Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites

We have used purified proteolytic fragments of von Willebrand factor (vWF) to characterize three related functional sites of the molecule that support interaction with platelet glycoprotein Ib, collagen, and heparin. A fragment of 116 kDa was found to be dimeric and consisted of disulfide-linked subunits which, after reduction and alkylation, corresponded to the previously described 52/48-kDa fragment extending from residue 449 to 728. Fragment III-T2, also a dimer, was composed of two pairs of disulfide-linked subunits, two 35-kDa heavy chains (residues 273-511) and two 10-kDa light chains (residues 674-728). The 116-kDa fragment, but not the constituent 52/48-kDa subunit, supported ristocetin-induced platelet aggregation and retained 20% (on a molar basis) of the ristocetin cofactor activity of native vWF; fragment III-T2 retained less than 5% activity. All three fragments, however, inhibited vWF interaction with glycoprotein Ib. Both 116-kDa and 52/48-kDa fragments inhibited vWF binding to heparin with similar potency, while fragment III-T2 had no effect in this regard. Only the 116-kDa fragment inhibited vWF binding to collagen. These results indicate that dimeric fragments containing two glycoprotein Ib-binding sites possess the minimal valency sufficient to support ristocetin-induced aggregation. The sequence comprising residues 512-673, missing in fragment III-T2, is necessary for binding to heparin and collagen and may be crucial for anchoring vWF to the subendothelium. Immunochemical and functional data suggest that the same sequence, although not essential for interaction with glycoprotein Ib, may influence the activity of the glycoprotein Ib-binding site. Only binding to collagen has absolute requirement for intact disulfide bonds. Thus, the three functional sites contained in the 116-kDa domain of vWF are structurally distinct.     

Backbone dynamics of the first, second, and third immunoglobulin modules of the neural cell adhesion molecule (NCAM)

The neural cell adhesion molecule (NCAM) is a cell surface multimodular protein, which plays an important role in cell-cell adhesion by homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. In the present study, the backbone dynamics of the first three immunoglobulin-like (Ig) modules of NCAM have been investigated by NMR spectroscopy. Ig1, Ig2, and Ig3 share low sequence identity but possess the same fold and have very similar three-dimensional structures. (15)N longitudinal and transverse relaxation rates and heteronuclear NOEs have been measured and subsequently analyzed by the axial symmetric Lipari-Szabo modelfree formalism to characterize fast (pico- to nanosecond) and slow (micro- to millisecond) motions in the three protein modules. We found that backbone motions of residues located in the beta-strand regions are generally restricted, while increased flexibility is observed in turns and loops. In all three modules, residues located in the segments connecting the C- and D-strand plus residues located in the segment connecting the E- and F-strand show significant chemical exchange on the micro- to millisecond time scale. In addition, a number of residues with small chemical exchange contribution seem to form contiguous regions in the beta sheets, suggesting that these motions might be correlated. Only few residues in the homophilic binding sites in the NCAM Ig1 and Ig2 modules show increased flexibility, indicating that the Ig1-Ig2-mediated NCAM homophilic binding does not depend on the local backbone mobility of the interacting modules.     

Tryptic digestion of bovine secretory IgA at elevated temperature and in urea. Isolation of SC domain 1 which is covalently bound to IgA dimer and binds non-covalently to IgM

1. Tryptic cleavage sites in bovine secretory component (SC) which become inaccessible when SC is bound to IgA dimer remained inaccessible at 60 degrees C and in 4 M urea at 37 degrees C. 2. This suggests the presence of strong interactions compatible with published affinity constants of ca 10(8) M-1. 3. In 5 M urea at 37 degrees C further cleavage of bound SC did occur to produce a fragment consisting of domain 1 which was disulphide bridged to the IgA dimer. 4. Binding studies on the isolated fragment showed that domain 1 did not account for all the binding by SC. 5. Cleavage of the isolated fragment with iodosobenzoic produced a smaller fragment consisting of the n-terminal third of domain 1 (residues 1-35). This N-terminal fragment showed significant binding.     

Identification of the actin and plasminogen binding regions of group B streptococcal phosphoglycerate kinase

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in sign ificantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.     

Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the first committed step in the biosynthesis of polyamines. ODC is a proven drug target for the treatment of African sleeping sickness. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface. Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association. Twenty-three mutant enzymes were analyzed by analytical ultracentrifugation, and none of the mutations were found to cause a greater than 1 kcal/mol decrease in dimer stability. These data suggest that the energetics of the interaction may be distributed across the interface. Most significantly, many of the mutations had large effects (DeltaDeltaG kcat/Km > 2.5 kcal/mol) on the catalytic efficiency of the enzyme. Residues that affected activity included those in or near the substrate binding site but also a number of residues that are distant (15-20 A) from this site. These data provide evidence that long-range energetic coupling of interface residues to the active site is essential for enzyme function, even though structural changes upon ligand binding to wild-type ODC are limited to local conformational changes in the active site. The ODC dimer interface appears to be optimized for catalytic function and not for dimer stability. Thus, small molecules directed to the ODC interfaces could impact biological function without having to overcome the difficult energetic barrier of dissociating the interacting partners.     

Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75A resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.     

Receptor-binding properties of a purified fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein.

A 208-amino-acid amino-terminal fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein contains all of the determinants required to recognize cell surface amphotropic receptors. This fragment was fused with a streptavidin-binding tag, expressed in Sf9 insect cells by using a baculovirus vector, and purified to homogeneity. The (125)I-labeled purified fragment (AS208) specifically bound various cell lines susceptible to amphotropic murine leukemia virus infection. The number of AS208-binding sites was in the range of 7 X 10(4) to 17 X 10(4) per cell. Quantitative analysis of binding revealed that AS208-binding sites are heterogeneous with regard to ligand binding affinity or that cooperativity exists between receptors. Competition experiments showed that the concentration of AS208 required to inhibit virus entry was lower than that required to inhibit the binding of virus particles at the cell surface. Taken together, these data suggested that amphotropic envelope-binding sites present at the cell surface do not act independently and do not participate equally in virus infection.     

Structure and interactions of NCAM Ig1-2-3 suggest a novel zipper mechanism for homophilic adhesion

The neural cell adhesion molecule, NCAM, mediates Ca(2+)-independent cell-cell and cell-substratum adhesion via homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. NCAM plays a key role in neural development, regeneration, and synaptic plasticity, including learning and memory consolidation. The crystal structure of a fragment comprising the three N-terminal Ig modules of rat NCAM has been determined to 2.0 A resolution. Based on crystallographic data and biological experiments we present a novel model for NCAM homophilic binding. The Ig1 and Ig2 modules mediate dimerization of NCAM molecules situated on the same cell surface (cis interactions), whereas the Ig3 module mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans interactions) through simultaneous binding to the Ig1 and Ig2 modules. This arrangement results in two perpendicular zippers forming a double zipper-like NCAM adhesion complex.     

Metal binding sites of a gamma-carboxyglutamic acid-rich fragment of bovine prothrombin.

The metal binding sites of a gamma-carboxyglutamic acid-rich fragment derived from bovine prothrombin were examined using paramagnetic lanthanide ions to evaluate the role of gamma-carboxyglutamic acid resideus in metal binding. A gamma-carboxyglutamic acid-rich peptide, fragment 12-44, was isolated from a tryptic digest of prothrombin. Using 153Gd(III), fragment 12-44 was found to contain one high affinity metal binding site (KD = 0.55 microM) and four to six lower affinity metal binding sites (KD approximately 4 to 8 microM). The S-carboxymethyl derivative of fragment 12-44, in which the disulfide bond in fragment 12-44 was reduced and alkylated, contained no high affinity metal binding site and four or five lower affinity sites (KD = 8 microM). The effects of paramagnetic lanthanide ions on fragment 12-44 and its S-carboxymethyl derivative were studied by natural abundance 13C NMR spectroscopy. The 13C NMR spectrum of fragment 12-44 was recorded at 67.88 MHz and the resonances were assigned by comparison to the chemical shift of carbon resonances of amino acids and peptides previously studied. The proximity between bound metal ions and carbon atoms in fragment 12-44 was estimated using Gd(III), based upon the strategy that the magnitude of the change in the transverse relaxation rate of resonances of carbon nuclei induced by bound metal ions is related in part to the interatomic distances between bound metal and carbon nuclei. Titration of fragment 12-44 with Gd(III) resulted in the selective broadening of the gamma-carboxyl carbon, C gamma, C beta, and C alpha resonances of gamma-carboxyglutamic acid, and the C epsilon of the arginines. S-Carboxymethyl fragment 12-44, which lacked the high affinity metal binding site, showed markedly decreased perturbation of the C epsilon of the arginine residues upon titration with Gd(III). These studies indicate that gamma-carboxyglutamic acid residues in prothrombin fragment 12-44 participate in metal liganding. A high affinity metal binding site in fragment 12-44 is in close proximity of Arg 16 and Arg 25 and is stabilized by the disulfide bond. On the basis of these data, a model of the metal binding sites is proposed in which the high affinity site is composed of two gamma-carboxyglutamic acid residues which participate in intramolecular metal-dependent bridging of two regions of the polypeptide chain. The lower affinity metal binding sites, formed by single or paired adjacent gamma-carboxyglutamic acid residues, then may participate in intermolecular metal-dependent protein . protein or protein . membrane complex formation.     

