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.     

Surface-exposed amino acid residues of HPV16 L1 protein mediating interaction with cell surface heparan sulfate

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.     

Characterization of the heparin binding sites in human apolipoprotein E

Apolipoprotein (apo) E mediates lipoprotein remnant clearance via interaction with cell-surface heparan sulfate proteoglycans. Both the 22-kDa N-terminal domain and 10-kDa C-terminal domain of apoE contain a heparin binding site; the N-terminal site overlaps with the low density lipoprotein receptor binding region and the C-terminal site is undefined. To understand the molecular details of the apoE-heparin interaction, we defined the microenvironments of all 12 lysine residues in intact apoE3 and examined their relative contributions to heparin binding. Nuclear magnetic resonance measurements showed that, in apoE3-dimyristoyl phosphatidylcholine discs, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pK(a) values, indicating high positive electrostatic potential around these residues. Binding experiments using heparin-Sepharose gel demonstrated that the lipid-free 10-kDa fragment interacted strongly with heparin and a point mutation K233Q largely abolished the binding, indicating that Lys-233 is involved in heparin binding and that an unusually basic lysine microenvironment is critical for the interaction with heparin. With lipidated apoE3, it is confirmed that the Lys-233 site is completely masked and the N-terminal site mediates heparin binding. In addition, mutations of the two heparin binding sites in intact apoE3 demonstrated the dominant role of the N-terminal site in the heparin binding of apoE even in the lipid-free state. These results suggest that apoE interacts predominately with cell-surface heparan sulfate proteoglycans through the N-terminal binding site. However, Lys-233 may be involved in the binding of apoE to certain cell-surface sites, such as the protein core of biglycan.     

Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA

The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.     

Characterization of the Interaction between the Chlamydial Adhesin OmcB and the Human Host Cell

In a previous study, we reported that the OmcB protein from Chlamydia pneumoniae mediates adhesion of the infectious elementary body to human HEp-2 cells by interacting with heparin/heparan sulfate-like glycosaminoglycans (GAGs) via basic amino acids located in the first of a pair of XBBXBX heparin-binding motifs (K. Moelleken and J. H. Hegemann, Mol. Microbiol. 67:403–419, 2008). In the present study, we show that the basic amino acid at position 57 (arginine) in the first XBBXBX motif, the basic amino acid at position 61 (arginine) in the second motif, and another amino acid (lysine 69) C terminal to it play key roles in the interaction. In addition, we show that discrimination between heparin-dependent and -independent adhesion by C. trachomatis OmcBs is entirely dependent on three variable amino acids in the so-called variable domain C terminal to the conserved XBBXBX motif. Here, the predicted conformational change in the secondary structure induced by the proline at position 66 seems to be crucial for heparin recognition. Finally, we performed neutralization experiments using different anti-heparan sulfate antibodies to gain insight into the nature of the GAGs recognized by OmcB. The results suggest that C. trachomatis serovar L2 OmcB interacts with 6-O-sulfated domains of heparan sulfate, while C. pneumoniae OmcB apparently interacts with domains of heparan sulfate harboring a diverse subset of O-sulfations.     

The heparin binding site of human extracellular-superoxide dismutase.

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the results. A previous report showed that both lysine and arginine residues probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a "cluster" of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.     

The heparin-binding domain of extracellular superoxide dismutase C and formation of variants with reduced heparin affinity.

A fundamental property of the secretory tetrameric extracellular superoxide dismutase (EC-SOD) is its affinity for heparin and analogues, in vivo, mediating attachment to heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. EC-SOD is in vivo heterogeneous with regard to heparin affinity and can be divided into subclasses; A which lacks heparin affinity, B with intermediate affinity, and C with strong heparin affinity. The EC-SOD C subunits contain 222 amino acids and among the last 20 carboxyl-terminal amino acids, 10 are positively charged and six of these are located in a cluster in positions 210-215. To analyze if this local accumulation of basic amino acids is responsible for heparin binding we produced three series of recombinant EC-SOD (rEC-SOD) variants, six containing amino acid exchanges in the carboxyl-terminal end, four with truncations, and two with both truncations and substitutions. Exchange of positively or negatively charged amino acids on the carboxyl-terminal side of the cluster results in only minor modifications in heparin affinity, whereas substitution of three of the amino acids in the cluster abrogates the heparin binding. Insertions of stop codons at different positions resulted in either C or A but not B class EC-SOD. In an attempt to produce EC-SODs with intermediate heparin affinities, plasmids defining C and A class EC-SOD were cotransfected into Chinese hamster ovary cells. In addition to the parental A and C class EC-SOD forms, two variants with intermediate heparin affinities were formed. Coincubation of EC-SOD C and A resulted in the appearance of one heterotetramer with intermediate affinity for heparin. We conclude that the cluster of six basic amino acids forms the essential part of the heparin-binding domain and that the composition of the four subunits in the EC-SOD tetramer determines the affinity for heparin. This domain is different from heparin-binding domains of other proteins, and its localization allows the distribution of EC-SOD in vivo to be regulated by proteolytic processing.     

