Some structural studies of heparin-binding proteins/peptides and their heparin or HS ligands

Heparin-Binding ProteinExperimentHeparin/HS
 ATNitrous acid degradation, 3-O-sulfatase treatment, paper electrophoresisHigh-affinity octasaccharide sequence containing pentasaccharide minimum motif; identification of 3-O-sulfated glucosamine (Lindahl et al., 1980)
Synthetic chemistry, NMR spectroscopyHigh-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 FXaCrystal structure of AT/FXa/synthetic pentasaccharideInteractions 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 FIXaCrystal 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)
  PCICrystal 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 1Crystal 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
 ThrombinCrystal 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-1Crystal 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 hexasaccharideFGF-1 interacts with the “one-sided” heparin mimetic: GlcNS-IdoA2S-GlcNAc6S-IdoA-GlcNS-IdoA2S (Muñoz-García et al., 2014)
 FGF-1/FGFR2cCrystal structure of FGF-1/FGFR2c/heparin (1E0O.pdb)FGF-1/FGFR2c/heparin decamer 2:2:1 stoichiometry (Pellegrini et al., 2000)
 FGF-2In 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/FGFR1Crystal 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-8bAffinity chromatography of HS fragments on immobilized FGFAll three FGFs bind to the same HS fragments with similar affinity; no evidence for differential binding to specific sequences (Kreuger et al., 2005)
 FGFRNMR spectroscopy and molecular modelingHeparin induces dimerization of FGFR heparin binding domain without FGF (Nieto et al., 2013)
 VEGFSPR on immobilized heparinHeparin octasaccharide minimum; the AT-binding motif not necessary (Zhao et al., 2012)
NMR titrations and molecular modelingInteractions predicted involve N-, 2-O-, and 6-O-sulfates (Robinson et al., 2006; Jeong et al., 2013)
 PDGF-AAFilter-trapping and affinity chromatography6–8 monosaccharides minimum, NS domains containing both 2-O- and 6-O-sulfate groups (Feyzi et al., 1997)
 HGF/SFX-ray crystallography of NK1 domain with tetrasaccharide (1GMN.pdb, 1GMO.pdb)Binding to N domain critical for biologic activity (Lietha et al., 2001)
 Gro-a (CXCL1)NMR of trapped dimer; titrations with octasaccharideHeparin 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 modelingMinimum 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 assaysNS 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 titrationHeparin binding site on the C-terminal α-helix and proximal loop (Schlorke et al., 2012; Möbius et al., 2013)
Ion mobilization MSA single dimeric form interacts with heparin octasaccharide (Seo et al., 2013)
 MIP-1α (CCL3)Human marrow LTC-IC maintenance assaysAn 8.3-kDa fragment consisting of two S domains separated by a short NA domain (Stringer et al., 2002)
 MIP-1β (CCL4)NMR spectroscopy, ultracentrifugationDisaccharide 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 MSOctasaccharides 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 MSMCP-1 can form two distinct dimers in interaction with heparin octasaccharide (Seo et al., 2013)
Sedimentation equilibrium ultracentrifugation, affinity chromatographyHeparin 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 dodecasaccharideHeparin dodecasaccharide binds orthogonally to the dimer interface (Ziarek et al., 2013)
NMR titration, STD with a 13C-labeled semisynthetic octasaccharide; restrained molecular modelingHeparin 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 enzyme9-kDa fragment, two S domains with short linking NA domain (Stringer and Gallagher, 1997).
 I-TAC (CXCL11)Affinity chromatography, NMR spectroscopy, molecular modelingTwo binding modes identified with differing affinities (Severin et al., 2010)
 IL-2ELISA binding and competition assaysFull-size heparin (17kDa) required for maximum ability to compete with mAb (Najjam et al., 1998)
 IL-4Heparin affinity chromatography, competition assays on immobilized IL-4S domains of HS required for interaction (Lortat-Jacob et al., 1997)
 IL-6ELISA competition assaysN- and 2-O-sulfation required but not 6-O-sulfation (Mummery and Rider, 2000)
 IL-7SPR with heparin oligosaccharides and other GAGsHeparin binds with higher affinity to human than to murine IL-7 (Zhang et al., 2012b)
 IL-10STD-NMR and transferred NOE techniquesSulfated disaccharide will bind, but minimum hexasaccharide required for high affinity (Künze et al., 2014)
 IL-12ELISA competition assaysAffinity increases evenly with length of oligosaccharide (Hasan et al., 1999)
 GMC-SFAffinity chromatographypH-dependent binding to helix C of GM-CSF due to histidine involvement (Sebollela et al., 2005)
 IFNγNMR titrationOctasaccharide minimum length for binding (Vanhaverbeke et al., 2004); two cationic sites on the protein surface involved (Saesen et al., 2013)
 SDF1α (CXCL12)SPR binding assays12–14 monosaccharide units, 2-O- and N-sulfate groups (Sadir et al., 2001)
