Insight from studies with recombinant fibrinogens.

Which knobs fit into which holes in fibrin polymerization?

The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site.

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.

Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.

Calcium binding to untreated and dephosphorylated porcine neurofilaments

Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca 2+ affinity of regulatory Ca 2+ sites in single channel recordings and [ 3H]ryanodine binding

The calcium-mediated interaction of factor VIIa with tissue factor is considered to be the primary trigger of blood coagulation. To determine the role of calcium ions in the action of factor VII, we prepared monoclonal antibodies whose binding to factor VII was calcium-dependent. A monoclonal antibody designated C6 strongly inhibited factor VII-induced clotting at a molar ratio of factor VII to antibody of 1:1. The half-maximal binding of factor VII to the C6 antibody was observed at a concentration of calcium ions of 80 microM. Proteolytic fragments of factor VII were assayed for their ability to inhibit competitively the binding of 125I-factor VII to immobilized C6 antibody. The binding was inhibited by increasing amounts of factor VII, by a fragment that contained the gamma-carboxyglutamic acid (Gla) domain linked to first epidermal growth factor-like domain, and by a Gla domain peptide (residues 1-41), over a range of concentration of 10(-9) to 10(-7) M. The antigenic site recognized by the monoclonal antibody C6, which was generated upon the high-affinity binding of calcium ions, was located in the Gla domain. The C6 antibody inhibited the activation of factor X and the amidolytic activity of factor VIIa in the presence of tissue factor. These results demonstrate that a high-affinity calcium-binding site(s) is located in the Gla domain of factor VII, which is concerned with the initiation of tissue factor-mediated blood coagulation by factor VIIa.

BackgroundProtein ligand-binding sites in the apo state exhibit structural flexibility. This flexibility often frustrates methods for structure-based recognition of these sites because it leads to the absence of electron density for these critical regions, particularly when they are in surface loops. Methods for recognizing functional sites in these missing loops would be useful for recovering additional functional information.ResultsWe report a hybrid approach for recognizing calcium-binding sites in disordered regions. Our approach combines loop modeling with a machine learning method (FEATURE) for structure-based site recognition. For validation, we compared the performance of our method on known calcium-binding sites for which there are both holo and apo structures. When loops in the apo structures are rebuilt using modeling methods, FEATURE identifies 14 out of 20 crystallographically proven calcium-binding sites. It only recognizes 7 out of 20 calcium-binding sites in the initial apo crystal structures.We applied our method to unstructured loops in proteins from SCOP families known to bind calcium in order to discover potential cryptic calcium binding sites. We built 2745 missing loops and evaluated them for potential calcium binding. We made 102 predictions of calcium-binding sites. Ten predictions are consistent with independent experimental verifications. We found indirect experimental evidence for 14 other predictions. The remaining 78 predictions are novel predictions, some with intriguing potential biological significance. In particular, we see an enrichment of beta-sheet folds with predicted calcium binding sites in the connecting loops on the surface that may be important for calcium-mediated function switches.ConclusionProtein crystal structures are a potentially rich source of functional information. When loops are missing in these structures, we may be losing important information about binding sites and active sites. We have shown that limited loop modeling (e.g. loops less than 17 residues) combined with pattern matching algorithms can recover functions and propose putative conformations associated with these functions.

The Ca2+-transporting ATPase has been labeled with N-cyclohexyl-N'-(4-dimethyl-amino-alpha-naphthyl)carbodiimide (NCD-4), a fluorescent carbodiimide which reacts with carboxyl groups of acidic residues. It has been reported that NCD-4 labels a transmembrane portion of the protein at the high-affinity calcium-binding sites. We have determined the depth of the calcium-sensitive probe by quenching the fluorescence by nitroxide-substituted fatty acids with its spin probe located at different carbons of the fatty acid chain (5, 7, 10, 12 and 16-nitroxide derivatives). We have found that all the calcium-sensitive fluorescence is quenched and that the efficiency of quenching decreases as the n-(4,4-dimethyl-3-oxazolinyloxy) (Doxyl) group is deeper in the membrane. We conclude that the NCD-4 label which is involved in the high-affinity calcium-binding site is located near the water/lipid interface. The fluorescence of the NCD-4 bound to that site can be quenched by acrylamide and Cu2+ but not by iodide, probably due to its anionic nature which will be repulsed by the abundance of negative charges of Glu and Asp residues of NCD-4 located at this site. The hydrophobic location of NCD-4 was confirmed by the fact that its fluorescence could be quenched by the spin label 2,2,6,6-tetramethyl-1-piperidine-N-oxyl but not by 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine-N-oxyl which is much less hydrophobic.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

Fibrinogen functions and fibrin assembly

The Influence of Troponin C Isoforms on Drosophils Stretch Activation and Power Generation

Calcium-dependent heart muscle contraction is regulated by the cardiac troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). cNTnC contains one calcium binding site (site II), and altered calcium binding in this site has been studied for decades. It has been previously shown that cNTnC mutants, which increase calcium sensitization may have therapeutic benefits, such as restoring cardiac muscle contractility and functionality post-myocardial infarction events. Here, we computationally characterized eight mutations for their potential effects on calcium binding affinity in site II of cNTnC. We utilized two distinct methods to estimate calcium binding: adaptive steered molecular dynamics (ASMD) and thermodynamic integration (TI). We observed a sensitizing trend for all mutations based on the employed ASMD methodology. The TI results showed excellent agreement with experimentally known calcium binding affinities in wild-type cNTnC. Based on the TI results, five mutants were predicted to increase calcium sensitivity in site II. This study presents an interesting comparison of the two computational methods, which have both been shown to be valuable tools in characterizing the impacts of calcium sensitivity in mutant cNTnC systems.

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.

Distinct interactions of Na+ and Ca2+ ions with the selectivity filter of the bacterial sodium channel NaVAb