Some structural studies of heparin-binding proteins/peptides and their heparin or HS ligands
| Heparin-Binding Protein | Experiment | Heparin/HS |
|---|---|---|
| Serpins | ||
| AT | Nitrous acid degradation, 3-O-sulfatase treatment, paper electrophoresis | High-affinity octasaccharide sequence containing pentasaccharide minimum motif; identification of 3-O-sulfated glucosamine (Lindahl et al., 1980) |
| Synthetic chemistry, NMR spectroscopy | High-affinity pentasaccharide sequence confirmed by synthesis, found to have high anti-Xa activity (Choay et al., 1983) | |
| Crystal structures of AT complexed with high-affinity synthetic pentasaccharide (1AZX.pdb, 1NQ9.pdb, 3EVJ.pdb 2GD4.pdb, 1EO3.pdb) | Studies exploring in detail the interaction between AT and a synthetic high-affinity pentasaccharide (Jin et al., 1997; Johnson and Huntington, 2003; McCoy et al., 2003; Johnson et al., 2006b; Langdown et al., 2009) | |
| AT with FXa | Crystal structure of AT/FXa/synthetic pentasaccharide | Interactions between AT and two exosites on FXa (Johnson et al., 2006b) |
| AT with thrombin (FIIa) | Crystal structure of AT/thrombin/heparin mimetic complex (1TB6.pdb) | Ternary complex, both AT and thrombin bound to same heparin molecule (Li et al., 2004) |
| Crystal structure of AT/thrombin/heparin mimetic complex (2B5T.pdb) | Native conformation of AT reactive center loop determined (Johnson et al., 2006a) | |
| Crystal structure of AT/anhydrothrombin/synthetic heparin 16-mer (1SR5.pdb) | 16-mer saccharide just long enough to bridge AT heparin binding site and thrombin exosite 2 (Dementiev et al., 2004) | |
| With FIXa | Crystal structure of AT/FIXa/heparin complex (3KCG.pdb) | Heparin-activated AT conformation interacts with FIXa at both active site and exosite (Johnson et al., 2010) |
| PCI | Crystal structure with thrombin and heparin tetradecasaccharide (3B9F.pdb); crystal structure of cleaved PCI with heparin octasaccharide (3DY0.pdb) | Multiple binding modes of bridging heparin; electron density for the disaccharide at thrombin exosite II only (Li et al., 2008) |
| Heparin binds to helix H, rather than helix D as for AT (Li and Huntington, 2008) | ||
| PN-1 protease nexin 1 | Crystal structure of PN-1 with heparin decamer (4DY0.pdb) and PN-1/heparin/thrombin complex (4DY7.pdb) | Suggests a two-stage sequence of an initial binding event followed by conformational rearrangement to form productive complex (Li and Huntington, 2012) |
| Serine protease | ||
| Thrombin | Crystal structure with heparin octasaccharide (1XMN.pdb) | Octasaccharide can engage with exosite II of more than one thrombin molecule (Carter et al., 2005) |
| Growth factors | ||
| FGF-1 | Crystal structure of FGF-1/heparin complex (1AXM.pdb, 2AXM.pdb) | Heparin 4-mer to 6-mer (DiGabriele et al., 1998); stoichiometry FGF-1/heparin 2:1 |
| Crystal structure of FGF-1 with four disaccharides (3UD7, 3UD8, 3UD9, 3UDA) | Of 48 synthetic disaccharides, 4 were identified that bind to FGF-1 (Hu et al., 2012) | |
| NMR structure of FGF-1 with a synthetic heparinoid hexasaccharide | FGF-1 interacts with the “one-sided” heparin mimetic: GlcNS-IdoA2S-GlcNAc6S-IdoA-GlcNS-IdoA2S (Muñoz-García et al., 2014) | |
| FGF-1/FGFR2c | Crystal structure of FGF-1/FGFR2c/heparin (1E0O.pdb) | FGF-1/FGFR2c/heparin decamer 2:2:1 stoichiometry (Pellegrini et al., 2000) |
| FGF-2 | In complex with heparin tetrasaccharide (1BFB.pdb) and hexasaccharide (1BFC.pdb) | FGF2/heparin 1:1 stoichiometry (Faham et al., 1996) |