 SclerostinNMR titration and molecular modeling (2K8P.pdb)Binding site can accommodate a heparin decamer (Veverka et al., 2009)
 NogginDisplacement of noggin from CHO cell surface; immunoprecipitationPrefers 10 or more monosaccharide units, with fully sulfated S domains (Viviano et al., 2004)
 Sonic hedgehogCrystal 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-selectinELISA assays, cell adhesion assaysHexasulfated tetrasaccharide (Nelson et al., 1993)
 PECAM-1Molecular modeling and SPR of PECAM-1 and domain deletion mutants with heparin and dodecasaccharideHeparin binding site involves domains 2 and 3; dodecasaccharide fractions have varying affinities (Coombe et al., 2008)
ECM proteins
 FibronectinInhibition of cell attachment14-mer minimum; high sulfation with GlcNS (Mahalingam et al., 2007)
 ThrombospondinCrystal 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 HAnalytical ultracentrifugation, X-ray scattering, SPRCooperative, bivalent binding to heparin of long oligosaccharides (dp18 and over) (Khan et al., 2012)
Affinity chromatography, microtiter plate binding assaysTwo GAG binding sites on factor H select different sulfation patterns (Clark et al., 2013)
 Human β-defensin 2Gel 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
 ECPNMR spectroscopy and crystal structure (2LVZ.pdb) with synthetic trisaccharideBinds to catalytic amino acid triad, explaining heparin inhibition of ribonucleolytic activity (García-Mayoral et al., 2013)
 EMBPCrystal structure with disaccharide (2BRS.pdb)Calcium-independent binding to this C-type lectin (Swaminathan et al., 2001)
 Glucuronyl C5-epimeraseCrystal structure with heparin hexasaccharide (4PXQ.pdb)Also evidence for close coupling of epimerase with OS enzymes (Qin et al., 2015)
 2-OSTCrystal structure with hexasaccharideNo publication yet
 3-OST-1Crystal 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-3Crystal structure with tetrasaccharide (1T8U.pdb)Structure of critical enzyme for biosynthesis of HS motif recognized by herpes simplex (Moon et al., 2004)
 Neutrophil elastaseIn vivo model of airway inflammation2- and 3-O-sulfation not necessary for elastase inhibition (Griffin et al., 2014)
Elastase release from neutrophils10 monosaccharides minimum length (Lever et al., 2007)
 TryptaseAnalysis of mast cells in HS6ST-deficient mice6-O-sulfation required for packing of tryptase in mast cell granules (Ferdous Anower-E-Khuda et al., 2012)
 Bacterial heparinase ICrystal 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 tuberculosisNMR spectroscopyMinimal binding length for heparin 8–10 monosaccharides; 14-mer causes structural rearrangement of the protein (Lebrun et al., 2012)
 Decorin-binding protein of Borrelia bergdorferiGel mobility shift assays, ITC, NMR/paramagnetic perturbation with hexasaccharidePrimary heparin binding site at the C terminus, secondary site near the N terminus (Morgan and Wang, 2013)
 Histone-like protein HlpSolid phase assays, NMR, and affinity chromatographyHeparin/HS binds Hlp at two distinct sites, conserved in mycobacterial homologs (Portugal et al., 2008)
Viral proteins
 HIV-1 gp120Inhibition of antibody binding and HIV-1 replicationO-sulfation important (Rider et al., 1994)
 HPV18 capsidCrystal structure of capsid with octa- and decasaccharideMultiple 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 capsidCrystal 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 HSVCell-based assays of viral entryRecognizes 3-O-sulfated sequence, product of 3-OST-3 (Shukla et al., 1999)
 vMIP-IIAffinity chromatography, NMR spectroscopy with disaccharide, molecular modelingHeparin binding site similar to that of human MIPs (Zhao and Liwang, 2010)
 Cyclophilin BOptical biosensor binding assays, electrophoretic mobility shift assaysRecognizes 3-O-sulfated sequence, product of 3-OST-3; N-unsubstituted GlcN (Vanpouille et al., 2007)
Amyloid and prion proteins
 APLP-1Crystal 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)
 PrPLight scattering, fluorescence, CD, NMRLMWH induces transient aggregation of PrP, but the soluble PrP/LMWH complex inhibits RNA-induced aggregation (Vieira et al., 2011)
 Aβ proteinSolid-state NMRHeparin 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)
 Annexin Vtetrasaccharide (1G5N.pdb)Calcium-dependent interaction (Capila et al., 2001)
Affinity chromatography, SPR, ITCOctasaccharide minimum length for binding (Capila et al., 1999)
 Annexin A2Crystal 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 proteinCrystal structure with heparin dodecasaccharide (1RID.pdb)Heparin binding site on subunit 4 (Ganesh et al., 2004)
 Cobra CTX A3Crystal structure with heparin hexasaccharide (1XT3.pdb)Cell surface lattice of heparin, citrate, and CTX proposed (Lee et al., 2005)
 CPPsITC, CD, NMR, MS, phase-contrast microscopyTryptophan content correlates with affinity for GAGs including heparin (Bechara et al., 2013)
 Arrestin-1NMR titration with LMWH and inositol hexaphosphateIP6 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.