Crystallographic Analysis of Calcium-dependent Heparin Binding to Annexin A2 (2006)

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2. Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2. Annexins are a family of Ca2+-dependent membrane-binding eukaryotic proteins that have been implicated in a myriad of physiological processes, including membrane trafficking, cell signaling, ion transport, inflammation, apoptosis, and hemostasis (1Gerke V. Creutz C.E. Moss S.E. Nat. Rev. Mol. Cell Biol. 2005; 6: 449-461Crossref PubMed Scopus (1130) Google Scholar). Most functional properties of annexins are attributed to their ability to bind membrane phospholipids and certain other anionic polymers, including glycosaminoglycans (GAGs) 2The abbreviations used are: GAGs, glycosaminoglycans; A2t, (annexin A2)2-(S100A10)2 heterotetramer; SPR, surface plasmon resonance; Δ4UAp2S, 4-deoxy-α-l-threo-2-sulfo-hex-4-enopyranosyluronic acid; IdoAp2S, 2-O-sulfo-α-l-idopyranosyluronic acid; GlcNpS6S, 2-deoxy-,2-sulfamido, 6-O-sulfo-α-d-glucopyranose; r.m.s.d., root mean square deviation. 2The abbreviations used are: GAGs, glycosaminoglycans; A2t, (annexin A2)2-(S100A10)2 heterotetramer; SPR, surface plasmon resonance; Δ4UAp2S, 4-deoxy-α-l-threo-2-sulfo-hex-4-enopyranosyluronic acid; IdoAp2S, 2-O-sulfo-α-l-idopyranosyluronic acid; GlcNpS6S, 2-deoxy-,2-sulfamido, 6-O-sulfo-α-d-glucopyranose; r.m.s.d., root mean square deviation. such as heparin and heparan sulfate, in a Ca2+-dependent manner. Human annexin A2 is abundantly expressed in various tissues such as brain, spleen, kidney, lung, placenta, intestine, and the adrenal glands, and in intracellular and extracellular locations. Putative intracellular roles for annexin A2 include membrane trafficking and cytoskeletal actin bundling; and extracellular annexin A2 has been proposed to play a role in the fibrinolytic pathway (1Gerke V. Creutz C.E. Moss S.E. Nat. Rev. Mol. Cell Biol. 2005; 6: 449-461Crossref PubMed Scopus (1130) Google Scholar). Annexin A2 often co-expresses with another protein, p11 (S100A10), forming an (annexin A2)2-(S100A10)2 heterotetramer (A2t). Monomeric and tetrameric annexin A2 share many of the same binding properties, e.g. the abilities to bind Ca2+, phospholipid membranes, and certain GAGs (including heparin and heparan sulfate), albeit typically with some modification in affinity. The p11 component alone, despite belonging to the E-F hand family of Ca2+-binding proteins, does not bind Ca2+ nor does it bind phospholipids or GAG. The association between annexin A2 and p11 does not require Ca2+ and utilizes the concave annexin molecular surface (2Waisman D.M. Mol. Cell. Biochem. 1995; 149: 301-322Crossref PubMed Scopus (262) Google Scholar). The putative role of annexin A2 in plasminogen activation through tissue plasminogen activator has been explored in previous studies (3Kang H.M. Choi K.S. Kassam G. Fitzpatrick S.L. Kwon M. Waisman D.M. Trends Cardiovasc. Med. 1999; 9: 92-102Crossref PubMed Scopus (52) Google Scholar, 4Hajjar K.A. Acharya S.S. Ann. N. Y. Acad. Sci. 2000; 902: 265-271Crossref PubMed Scopus (66) Google Scholar), as has the function of heparin in fibrinolysis (5Liang J.F. Li Y. Yang V.C. Thromb. Res. 2000; 97: 349-358Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 6Bell J. Duhon S. Doctor V.M. Blood Coagul. Fibrinolysis. 