| In complex with three heparin disaccharides (4OEE.pdb, 4OEF.pdb, 4OEG.pdb) | Water-mediated interactions between disaccharide and protein (Li et al., 2008) | |
| FGF-2/FGFR1 | Crystal structure of FGF-2/FGFR1/heparin (1FQ9.pdb) | FGF-2/FGFR1/heparin decamer 2:2:2 (Schlessinger et al., 2000) |
| FGF-4, FGF-7, FGF-8b | Affinity chromatography of HS fragments on immobilized FGF | All three FGFs bind to the same HS fragments with similar affinity; no evidence for differential binding to specific sequences (Kreuger et al., 2005) |
| FGFR | NMR spectroscopy and molecular modeling | Heparin induces dimerization of FGFR heparin binding domain without FGF (Nieto et al., 2013) |
| VEGF | SPR on immobilized heparin | Heparin octasaccharide minimum; the AT-binding motif not necessary (Zhao et al., 2012) |
| NMR titrations and molecular modeling | Interactions predicted involve N-, 2-O-, and 6-O-sulfates (Robinson et al., 2006; Jeong et al., 2013) | |
| PDGF-AA | Filter-trapping and affinity chromatography | 6–8 monosaccharides minimum, NS domains containing both 2-O- and 6-O-sulfate groups (Feyzi et al., 1997) |
| HGF/SF | X-ray crystallography of NK1 domain with tetrasaccharide (1GMN.pdb, 1GMO.pdb) | Binding to N domain critical for biologic activity (Lietha et al., 2001) |
| Chemokines | ||
| Gro-a (CXCL1) | NMR of trapped dimer; titrations with octasaccharide | Heparin binds orthogonally to interhelical axis of disulphide-trapped dimer; octasaccharide is not long enough to span completely both heparin binding sites in the dimer (Poluri et al., 2013). |
| RANTES (CCL5) | SPR binding assays and molecular modeling | Minimum 14 monosaccharide units needed for high-affinity dimer complexed with heparin heptadecasaccharide modeled (Vives et al., 2002) |
| Crystal structure with disaccharides (1U4L.pdb, 1U4M.pdb) | Only disaccharides with 2-sulfation of uronic acid cocrystallized (Shaw et al., 2004) | |
| IL-8 (CXCL8) | Filter-trapping binding assays | NS block of about 6 monosaccharide units within an approximately 22- to 24-mer sequence, separated by a region of ≤14 monosaccharide residues that may be fully NA (Spillmann et al., 1998) |
| NMR titration | Heparin binding site on the C-terminal α-helix and proximal loop (Schlorke et al., 2012; Möbius et al., 2013) | |
| Ion mobilization MS | A single dimeric form interacts with heparin octasaccharide (Seo et al., 2013) | |
| MIP-1α (CCL3) | Human marrow LTC-IC maintenance assays | An 8.3-kDa fragment consisting of two S domains separated by a short NA domain (Stringer et al., 2002) |
| MIP-1β (CCL4) | NMR spectroscopy, ultracentrifugation | Disaccharide can support dimerization (McCornack et al., 2004) |
| CCR2 ligands: MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), Eotaxin-1 (CCL-11) | Filtration trapping and FTICR MS | Octasaccharides with at least 10 sulfates are required; MCP-1 and MCP-2 dimerize in presence of octasaccharide, the others remain monomeric (Yu et al., 2005) |
| Ion mobilization MS | MCP-1 can form two distinct dimers in interaction with heparin octasaccharide (Seo et al., 2013) | |
| Sedimentation equilibrium ultracentrifugation, affinity chromatography | Heparin binding site is a continuous ring of basic residues round tetramer (Lau et al., 2004) | |
| SDF-1 (CXCL12) | NMR spectroscopy and crystal structure (2NWG.pdb) | Two distinct binding sites for the disaccharide on the CXCL12 dimer (Murphy et al., 2007) |
