Backbone and side-chain dynamics of residues in a partially folded beta-sheet peptide from platelet factor-4.

Structurally characterizing partially folded states is problematic given the nature of these transient species. A peptide 20mer, T38AQLIATLKNGRKISLDLQA57 (P20), which has been shown to partially fold in a relatively stable turn/loop conformation (LKNGR) and transient beta-sheet structure, is a good model for studying backbone and side-chain mobilities in a transiently folded peptide by using 13C-NMR relaxation. Here, four residues in P20, A43, T44, G48, and 151, chosen for their positions in or near the loop conformation and for compositional variety, have been selectively 13C-enriched. Proton-coupled and decoupled 13C-NMR relaxation experiments have been performed to obtain the temperature dependencies (278 K to 343 K) of auto- and cross-correlation motional order parameters and correlation times. In order to differentiate sequence-neighbor effects from folding effects, two shorter peptides derived from P20, IATLK (P5) and NGRKIS (P6), were similarly 13C-enriched and investigated. For A43, T44, G48, and 151 residues in P20 relative to those in P5/P6, several observations are consistent with partial folding in P20: (1) C alpha H motional tendencies are all about the same, vary less with temperature, and are relatively more restricted, (2) G48 C alpha H2 phi (t) psi (t) rotations are more correlated, and (3) methyl group rotations are slower and yield lower activation energies consistent with formation of hydrophobic "pockets." In addition, T44 and 151 C beta H mobilities in P20 are more restricted at lower temperature than those of their C alpha H and display significantly greater sensitivity to temperature suggesting a larger enthalpic contribution to side-chain mobility. Moreover, at higher temperatures, side-chain methyls and methylenes in P20 are more motionally restricted than those in P5/P6, suggesting that some type of "folded" or "collapsed" structure remains in P20 for what normally would be considered an "unfolded" state.     

Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state.

We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild-type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2-state process. Acid denaturation, however, reveals a cooperative transition at approximately pH 1.8 to a partially folded state. This acid state can be further denatured in a reversible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence. Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric. Although compact, the acid state does not resemble the native protein: the acid state displays a near-UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1-anilinonaphthalene, 8-sulfonate much more than either the native or unfolded states. Therefore, the acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core.     

The intracellular region of ClC-3 chloride channel is in a partially folded state and a monomer

In contrast to bacterial ClC chloride channels, all eukaryotic ClC chloride channels have a conserved long intracellular region that makes up of the carboxyl terminus of the protein and is necessary for channel functions as a channel gate. Little is known, however, about the molecular structure of the intracellular region of ClC chloride channels so far. Here, for the first time, we have expressed and purified the intracellular region of the rat ClC-3 chloride channel (C-ClC-3) as a water-soluble protein under physiological conditions, and investigated its structural characteristics and assembly behavior by means of circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), size exclusion chromatography and analytical ultracentrifugation. The far-UV CD spectra of C-ClC-3 in the native state and in the presence of urea clearly show that the protein has a significantly folded secondary structure consisting of alpha-helices and beta-sheets, while the near-UV CD spectra and DSC experiments indicate the protein is deficient in well-defined tertiary packing. Its Stokes radius is larger than its expected size as a folded globular protein, as determined on size exclusion chromatography. Furthermore, the DisEMBL program, a useful computational tool for the prediction of disordered/unstructured regions within a protein sequence, predicts that the protein is in a partially folded state. Based on these results, we conclude that C-ClC-3 is partially folded. On the other hand, both size exclusion chromatography and sedimentation equilibrium analysis show that C-ClC-3 exists as a monomer in solution, not a dimer like the whole ClC-3 molecule.     

A study of beta-thromboglobulin and platelet factor-4 plasma levels in steady state sickle cell patients

To evaluate the platelet function in sickle cell syndromes we measured the beta-thromboglobulin (beta-TG) and platelet factor 4 (PF-4) plasma values of 45 patients suffering from homozygous sickle cell anaemia (10) and sickle cell beta-thalassaemia (35) in steady state. The results were compared to those of 32 normal controls. Both the beta-TG and PF-4 levels were found to be significantly higher in patients than in controls but the beta-TG:PF-4 ratio was significantly lower in the patients group. This finding and the absence of any statistical correlation between platelet number and beta-TG or PF-4 indicate that platelets seem to be somehow activated in sickle cell syndromes, both in homozygotes and sickle cell/beta-thalassaemia heterozygotes. This platelet activation seems to exist even in steady state sickle cell disease patients, regardless of the functional status of the spleen.     

Structural and dynamic characteristics of a partially folded state of ubiquitin revealed by hydrogen exchange mass spectrometry

Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Convoluted exchange patterns are interpreted with the aid of HDX simulations in a three-state system (highly structured, partially unstructured, and fully unstructured states). The data clearly indicate a highly dynamic character of the non-native state. Furthermore, combination of HDX and protein ion fragmentation in the gas phase [by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. The highest degree of structural disorder is displayed by the C-terminal segment (Gly(53)-Gly(76)), which was previously suggested to form a transient alpha-helix. The least dynamic segment of ubiquitin in the A-state is Thr(9)-Glu(18) (which was previously suggested to form a stable nativelike beta-strand), with the adjacent segments exhibiting somewhat diminished conformational stability. The study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium.     