2003; 14: 229-234PubMed Google Scholar). The identification of annexin A2 as a heparin-binding protein (7Kassam G. Manro A. Braat C.E. Louie P. Fitzpatrick S.L. Waisman D.M. J. Biol. Chem. 1997; 272: 15093-15100Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) has raised the possibility that this protein-GAG interaction may participate in the regulation of thrombotic processes. To characterize the heparin-annexin A2 interaction and to determine the basis for its Ca2+ dependence, crystallographic studies were initiated on human annexin A2 in complex with heparin-derived oligosaccharides of varying lengths. Binding measurements in solution were also performed using surface plasmon resonance (SPR). Annexin A2 Expression and Purification—Wild type human annexin A2 in vector pAED4.91 was transformed into Escherichia coli BL21 (DE3). Transformed cells were grown in LB Broth, Miller (American Bioanalytical). Cells were harvested by centrifugation at 6,000 × g for 15 min and resuspended in lysis buffer containing 100 mm Tris, pH 7.5, 200 mm NaCl, 2 mm dithiothreitol, 1 mm EGTA, and protease inhibitors (Sigma), including 0.5 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin, 0.2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 μg/ml aprotinin, 0.5 mm benzamidine, and 5 μm E-64. The cell solution was sonicated for 6 min and centrifuged at 100,000 × g for 1 h. The supernatant was precipitated with 50% (NH4)2SO4 and centrifuged at 27,000 × g for 20 min. This supernatant was then applied to a butyl-Sepharose column (Amersham Biosciences), and annexin A2 was eluted with a linear gradient from 50 to 0% (NH4)2SO4 in the lysis buffer. The fractions containing annexin A2 were then dialyzed against sample buffer (20 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm EGTA, and 1 mm dithiothreitol). The dialyzed sample then was applied to a DEAE-Sepharose column (Amersham Biosciences). Pure annexin A2 protein was in the flow-through from the column. SDS-PAGE and matrix-assisted laser desorption ionization-mass spectrometry confirmed the presence of the intact full-length annexin A2 polypeptide. Heparin-derived Oligosaccharide Isolation—Three oligosaccharides were co-crystallized with annexin A2 as follows: heparin-derived tetrasaccharide, hexasaccharide, and octasaccharide. Homogenous heparin oligosaccharides were purified as described previously (8Capila I. Hernaiz M.J. Mo Y.D. Mealy T.R. Campos B. Dedman J.R. Linhardt R.J. Seaton B.A. Structure (Camb.). 2001; 9: 57-64Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Crystallographic Analysis—Crystals of annexin A2 in the absence of heparin were grown at 17 °C by vapor diffusion against a reservoir solution containing 14% PEG8000, 0.1 m Tris, pH 8.5, 10 mm CaCl2, and 15% glycerol was added for cryo-protection. For the complexes, 10 mg/ml annexin A2 and 20 mm of each of three heparin-derived oligosaccharides were mixed and co-crystallized similarly by vapor diffusion at 17 °C against solutions containing 0.8-1.0 m 1,6-hexanediol, 0.1 m sodium acetate, pH 4.8-5.0, and 10-15 mm CaCl2. Evaporation of the droplet provided sufficient cryo-protection from the concentrated mother liquor. Crystallographic data from the tetrasaccharide-annexin A2 complex crystal was collected at the Brookhaven National Laboratory Synchrotron X8C beam-line. Data from the uncomplexed annexin A2 crystal, hexasaccharide-bound and octasaccharide-bound annexin A2 complex crystals were collected on an R-axis IV imaging plate detector mounted on a Rigaku RU-300 rotating anode generator. To collect high resolution data of the hexasaccharide-bound annexin A2 crystal, the 2θ angle was set at 10°. All crystals were directly cooled to 80-100 K by vapor nitrogen stream prior to collection. Data were indexed, integrated, and scaled using the DENZO and SCALE-PACK software packages (9Otwinowksi Z. Minor W. Methods Enzymol. 