| NMR titration with heparin dodecasaccharide | Heparin dodecasaccharide binds orthogonally to the dimer interface (Ziarek et al., 2013) | |
| NMR titration, STD with a 13C-labeled semisynthetic octasaccharide; restrained molecular modeling | Heparin binding site and dimerization confirmed, NMR restrained and unrestrained molecular modeling in good agreement (Laguri et al., 2011) | |
| PF4 (CXCL4) | Filter-trapping assays, protection of HS fragment against enzyme | 9-kDa fragment, two S domains with short linking NA domain (Stringer and Gallagher, 1997). |
| I-TAC (CXCL11) | Affinity chromatography, NMR spectroscopy, molecular modeling | Two binding modes identified with differing affinities (Severin et al., 2010) |
| Cytokines | ||
| IL-2 | ELISA binding and competition assays | Full-size heparin (17kDa) required for maximum ability to compete with mAb (Najjam et al., 1998) |
| IL-4 | Heparin affinity chromatography, competition assays on immobilized IL-4 | S domains of HS required for interaction (Lortat-Jacob et al., 1997) |
| IL-6 | ELISA competition assays | N- and 2-O-sulfation required but not 6-O-sulfation (Mummery and Rider, 2000) |
| IL-7 | SPR with heparin oligosaccharides and other GAGs | Heparin binds with higher affinity to human than to murine IL-7 (Zhang et al., 2012b) |
| IL-10 | STD-NMR and transferred NOE techniques | Sulfated disaccharide will bind, but minimum hexasaccharide required for high affinity (Künze et al., 2014) |
| IL-12 | ELISA competition assays | Affinity increases evenly with length of oligosaccharide (Hasan et al., 1999) |
| GMC-SF | Affinity chromatography | pH-dependent binding to helix C of GM-CSF due to histidine involvement (Sebollela et al., 2005) |
| IFNγ | NMR titration | Octasaccharide minimum length for binding (Vanhaverbeke et al., 2004); two cationic sites on the protein surface involved (Saesen et al., 2013) |
| SDF1α (CXCL12) | SPR binding assays | 12–14 monosaccharide units, 2-O- and N-sulfate groups (Sadir et al., 2001) |
| Morphogens | ||
| Sclerostin | NMR titration and molecular modeling (2K8P.pdb) | Binding site can accommodate a heparin decamer (Veverka et al., 2009) |
| Noggin | Displacement of noggin from CHO cell surface; immunoprecipitation | Prefers 10 or more monosaccharide units, with fully sulfated S domains (Viviano et al., 2004) |
| Sonic hedgehog | Crystal structure with heparin 18-mer (4C4N.pdb) and with chondroitin 4-sulfate (4C4M.pdb) | Heparin 15-mer required to induce dimerization; orientation of GAG chains in complex depends on sulfation pattern (Whalen et al., 2013) |
| Heparin binding: adhesion molecules | ||
| Integrins (αvβ3) | Molecular modeling (docking) | A conserved heparin binding site identified in RGD integrins but not in non-RGD integrins (Ballut et al., 2013) |
| P-selectin, L-selectin | ELISA assays, cell adhesion assays | Hexasulfated tetrasaccharide (Nelson et al., 1993) |
| PECAM-1 | Molecular modeling and SPR of PECAM-1 and domain deletion mutants with heparin and dodecasaccharide | Heparin binding site involves domains 2 and 3; dodecasaccharide fractions have varying affinities (Coombe et al., 2008) |
| ECM proteins | ||
| Fibronectin | Inhibition of cell attachment | 14-mer minimum; high sulfation with GlcNS (Mahalingam et al., 2007) |
| Thrombospondin | Crystal structure with heparin octasaccharide (2OUJ.pdb), decasaccharide (2OUH.pdb) and AT-binding pentasaccharide (1ZA4.pdb) | Octasaccharide and decasaccharide bind thrombospondin similarly to synthetic pentasaccharide (Tan et al., 2006) and induce formation of trans- and cis-dimers respectively (Tan et al., 2008) |