Proteolytic formation and properties of a fragment of protein C containing the gamma-carboxyglutamic acid rich domain and the EGF-like region

The function of the epidermal growth factor (EGF) like domains in the vitamin K dependent plasma proteins is largely unknown. In order to elucidate the function of these domains in protein C, we have devised a method to isolate the EGF-like region from the light chain connected to the NH2-terminal region, containing the gamma-carboxyglutamic acid (Gla) residues. This was accomplished by tryptic cleavage of protein C that had been reversibly modified with citraconic anhydride to prevent cleavage at the lysine residue (in position 43) that is located between the two regions. The isolated fragment consists of residues 1-143 from the light chain of protein C connected by a disulfide bond to residues 108-131 from the heavy chain. Upon Ca2+ binding to the isolated Gla-EGF fragment from bovine protein C, the tryptophan fluorescence emission was quenched in a manner indicating binding to at least two classes of binding sites. These were presumably the Gla-independent Ca2(+)-binding site located in the EGF-like region and the lower affinity sites in the Gla region. A comparison with the tryptophan fluorescence quenching that occurred upon Ca2+ binding to the separately isolated EGF-like and Gla regions suggested that the EGF-like region influenced the structure and Ca2+ binding of the Gla region. The isolated Gla-EGF fragment functioned as an inhibitor of the anticoagulant effect of activated protein C in a clotting assay, whereas no inhibition was observed with either the Gla region or the EGF-like region.     

Structural basis for the activation of FGFR by NCAM

The fibroblast growth factor receptor (FGFR) can be activated through direct interaction with the neural cell adhesion molecule (NCAM). The extracellular part of the FGFR consists of three immunoglobulin-like (Ig) modules, and that of the NCAM consists of five Ig and two fibronectin type III (F3) modules. NCAM-FGFR interactions are mediated by the third FGFR Ig module and the second NCAM F3 module. Using surface plasmon resonance and nuclear magnetic resonance analyses, the present study demonstrates that the second Ig module of FGFR also is involved in binding to the NCAM. The second Ig module residues involved in binding were identified and shown to be localized on the "opposite sides" of the module, indicating that when NCAMs are clustered (e.g., due to homophilic binding), high-affinity FGFR binding sites may be formed by the neighboring NCAMs.     

Fragments of bovine insulin-like growth factors I and II stimulate proliferation of rat L6 myoblast cells

The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells. Two fragments of IGF I (residues 21-45 and 31-55) and two fragments of IGF II (residues 20-44 and 30-54) were active while the other fragments were inactive in stimulating cell proliferation. Although the activity of these fragments was observed only at a high concentration of 0.1 mM, the results imply that the active site is located around residues 31-45 for IGF I fragments and residues 30-44 for IGF II fragments. Consequently, an IGF I fragment (residues 26-50) having a five-residue extension to both the N- and C-terminal sites of residues 31-45 also stimulated the proliferation of L6 myoblast cells. Furthermore, the substitution of Ile-35 in two IGF II fragments (residues 21-45 and 31-55) by Ser inactivated these fragments. This suggests that Ile-35 is an essential residue for IGF II fragment activity. Ser-35, which was reported in the original sequencing of bovine IGF II, is incorrect in the sequence and furthermore has been consistently found to be an Ile-35 in our hands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.     

Triple effect of mimetic peptides interfering with neural cell adhesion molecule homophilic cis interactions

The neural cell adhesion molecule (NCAM) is pivotal in neural development, regeneration, and learning. Here we characterize two peptides, termed P1-B and P2, derived from the homophilic binding sites in the first two N-terminal immunoglobulin (Ig) modules of NCAM, with regard to their effects on neurite extension and adhesion. To evaluate how interference of these mimetic peptides with NCAM homophilic interactions in cis influences NCAM binding in trans, we employed a coculture system in which PC12-E2 cells were grown on monolayers of fibroblasts with or without NCAM expression and the rate of neurite outgrowth subsequently was analyzed. P2, but not P1-B, induced neurite outgrowth in the absence of NCAM binding in trans. When PC12-E2 cells were grown on monolayers of NCAM-expressing fibroblasts, the effect of both P1-B and P2 on neurite outgrowth was dependent on peptide concentrations. P1-B and P2 acted as conventional antagonists, agonists, and reverse agonists of NCAM at low, intermediate, and high peptide concentrations, respectively. The demonstrated in vitro triple pharmacological effect of mimetic peptides interfering with the NCAM homophilic cis binding will be valuable for the understanding of the actions of these mimetics in vivo.     

The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex

Stable maintenance of P1 plasmids in Escherichia coli is mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and the E. coli integration host factor (IHF) bound specifically to the P1 parS site. IHF strongly stimulates ParB binding to parS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex. A fragment missing only the last 16 amino acids of ParB bound specifically to parS, but binding was weak and was no longer stimulated by IHF. The ability of IHF to stimulate ParB binding to parS correlated with the ability of ParB to dimerize via its C terminus. Using full and partial parS sites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences within parS. Based on these data, we have proposed a model of how the ParB dimer binds parS to form the minimal partition complex.     

