Chromatin-independent binding of serum amyloid P component to apoptotic cells.

Human serum amyloid P component (SAP) is a glycoprotein structurally belonging to the pentraxin family of proteins, which has a characteristic pentameric organization. Mice with a targeted deletion of the SAP gene develop antinuclear Abs, which was interpreted as evidence for a role of SAP in controlling the degradation of chromatin. However, in vitro SAP also can bind to phosphatidylethanolamine, a phospholipid which in normal cells is located mainly in the inner leaflet of the cell membrane, to be translocated to the outer leaflet of the cell membrane during a membrane flip-flop. We hypothesized that SAP, because of its specificity for phosphatidylethanolamine, may bind to apoptotic cells independent of its nuclear binding. Calcium-dependent binding of SAP to early, nonpermeable apoptotic Jurkat, SKW, and Raji cells was indeed observed. Experiments with flip-flopped erythrocytes confirmed that SAP bound to early apoptotic cells via exposed phosphatidylethanolamine. Binding of SAP was stronger to late, permeable apoptotic cells. Experiments with enucleated neutrophils, with DNase/RNase treatment of late apoptotic Jurkat cells, and competition experiments with histones suggested that binding of SAP to late apoptotic cells was largely independent of chromatin. Confocal laser microscopic studies indeed suggested that SAP bound to these apoptotic cells mainly via the blebs. Thus, this study shows that SAP binds to apoptotic cells already at an early stage, which raises the possibility that SAP is involved in dealing with apoptotic cells in vivo.     

Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod

cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0-C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in beta myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a K(d) of 3.52 microM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.     

Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis

Serum amyloid P component (SAP) is a member of the pentraxin family of proteins. These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. We previously determined that the binding of CRP to leukocytes occurs through Fc receptors for IgG (FcgammaR). We now report that SAP also binds to FcgammaR and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to FcgammaRI, FcgammaRIIa, and FcgammaRIIIb expressed on transfected COS cells was detected using SAP-biotin and PE-streptavidin. Zymosan was used to test the functional consequences of SAP and CRP binding to FcgammaR. Both SAP and CRP bound to zymosan and enhanced its uptake by PMN. This enhanced phagocytosis was abrogated by treatment of PMN with wortmannin, a phosphatidylinositol-3 kinase inhibitor, or with piceatannol, a Syk inhibitor, consistent with uptake through FcgammaR. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove FcgammaRIIIb also decreased phagocytosis of SAP-opsonized zymosan, but not CRP-opsonized zymosan. These results suggest that SAP may function in host defense. In addition, as SAP binds to chromatin, a major immunogen in systemic lupus erythematosus, it may provide a clearance mechanism for this Ag through FcgammaR bearing cells.     

Localization of a heparin binding site in the catalytic domain of factor XIa.

Inhibition of factor XIa by protease nexin II (K(i) approximately 450 pM) is potentiated by heparin (K(I) approximately 30 pM). The inhibition of the isolated catalytic domain of factor XIa demonstrates a similar potentiation by heparin (K(i) decreasing from 436 +/- 62 to 88 +/- 10 pM) and also binds to heparin on surface plasmon resonance (K(d) 11.2 +/- 3.2 nM vs K(d) 8.63 +/- 1.06 nM for factor XIa). The factor XIa catalytic domain contains a cysteine-constrained alpha-helix-containing loop: (527)CQKRYRGHKITHKMIC(542), identified as a heparin-binding region in other coagulation proteins. Heparin-binding studies of coagulation proteases allowed a grouping of these proteins into three categories: group A (binding within a cysteine-constrained loop or a C-terminal heparin-binding region), factors XIa, IXa, Xa, and thrombin; group B (binding by a different mechanism), factor XIIa and activated protein C; and group C (no binding), factor VIIa and kallikrein. Synthesized peptides representative of the factor XIa catalytic domain loop were used as competitors in factor XIa binding and inhibition studies. A native sequence peptide binds to heparin with a K(d) = 86 +/- 15 nM and competes with factor XIa in binding to heparin, K(i) = 241 +/- 37 nM. A peptide with alanine substitutions at (534)H, (535)K, (538)H, and (539)K binds and competes with factor XIa for heparin-binding in a manner nearly identical to that of the native peptide, whereas a scrambled peptide is approximately 10-fold less effective, and alanine substitutions at residues (529)K, (530)R, and (532)R result in loss of virtually all activity. We conclude that residues (529)K, (530)R, and (532)R comprise a high-affinity heparin-binding site in the factor XIa catalytic domain.     

Fibronectin binding to complement subcomponent C1q. Localization of their respective binding sites.

The interaction of purified human plasma fibronectin with the C1q subcomponent of complement was investigated by using a solid-phase radiobinding assay. 125I-fibronectin binding to native C1q, purified collagen domain (C1q-c) or globular domain (C1q-g) was compared. When the purified domains were insolubilized by binding to plastic, the C1q-c exhibited 59% of the binding demonstrated with intact C1q, whereas the C1q-g exhibited 35% of the binding. N-Terminal sequencing of the globular domain showed that a sequence of seven collagen-like amino acids was retained on each chain of the C1q-g fragment. 125I-fibronectin binding to C1q could be inhibited equally well by fluid-phase C1q and C1q-c, but not by fluid-phase C1q-g, implying that the collagen-like region retained on the C1q-g is masked in the fluid phase. In addition, studies were performed to determine which subunit(s) of C1q bind(s) fibronectin. The percentages of fibronectin bound by the A, B, and C chain of C1q were found to be 38, 21 and 41% respectively. Inhibition studies with purified 200-180 kDa, 50 kDa or 29 kDa fragments of fibronectin show that the binding site on fibronectin for C1q is the 50 kDa gelatin-binding domain.     