| Complement system and innate immunity | ||
| Factor H | Analytical ultracentrifugation, X-ray scattering, SPR | Cooperative, bivalent binding to heparin of long oligosaccharides (dp18 and over) (Khan et al., 2012) |
| Affinity chromatography, microtiter plate binding assays | Two GAG binding sites on factor H select different sulfation patterns (Clark et al., 2013) | |
| Human β-defensin 2 | Gel mobility shift, NMR titration with synthetic heparin pentasaccharide (and DS hexasaccharide) | Heparin binding site mapped; heparin pentasaccharide induced dimerization (unlike DS hexasaccharide) (Seo et al., 2010) |
| NMR titration and high pressure NMR | “Sandwich-like” dimerization mode proposed (De Paula et al., 2014) | |
| Cytotoxic mediators | ||
| ECP | NMR spectroscopy and crystal structure (2LVZ.pdb) with synthetic trisaccharide | Binds to catalytic amino acid triad, explaining heparin inhibition of ribonucleolytic activity (García-Mayoral et al., 2013) |
| EMBP | Crystal structure with disaccharide (2BRS.pdb) | Calcium-independent binding to this C-type lectin (Swaminathan et al., 2001) |
| Enzymes | ||
| Glucuronyl C5-epimerase | Crystal structure with heparin hexasaccharide (4PXQ.pdb) | Also evidence for close coupling of epimerase with OS enzymes (Qin et al., 2015) |
| 2-OST | Crystal structure with hexasaccharide | No publication yet |
| 3-OST-1 | Crystal structure with heptasaccharide (3UAN.pdb) | Comparison with the 3-OST-3 structure (see below) allows rationalization of 3-OST isoform specificity (Moon et al., 2012) |
| 3-OST-3 | Crystal structure with tetrasaccharide (1T8U.pdb) | Structure of critical enzyme for biosynthesis of HS motif recognized by herpes simplex (Moon et al., 2004) |
| Neutrophil elastase | In vivo model of airway inflammation | 2- and 3-O-sulfation not necessary for elastase inhibition (Griffin et al., 2014) |
| Elastase release from neutrophils | 10 monosaccharides minimum length (Lever et al., 2007) | |
| Tryptase | Analysis of mast cells in HS6ST-deficient mice | 6-O-sulfation required for packing of tryptase in mast cell granules (Ferdous Anower-E-Khuda et al., 2012) |
| Bacterial heparinase I | Crystal structure with disaccharide (3IN9.pdb) and dodecasaccharide (3INA.pdb) | Strained conformation of the heparin dodecamer induced by binding allows enzyme to act endolytically on longer heparin chains (Han et al., 2009) |
| Microbial adhesins | ||
| Heparin-binding hemagglutinin of Mycobacterium tuberculosis | NMR spectroscopy | Minimal binding length for heparin 8–10 monosaccharides; 14-mer causes structural rearrangement of the protein (Lebrun et al., 2012) |
| Decorin-binding protein of Borrelia bergdorferi | Gel mobility shift assays, ITC, NMR/paramagnetic perturbation with hexasaccharide | Primary heparin binding site at the C terminus, secondary site near the N terminus (Morgan and Wang, 2013) |
| Histone-like protein Hlp | Solid phase assays, NMR, and affinity chromatography | Heparin/HS binds Hlp at two distinct sites, conserved in mycobacterial homologs (Portugal et al., 2008) |
| Viral proteins | ||
| HIV-1 gp120 | Inhibition of antibody binding and HIV-1 replication | O-sulfation important (Rider et al., 1994) |
