genes were arranged tandemly along the genomic fragments. Three new members of the calflagin family were sequenced from a cDNA clone and the two genomic clones. Two unrelated families of 3' flanking sequences were downstream from the calflagin genes. An open reading frame that was unrelated to any calflagin sequence was at the 5' end of the 2.5 kb genomic fragment. The deduced amino acid sequences of the genomic clones (called Tb-24 and Tb-1.7g) were similar to the previously described Tb-17. Each encoded an approximately 24 kDa protein which contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. The cDNA encoded a protein (called Tb-44A) which was approximately twice as large as the other calflagins. The large size resulted from a nearly direct repeat of 186 amino acids. In general, variability among the T. brucei calflagins was greater than observed for related proteins from Trypanosoma cruzi. We demonstrate that this variability resulted from amino acid substitutions at the N-terminus, C-terminal extensions, and duplication of internal segments.

Primary structure and binding properties of calgranulin C, a novel S100-like calcium-binding protein from pig granulocytes.

In this paper we report the biochemical characterization of calgranulin C, a new member of the S100 protein family. The protein is highly abundant in the cytosol of pig granulocytes, with relatively small amounts in lymphocytes. A simple protocol for the rapid purification of calgranulin C is described. The purified protein migrates as a single entity on SDS-polyacrylamide gel electrophoresis while it has two isoforms focusing at pH 5.8 and 5.5. Gel filtration and cross-linking experiments indicate that calgranulin C is capable of dimerization. The complete amino acid sequence was determined by Edman degradation of peptides generated by trypsin and V8 protease digestion. Calgranulin C consists of 91 residues and has a calculated molecular mass of 10,614 daltons. This value is virtually identical to that obtained by electrospray mass spectrometry. Sequence analysis predicts two EF-hand calcium-binding motifs, the first having an extended loop that is distinctive of the S100 protein family. The metal-binding properties were studied by means of a direct 45Ca(2+)-binding assay and by tyrosine fluorescence titration. Calgranulin C binds not only calcium but also zinc ions. A single high affinity Zn(2+)-binding site per monomer was evidenced by fluorimetric titration. Zinc binding to calgranulin C induces a remarkable increase in the protein affinity for calcium; in the absence of zinc, the protein binds 1 Ca2+/monomer with a binding constant of about 2 x 10(4) M-1, whereas the Zn(2+)-loaded form binds 2 Ca2+/monomer with Ka values of approximately 3 x 10(7) and 6 x 10(4) M-1. Circular dichroism analysis showed that the binding of calcium to calgranulin C induces a 15% decrease in the apparent alpha-helix content. This result and the calcium-dependent binding of the protein to a phenyl-Superose column strongly suggest that calgranulin C undergoes a gross conformational change upon calcium binding, thus supporting the idea that this protein may be involved in Ca(2+)-dependent signal transduction events.

Identification of the vinculin-binding site in the cytoskeletal protein alpha-actinin.

Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both 'lollipop'- and 'dumbell'-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.

Hidden Markov Models in Computational Biology: Applications to Protein Modeling

Hidden Markov Models (HMMs) are applied to the problems of statistical modeling, database searching and multiple sequence alignment of protein families and protein domains. These methods are demonstrated on the globin family, the protein kinase catalytic domain, and the EF-hand calcium binding motif. In each case the parameters of an HMM are estimated from a training set of unaligned sequences. After the HMM is built, it is used to obtain a multiple alignment of all the training sequences. It is also used to search the SWISS-PROT 22 database for other sequences that are members of the given protein family, or contain the given domain. The HMM produces multiple alignments of good quality that agree closely with the alignments produced by programs that incorporate three-dimensional structural information. When employed in discrimination tests (by examining how closely the sequences in a database fit the globin, kinase and EF-hand HMMs), the HMM is able to distinguish members of these families from non-members with a high degree of accuracy. Both the HMM and PROFILESEARCH (a technique used to search for relationships between a protein sequence and multiply aligned sequences) perform better in these tests than PROSITE (a dictionary of sites and patterns in proteins). The HMM appears to have a slight advantage over PROFILESEARCH in terms of lower rates of false negatives and false positives, even though the HMM is trained using only unaligned sequences, whereas PROFILESEARCH requires aligned training sequences. Our results suggest the presence of an EF-hand calcium binding motif in a highly conserved and evolutionary preserved putative intracellular region of 155 residues in the α-1 subunit of L-type calcium channels which play an important role in excitation-contraction coupling. This region has been suggested to contain the functional domains that are typical or essential for all L-type calcium channels regardless of whether they couple to ryanodine receptors, conduct ions or both.