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.     

Elucidation of the structural features of heparan sulfate important for interaction with the Hep-2 domain of fibronectin

The interaction of fibronectin with cell surface heparan sulfate proteoglycans is important biologically in inducing reorganization of the cytoskeleton and the assembly of focal adhesions. The major heparan sulfate-binding site in fibronectin, which is also implicated in these morphological events, is the COOH-terminal Hep-2 domain. We describe the first extensive study of the structural determinants required for the interaction between heparan sulfate/heparin and Hep-2. It is clear that, in heparan sulfate, there is a very prominent role for N-sulfate groups, as opposed to a relatively small apparent contribution from carboxyl groups. Furthermore, a minimal octasaccharide binding sequence appeared to contain at least two 2-O-sulfated iduronate residues, but no 6-O-sulfate groups. However, affinity was enhanced by the presence of 6-O-sulfates, and the interaction with Hep-2 also increased progressively with oligosaccharide size up to a maximum length of a tetradecasaccharide. This overall specificity is compatible with recent information on the structure of Hep-2 (Sharma, A., Askari, J. A., Humphries, M. J., Jones, E. Y., and Stuart, D. I. (1999) EMBO J. 18, 1468-1479) in which two separate, positively charged clusters, involving up to 11 basic amino acid residues (mostly arginines with their preferential ability to co-ordinate sulfate groups), could form a single extended binding site.     

Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Heparin binds to the laminin alpha4 chain LG4 domain at a site different from that found for other laminins

We previously reported that the LG4 domain of the laminin alpha4 chain is responsible for high-affinity heparin binding. To specify the amino acid residues involved in this activity, we produced a series of alpha4 LG4-fusion proteins in which each of the 27 basic residues (arginine, R; histidine; lysine, K) were replaced one by one with alanine (A). When the effective residues R1520A, K1531A, K1533A, and K1539A are mapped on a structural model, they form a track on the concave surface of the beta-sandwich, suggesting that they interact with adjacent sulfate groups along the heparin chain. Whereas low-affinity heparin-binding sites of other LG domains have been located at the top of the beta-sheet sandwich opposite the N and C termini, the residues for high-affinity heparin binding of alpha4 LG4 reveal a new topological area of the LG module.     

Identification of a heparin-binding motif on adeno-associated virus type 2 capsids

Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.     

Hepatitis C virus attachment mediated by apolipoprotein E binding to cell surface heparan sulfate

Viruses are known to use virally encoded envelope proteins for cell attachment, which is the very first step of virus infection. In the present study, we have obtained substantial evidence demonstrating that hepatitis C virus (HCV) uses the cellular protein apolipoprotein E (apoE) for its attachment to cells. An apoE-specific monoclonal antibody was able to efficiently block HCV attachment to the hepatoma cell line Huh-7.5 as well as primary human hepatocytes. After HCV bound to cells, however, anti-apoE antibody was unable to inhibit virus infection. Conversely, the HCV E2-specific monoclonal antibody CBH5 did not affect HCV attachment but potently inhibited HCV entry. Similarly, small interfering RNA-mediated knockdown of the key HCV receptor/coreceptor molecules CD81, claudin-1, low-density lipoprotein receptor (LDLr), occludin, and SR-BI did not affect HCV attachment but efficiently suppressed HCV infection, suggesting their important roles in HCV infection at postattachment steps. Strikingly, removal of heparan sulfate from the cell surface by treatment with heparinase blocked HCV attachment. Likewise, substitutions of the positively charged amino acids with neutral or negatively charged residues in the receptor-binding region of apoE resulted in a reduction of apoE-mediating HCV infection. More importantly, mutations of the arginine and lysine to alanine or glutamic acid in the receptor-binding region ablated the heparin-binding activity of apoE, as determined by an in vitro heparin pulldown assay. HCV attachment could also be inhibited by a synthetic peptide derived from the apoE receptor-binding region. Collectively, these findings demonstrate that apoE mediates HCV attachment through specific interactions with cell surface heparan sulfate.     