| HPV18 capsid | Crystal structure of capsid with octa- and decasaccharide | Multiple binding sites found at top rim and side walls of capsid pentamer may indicate sites of interaction with extended heparin chain (Dasgupta et al., 2011) |
| FMDV capsid | Crystal structure with heterogenous heparin (1QQP.pdb, 1ZBA.pdb) | Fully sulfated oligosaccharide motif picked out by binding site at the junction of three capsid proteins (Fry et al., 1999;Fry et al., 2005) |
| gD glycoprotein of HSV | Cell-based assays of viral entry | Recognizes 3-O-sulfated sequence, product of 3-OST-3 (Shukla et al., 1999) |
| vMIP-II | Affinity chromatography, NMR spectroscopy with disaccharide, molecular modeling | Heparin binding site similar to that of human MIPs (Zhao and Liwang, 2010) |
| Cyclophilin B | Optical biosensor binding assays, electrophoretic mobility shift assays | Recognizes 3-O-sulfated sequence, product of 3-OST-3; N-unsubstituted GlcN (Vanpouille et al., 2007) |
| Amyloid and prion proteins | ||
| APLP-1 | Crystal structure of APLP-1 E2 domain with heparin hexasaccharide (3QMK.pdb) (also ITC and affinity chromatography) | Heparin stabilizes the dimer, in both solid and solution states, with 2:1 protein/heparin stoichiometry (Xue et al., 2011) |
| PrP | Light scattering, fluorescence, CD, NMR | LMWH induces transient aggregation of PrP, but the soluble PrP/LMWH complex inhibits RNA-induced aggregation (Vieira et al., 2011) |
| Aβ protein | Solid-state NMR | Heparin has a lower affinity for Aβ(1–40) fibrils having 2-fold molecular symmetry than for fibrils with 3-fold symmetry (Madine et al., 2012) |
| Miscellaneous | ||
| Annexin V | tetrasaccharide (1G5N.pdb) | Calcium-dependent interaction (Capila et al., 2001) |
| Affinity chromatography, SPR, ITC | Octasaccharide minimum length for binding (Capila et al., 1999) | |
| Annexin A2 | Crystal structures with tetrasaccharide (2HYU.pdb) and hexasaccharide (2HYV.pdb) | Calcium binds to both protein and ligand (Shao et al., 2006) |
| Peptidoglycan recognition protein (camel) | Crystal structure (3OGX.pdb) | Disaccharide bound tightly to subsite S1 of this antibacterial protein (Sharma et al., 2012) |
| Vaccinia complement protein | Crystal structure with heparin dodecasaccharide (1RID.pdb) | Heparin binding site on subunit 4 (Ganesh et al., 2004) |
| Cobra CTX A3 | Crystal structure with heparin hexasaccharide (1XT3.pdb) | Cell surface lattice of heparin, citrate, and CTX proposed (Lee et al., 2005) |
| CPPs | ITC, CD, NMR, MS, phase-contrast microscopy | Tryptophan content correlates with affinity for GAGs including heparin (Bechara et al., 2013) |
| Arrestin-1 | NMR titration with LMWH and inositol hexaphosphate | IP6 and LMWH bind to the same site; both induce the release of the arrestin C-tail (Zhuang et al., 2010) |
APLP, amyloid precursor-like protein; CD, circular dichroism; CHO, Chinese hamster ovary; CPP, cell-penetrating peptide; CTX, cardiotoxin; DS, (structure given in Petitou et al. 2009); ELISA, enzyme-linked immunosorbent assay; FMDV, foot-and-mouth disease virus; FTICR, (structure given in Petitou et al. 2009); GMC-SF, granulocyte macrophage colony-stimulating factor; I-TAC, (structure given in Petitou et al. 2009); IFN, interferon; IP6, (structure given in Petitou et al. 2009); ITC, isothermal titration calorimetry; LTC-IC, (structure given in Petitou et al. 2009); NOE, nuclear Overhauser effect; OS, (structure given in Petitou et al. 2009); PDGF, platelet-derived growth factor; PrP, prion protein; vMIP, viral macrophage inflammatory protein.