The C-terminal binding domain of hirullin P18. Antithrombin activity and comparison to hirudin peptides

Hirullin P18 is a 61-amino acid hirudin-related protein having potent antithrombin activity. Similar to hirudin, it contains a highly acidic C-terminus, but has a significantly different sequence from any other known hirudin variant. The present study demonstrates that the C-terminal fragment acetyl-hirullin P18(41-62) [corrected] possesses an antithrombin potency similar to that of acetyl-desulfatohirudin(45-65). Additionally, like the hirudin fragment analog, it inhibits fibrin-clot formation by binding to a non-catalytic site on thrombin. Sequential shortening of the hirullin P18 C-terminal fragment demonstrates the critical nature of Phe51, which corresponds to the important Phe56 residue of hirudin. Although the sequences of hirullin P18(54-61) and hirudin(59-65) have substantial differences, the C-terminal functional domain represented by hirullin P18(50-61) appears to be comparable to hirudin(55-65) in terms of its functional role in antithrombin activity.     

Protein Z-dependent protease inhibitor binds to the C-terminal domain of protein Z

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K(D)) of approximately 7 nm. However, the apparent K(D) for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes approximately 6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.     

Localization of the conglutinin binding site on the third component of human complement

The binding site on the human third complement component for bovine conglutinin has been located. C3 fragments were purified to homogeneity by preparative SDS-polyacrylamide-gel electrophoresis. Only the N-terminal 27,000 dalton (Da) fragment of the alpha'-chain and the beta-chain were found to be glycosylated, and the carbohydrate was susceptible to endo-beta-N-acetylglucosaminidase H. This finding indicates that only high mannose or hybrid-type oligosaccharide chains are present on the C3 molecule. Binding to conglutinin was determined by an enzyme-linked immunosorbent assay and occurred with C3b, iC3b, C3c, the alpha-chain, and the 27,000 Da fragment of the alpha'-chain, but not with C3d or the C-terminal 40,000 Da fragment of the alpha'-chain. The beta-chain displayed very weak interaction. Binding to conglutinin could be inhibited by EDTA, N-acetylglucosamine, and to a lesser degree by mannose. Enzymatic removal of the carbohydrate from the C3 molecule abolished binding to conglutinin. It is concluded that bovine conglutinin binds to the carbohydrate moiety located on the N-terminal 27,000 Da polypeptide of the alpha-chain.     

Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.     

The C-terminal end of AraC tightly binds to the rest of its domain

Genes were synthesized to express two DNA binding domains of AraC connected by short linkers. The abilities of the resulting proteins to bind to DNA containing AraC half-sites separated by the usual four bases as well as an additional two or three helical turns of the DNA were measured. The inability of some of the protein constructs to bind to widely separated half-sites indicates that the C-terminal 14 amino acids of AraC are firmly bound to the rest of the DNA binding domain.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.     

Localization of the human complement component C3 binding site on the IgG heavy chain

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.     

Serum amyloid P component is the major calcium-dependent specific DNA binding protein of the serum

Serum amyloid P component (SAP), a member of the highly conserved pentraxin family of plasma proteins, was found to be the only protein in whole normal or acute phase serum which underwent specific calcium-dependent binding to either single or double-stranded DNA immobilised on gel. Isolated purified SAP also bound to long chromatin, to H1-stripped chromatin and to native DNA in solution at physiological ionic strength. Pure SAP which had been immobilised on gel, specifically bound nucleosome core particles from solution. These observations strongly suggest that SAP may bind to extracellular chromatin and DNA in vivo and that this may be its physiological role.     

Identification of an atypical calcium-dependent calmodulin binding site on the C-terminal domain of GluN2A

N-methyl-d-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875–1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca²⁺/calmodulin binds GluN2A with high affinity (5.2 ± 2.4 nM) in vitro. Direct interaction of Ca²⁺/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca²⁺/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca²⁺/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association.     

Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic, antimetastatic, and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA), and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase, implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations, indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.     

A Nucleotide-dependent molecular switch controls ATP binding at the C-terminal domain of Hsp90. N-terminal nucleotide binding unmasks a C-terminal binding pocket.

In vivo function of the molecular chaperone Hsp90 is ATP-dependent and requires the full-length protein. Our earlier studies predicted a second C-terminal ATP-binding site in Hsp90. By applying direct biochemical approaches, we mapped two ATP-binding sites and unveiled the C-terminal ATP-binding site as the first example of a cryptic chaperone nucleotide-binding site, which is opened by occupancy of the N-terminal site. We identified an N-terminal gamma-phosphate-binding motif in the middle domain of Hsp90 similar to other GHKL family members. This motif is adjacent to the phosphate-binding region of the C-terminal ATP-binding site. Whereas novobiocin disrupts both C- and N-terminal nucleotide binding, we found a selective C-terminal nucleotide competitor, cisplatin, that strengthens the Hsp90-Hsp70 complex leaving the Hsp90-p23 complex intact. Cisplatin may provide a pharmacological tool to dissect C- and N-terminal nucleotide binding of Hsp90. A model is proposed on the interactions of the two nucleotide-binding domains and the charged region of Hsp90.