On the possibility for a protein to exist in a manifold of partially folded states

With the help of the methods of tryptophan fluorescence and room-temperature phosphorescence and using Escherichia coli alkaline phosphatase as an example, the ability of a protein to exist in a manifold of partially folded thermodynamically stable states differing in conformation, the internal dynamics, and functional activity was shown. Such intermediate (between native and unfolded) structures may form during unfolding or folding of the protein. It was shown that the degree of destruction of the native structural organization of the globule depends on both the nature and the mode of action of the destroying agent and the structure of the protein. Conformational transitions of the globule can change the kind of the internal dynamics (fast, slow), and shifts of dynamics can initiate conformation changes of the protein and precede them. A scheme of the structural and functional transformations of the protein during denaturation is presented, which takes into account the possibility of globule transitions into a manifold of functional active and inactive partially folded states. The role of partially folded forms of cell proteins in the development of pathology is discussed.     

Comparison between different laboratory tests for the detection and prevention of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a possible complication of heparin therapy that can evolve with life-threatening thromboembolism, for which early diagnosis is essential. However, the specific laboratory approach to the diagnosis of HIT is still controversial. METHODS: Sera from 13 patients with HIT, from 15 patients with non-HIT thrombocytopenia, and from 10 normal subjects were used to compare nonfunctional and functional methods to detect anti-heparin:PF-4 antibodies and platelet activation. We used three enzyme-linked immunosorbent assays (ELISAs) and the particle gel immunoassay as nonfunctional tests, and platelet aggregometry, CD62p (p-selectin) phenotypical expression, and Annexin V binding as functional assays. RESULTS: CD62p expression was positive in 85% of the cases and Annexin V was positive in 40% of the HIT cases examined. Aggregometry gave variable results that depend strongly on the donor. CONCLUSION: Functional tests for platelet activation are more reliable for HIT diagnosis than the nonfunctional tests. We conclude that the phenotypical expression of p-selectin detected by flow cytometry on activated platelets appears to be a good functional marker for the diagnosis of HIT and its possible thromboembolic complications.     

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.     

Solution structure of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA binding

The structure of a CCHHC zinc-binding domain from neural zinc finger factor-1 (NZF-1) has been determined in solution though the use of NMR methods. This domain is a member of a family of domains that have the Cys-X(4)-Cys-X(4)-His-X(7)-His-X(5)-Cys consensus sequence. The structure determination reveals a novel fold based around a zinc(II) ion coordinated to three Cys residues and the second of the two conserved His residues. The other His residue is stacked between the metal-coordinated His residue and a relatively conserved aromatic residue. Analysis of His to Gln sequence variants reveals that both His residues are required for the formation of a well-defined structure, but neither is required for high-affinity metal binding at a tetrahedral site. The structure suggests that a two-domain protein fragment and a double-stranded DNA binding site may interact with a common two-fold axis relating the two domains and the two half-sites of the DNA-inverted repeat.     

Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.     

Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state.

Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F). Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways. We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs). Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs. We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E. Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif. These data provide a quantitative framework for a new molecular model of translational regulation.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.     

Modulation of eucaryotic initiation factor-4E binding to 5'-capped oligoribonucleotides by modified anti-sense oligonucleotides.

The 5' cap structure of eucaryotic mRNA plays a pivotal role in mRNA metabolism. This report demonstrates that anti-sense oligonucleotides equipped with 3'-overhanging nucleotides modulate the amount of recombinant human eucaryotic initiation factor-4E that binds to a 5'-capped oligoribonucleotide. The degree of inhibition or enhancement of protein binding is dependent upon the number and sequence of overhanging nucleotides. A 45% inhibition of complexation was observed by the addition of one 3'-overhanging guanosine residue. Addition of a second residue (+2/GN) resulted in a higher degree of inhibition, 77-88%. In contrast, addition of one adenosine residue enhanced the formation of the eucaryotic initiation factor-4E-m7GpppRNA complex by 213%. Modulation of protein interactions with the 5'-cap structure is likely to effect several biological events, including pre-mRNA processing, transport of the mRNA from the nucleus to the cytoplasm and translation of the target mRNA. This targeting strategy in anti-sense chemistry may have practical applications in experimental biology and medicine.     