The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity

BACKGROUND: One strategy that cells employ to respond to environmental stresses (temperature, oxidation, and pathogens) is to increase the expression of heat shock proteins necessary to maintain viability. Several heat shock proteins function as molecular chaperones by binding unfolded polypeptides and preventing their irreversible aggregation. Hsp33, a highly conserved bacterial heat shock protein, is a redox-regulated molecular chaperone that appears to protect cells against the lethal effects of oxidative stress. RESULTS: The 2.2 A crystal structure of a truncated E. coli Hsp33 (residues 1-255) reveals a domain-swapped dimer. The core domain of each monomer (1-178) folds with a central helix that is sandwiched between two beta sheets. The carboxyl-terminal region (179-235), which lacks the intact Zn binding domain of Hsp33, folds into three helices that pack on the other subunit. The interface between the two core domains is comprised of conserved residues, including a rare Glu-Glu hydrogen bond across the dyad axis. Two potential polypeptide binding sites that span the dimer are observed: a long groove containing pockets of conserved and hydrophobic residues, and an intersubunit 10-stranded beta sheet "saddle" with a largely uncharged or hydrophobic surface. CONCLUSIONS: Hsp33 is a dimer in the crystal structure. Solution studies confirmed that this dimer reflects the structural changes that occur upon activation of Hsp33 as a molecular chaperone. Patterns of conserved residues and surface charges suggest that two grooves might be potential binding sites for protein folding intermediates.     

Human apolipoprotein B-100 heparin-binding sites

Seven distinct heparin-binding sites have been demonstrated on human apolipoprotein (apo) B-100 by using a combination of digestion with cyanogen bromide or Staphylococcus aureus V-8 protease and heparin-Sepharose affinity chromatography. Based on fragment analysis, the approximate boundaries of the seven binding sites are as follows: site A, residues 5-99; site B, residues 205-279; site C, residues 875-932; site D, residues 2016-2151; site E, residues 3134-3209; site F, 3356-3489; and site G, residues 3659-3719. In sites E and F, two short regions enriched in basic amino acids have been identified, and it is likely that they are responsible for a major portion of the heparin-binding properties of these sites. The relative binding affinity of each of the seven sites was estimated in two ways. First, the affinity was assessed in a ligand blot assay using a 125I-labeled high-reactive heparin subfraction. Second, apoB-100 fragments generated by cyanogen bromide or S. aureus V-8 protease were separated into low- and high-affinity fractions by gradient salt elution of a heparin-Sepharose column. The distribution of the seven binding sites in the two fractions was determined in an immunoblotting assay using antibodies specific to each site, i.e. antibodies raised against synthetic peptide sequences found within each of the seven sites. The results of these two approaches demonstrate that site E and, to a somewhat lesser extent, site F bind to heparin with the highest affinity. Based on the analogy with apoE, in which the high-affinity heparin-binding site coincides with the domain of the protein that interacts with apoB,E (low density lipoprotein) receptors, the results of this study indicate that site E and site F, either singly or in combination, might constitute the receptor binding domain of apoB-100.     

Molecular modeling and mutagenesis studies of the N-terminal domains of galectin-3: evidence for participation with the C-terminal carbohydrate recognition domain in oligosaccharide binding

A model structure (Henrick,K., Bawumia,S., Barboni,E.A.M., Mehul,B. and Hughes, R.C. (1998) Glycobiology:, 8, 45-57) of the carbohydrate recognition domain (CRD, amino acid residues 114-245) of hamster galectin-3 has been extended to include N-terminal domain amino acid residues 91-113 containing one of the nine proline-rich motifs present in full-length hamster galectin-3. The modeling predicts two configurations of the N-terminal tail: in one the tail turns toward the first (SI) and last (S12) beta-strands of the CRD and lies at the apolar dimer interface observed for galectins -1 and -2. In the second folding arrangement the N-terminal tail lies across the carbohydrate-binding pocket of the CRD where it could participate in sugar-binding: in particular tyrosine 102 and adjacent residues may interact with the partly solvent exposed nonreducing N-acetylgalactosamine and fucose substituents of the A-blood group structure GalNAcalpha1,3 [Fucalpha1,2]Galbeta1,4GlcNAc-R. Binding studies using surface plasmon resonance of a recombinant fragment Delta1-93 protein containing residues 94-245 of hamster galectin-3 and a collagenase-derived fragment Delta1-103 containing residues 104-245, as well as alanine mutagenesis of residues 101-105 in Delta1-93 protein, support the prediction that Tyr102 and adjacent residues make significant contributions to oligosaccharide binding.