Structural determinants in the C-terminal domain of apolipoprotein E mediating binding to the protein core of human aortic biglycan

Apolipoprotein (apo) E-containing high density lipoprotein particles were reported to interact in vitro with the proteoglycan biglycan (Bg), but the direct participation of apoE in this binding was not defined. To this end, we examined the in vitro binding of apoE complexed with dimyristoylphosphatidylcholine (DMPC) to human aortic Bg before and after glycosaminoglycan (GAG) depletion. In a solid-phase assay, apoE.DMPC bound to Bg and GAG-depleted protein core in a similar manner, suggesting a protein-protein mode of interaction. The binding was decreased in the presence of 1 m NaCl and was partially inhibited by either positively (0.2 m lysine, arginine) or negatively charged (0.2 m aspartic, glutamic) amino acids. A recombinant apoE fragment representing the C-terminal 10-kDa domain, complexed with DMPC, bound as efficiently as full-length apoE, whereas the N-terminal 22-kDa domain was inactive. Similar results were obtained with a gel mobility shift assay. Competition studies using a series of recombinant truncated apoEs showed that the charged segment in the C-terminal domain between residues 223 and 230 was involved in the binding. Overall, our results demonstrate that the C-terminal domain contains elements critical for the binding of apoE to the Bg protein core and that this binding is ionic in nature and independent of GAGs.     

Characterization of cytoplasmic arginine-rich basic protein of Guerin epithelioma

Summary Arginine-rich basic protein from cytoplasma of Guerin epitheliomas has been isolated and characterized. It contains five amino acids: arginine, lysine, glycine, alanine and glutamic acid which make together 74 per cent of all amino acid residues. The protein has a cationic character with an isoelectric point of 8.2. No carbohydrate component was found in this protein. The significance of arginine-rich basic protein in the cytoplasma of Guerin epithelioma is discussed briefly.     

Interferon-gamma binds to heparan sulfate by a cluster of amino acids located in the C-terminal part of the molecule

Using three different approaches (domain mapping with monoclonal antibodies, limited enzymatic digestion and competition with synthetic peptides), we demonstrated that a cluster of basic amino acids on interferon-gamma is involved in its binding to heparan sulfate. This cluster (Lys125-Arg131) is localized in the C-terminal part of IFN-gamma. Once bound to heparin sulfate, IFN-gamma is protected against protease attack.     

Characterization of heparin binding of human extracellular superoxide dismutase

The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K(d), of 0.12 microM, which is 700 and 10-20 times lower than the K(d) values for the synthetic peptide and the fusion proteins, respectively. However, when an alpha-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K(d) decreased to 0.64 microM. The differences in the K(d) values were mainly governed by differences in the association rate constants (k(ass)). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s(-)(1)), and a very high association rate constant (8 x 10(5) M(-)(1) s(-)(1)), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an alpha-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.     

Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins

Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 Å spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection. BioEssays 20:156–167, 1998. © 1998 John Wiley & Sons, Inc.     

Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.     

Characterization of the stromal cell-derived factor-1alpha-heparin complex

The binding of chemokines to glycosaminoglycans is thought to play a crucial role in chemokine functions. It has recently been shown that stromal cell-derived factor-1alpha (SDF-1alpha), a CXC chemokine with potent anti-human immunodeficiency virus activity, binds to heparan sulfate through a typical consensus sequence for heparin recognition (BBXB, where B is a basic residue KHLK, amino acids 24-27). Calculation of the accessible surface, together with the electrostatic potential of the SDF-1alpha dimer, revealed that other amino acids (Arg-41 and Lys-43) are found in the same surface area and contribute to the creation of a positively charged crevice, located at the dimer interface. GRID calculations confirmed that this binding site will be the most energetically favored area for the interaction with sulfate groups. Site-directed mutagenesis and surface plasmon resonance-based binding assays were used to investigate the structural basis for SDF-1alpha binding to heparin. Among the residues clustered in this basic surface area, Lys-24 and Lys-27 have dominant roles and are essential for interaction with heparin. Amino acids Arg-41 and Lys-43 participate in the binding but are not strictly required for the interaction to take place. Direct binding assays and competition analysis with monoclonal antibodies also permitted us to show that the N-terminal residue (Lys-1), an amino acid critical for receptor activation, is involved in complex formation. Binding studies with selectively desulfated heparin, heparin oligosaccharides, and heparitinase-resistant heparan sulfate fragments showed that a minimum size of 12-14 monosaccharide units is required for efficient binding and that 2-O- and N-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1alpha, and a docking study was undertaken. During the energy minimization process, heparin lost its perfect ribbon shape and fitted the protein surface perfectly. In the model, Lys-1, Lys-24, Lys-27, and Arg-41 were found to have the major role in binding a polysaccharide fragment consisting of 13 monosaccharide units.