Engineering, biophysical characterisation and binding properties of a soluble mutant form of annexin A2 domain IV that adopts a partially folded conformation

The four approximately 75-residue domains (repeats) that constitute the annexin core structure all possess an identical five-alpha-helix bundle topology, but the physico-chemical properties of the isolated domains are different. Domain IV of the annexins has previously been expressed only as inclusion bodies, resistant to solubilisation. Analysis of the conserved, exposed hydrophobic residues of the four annexin domains reveals that domain IV contains the largest number of hydrophobic residues involved in interfacial contacts with the other domains. We designed five constructs of domain IV of annexin A2 in which several interfacial hydrophobic residues were substituted by hydrophilic residues. The mutant domain, in which all fully exposed hydrophobic interfacial residues were substituted, was isolated as a soluble protein. Circular dichroism measurements indicate that it harbours a high content of alpha-helical secondary structure and some tertiary structure. The CD-monitored (lambda=222 nm) thermal melting profile suggests a weak cooperative transition. Nuclear magnetic resonance (1H-15N) correlation spectroscopy reveals heterogeneous line broadening and an intermediate spectral dispersion. These properties are indicative of a partially folded protein in which some residues are in a fairly structured conformation, whereas others are in an unfolded state. This conclusion is corroborated by 1-anilinonaphthalene-8-sulfonate fluorescence (ANS) analyses. Surface plasmon resonance measurements also indicate that this domain binds heparin, a known ligand of domain IV in the full-length annexin A2, although with lower affinity.     

Differences between the prion protein and its homolog Doppel: a partially structured state with implications for scrapie formation

The key event in the pathogenesis of prion diseases is a conformational change in the prion protein (PrP). Models for conversion of PrP(C) into PrP(Sc) typically implicate an, as yet, unidentified intermediate. In an attempt to identify such an intermediate, we used native-state hydrogen exchange monitored with NMR. Although we were unable to detect an intermediate directly, we observed substantial protection above that expected based upon measurements of the global stability of PrP (>2 kcal mol(-1) super protection). This super protection implicates either structure in the denatured state or the presence of an intermediate. Similar experiments with Doppel, a homolog of PrP that does not form infectious prions, failed to demonstrate such super protection. This suggests that the partially structured state of PrP encompassing portions of the B and C helices, may be a significant factor in the ability of PrP to convert from PrP(C) to PrP(Sc).     

Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells.

Heparin is required for the binding of basic fibroblast growth factor (bFGF) to high-affinity receptors on cells deficient in cell surface heparan sulfate proteoglycan. So that this heparin requirement could be evaluated in the absence of other cell surface molecules, we designed a simple assay based on a genetically engineered soluble form of murine FGF receptor 1 (mFR1) tagged with placental alkaline phosphatase. Using this assay, we showed that FGF-receptor binding has an absolute requirement for heparin. By using a cytokine-dependent lymphoid cell line engineered to express mFR1, we also showed that FGF-induced mitogenic activity is heparin dependent. Furthermore, we tested a series of small heparin oligosaccharides of defined lengths for their abilities to support bFGF-receptor binding and biologic activity. We found that a heparin oligosaccharide with as few as eight sugar residues is sufficient to support these activities. We also demonstrated that heparin facilitates FGF dimerization, a property that may be important for receptor activation.     

In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk.

Gram-negative bacteria release LPS, which activates Toll-like-receptor-4 (TLR4) in the host, initiating an inflammatory response to infection. Infection increases risk for thrombosis. Platelets contribute to defense from infection and to thrombosis. Experiments were designed to determine whether LPS, through TLR4 signaling, affects platelet phenotype. Platelet responses in wild-type (WT) mice and mice that lack the TLR4 gene (dTLR4) were compared following a single nonlethal injection of LPS (0.2 mg/kg iv). Compared with WT mice, mice without TLR4 had fewer circulating platelets with lower RNA content and were less responsive to thrombin-activated expression of P-selectin but were equally sensitive to aggregation or ATP secretion. One week following the LPS injection, the time it takes for the circulating platelet pool to turnover, the number of circulating platelets, thrombin-induced expression of P-selectin, and collagen-activated aggregation were increased comparably in both groups of mice. Therefore, the change of the platelet pool to an activated phenotype 1 wk after a single exposure to LPS appears to arise from a process that is independent of TLR4. The persistence of the effect 1 wk after the injection suggests that the changes reflect an action of LPS on megakaryocytes and their platelet progeny rather than on circulating platelets, which would have been cleared.     

Mechanisms of lipopolysaccharide-initiated rabbit platelet responses. II. Evidence that lipid A is responsible for binding of lipopolysaccharide to the platelet.

The mechanism of bacterial lipopolysaccharide-(LPS) initiated, complement-(C) mediated rabbit platelet lysis has been examined. The results of these studies support our previous observations that activation of the alternative C pathway is required for platelet lysis and that preparations of LPS that activate only the classical pathway (e.g., lipid A) do not cause lysis. The temporal relationship of the interaction of the LPS with the platelet before the addition of plasma suggests a time-dependent association of the LPS with the platelet. On the basis of a number of experiments, including inhibition with polymyxin B, treatment of LPS with alkali, and blocking experiments with polysaccharide-free LPS preparations, it is concluded that the lipid A region of the LPS molecule is responsible for attaching the LPS to the platelet. Finally, a comparison of the activity of lipid A-associated protein-LPS complexes with protein-free LPS demonstrated that an equivalent extent of platelet lysis was achieved with one-one hundredth the concentration of the former as that required for protein-free LPS. The data suggest that LAP facilitates attachment of the LPS to the platelet.