1997; 27: 307-326Crossref Scopus (38523) Google Scholar). Phases were provided by molecular replacement using the program EPMR (10Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (690) Google Scholar) and previous structures of annexin A2 (Protein Data Bank entry 1W7B) and annexin A5 (Protein Data Bank entry 1G5N) as starting models. Model building and refinement steps were carried out using O (11Jones T.A. Zou J.Y. Cowan S.W. Kjelgaard M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar) and CNS (12Bru¨nger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar), respectively. For the complexes, initial Fo - Fc difference electron density maps provided the locations of the bound heparin saccharides, which were included in subsequent refinement using parameters employed previously (8Capila I. Hernaiz M.J. Mo Y.D. Mealy T.R. Campos B. Dedman J.R. Linhardt R.J. Seaton B.A. Structure (Camb.). 2001; 9: 57-64Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). For all structures, individual B-factors were refined for all atoms. Data collection and refinement statistics of the crystallization work are presented in Table 1. Figures were all made through Pymol (13DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar).TABLE 1Crystallographic data and refinement statisticsAN2aAN2 indicates annexin A2.AN2 + tetrasaccharideAN2 + hexasaccharideDiffraction dataResolution (Å)2.101.861.42Space groupP21P21212P21212Unit cella (Å)60.5107.9107.7b (Å)64.354.354.3c (Å)104.268.968.6β91.73°90.0°90.0°No. of reflections45,42332,15375,286CompletenessbCompleteness is reported for all reflections and for the highest resolution shell (values in parentheses). (%)96.7 (93.6)92.6 (94.8)97.9 (83.5)Redundancy3.64.35.0RmergecRmerge reported for all reflections and for the highest resolution shell (values in parentheses); Rmerge = ∑|Ii - 〈I 〉|/∑Ii, where Ii is the intensity of an individual reflection, and 〈I 〉 is the mean intensity of that reflection. (%)6.7 (33.1)7.5 (34.9)5.5 (37.5)RefinementResolution range (Å)40-2.1050-1.8650-1.42RcrystdRcryst = ∑|Fp| - |Fcalc|/∑|Fp|, where |Fcalc| and |Fp| are the calculated and observed structure factors, respectively.0.21420.19860.2118RfreeeRfree is defined as in Bru¨nger et al. (12).0.25630.23220.2227No. of monomers in arbitrary units211No. of protein atoms495624782478No. of ions (Ca2+)1455No. of heparin atoms07085No. of water molecules438432518No. of atoms total540829853086R.m.s.d.fr.m.s.d. indicates root mean square deviation. angles1.0534°0.9901°1.0404°R.m.s.d. bonds (Å)0.00530.00460.0045Mean 〈B 〉 overall (Å2)30.120.822.3Mean 〈B 〉 protein (Å2)29.618.719.6Mean 〈B 〉 heparin (Å2)27.928.2Ramachandran plot (%)gData were calculated using Procheck (23). The sole residue in either generously allowed or disallowed regions corresponds to Val50 in the I-AB Ca2+ -binding loop.Most favored93.794.794.7Additional allowed6.04.94.9Generously allowed0.40.00.0Disallowed0.00.40.4a AN2 indicates annexin A2.b Completeness is reported for all reflections and for the highest resolution shell (values in parentheses).c Rmerge reported for all reflections and for the highest resolution shell (values in parentheses); Rmerge = ∑|Ii - 〈I 〉|/∑Ii, where Ii is the intensity of an individual reflection, and 〈I 〉 is the mean intensity of that reflection.d Rcryst = ∑|Fp| - |Fcalc|/∑|Fp|, where |Fcalc| and |Fp| are the calculated and observed structure factors, respectively.e Rfree is defined as in Bru¨nger et al. (12Bru¨nger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar).f r.m.s.d. indicates root mean square deviation.g Data were calculated using Procheck (23Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The sole residue in either generously allowed or disallowed regions corresponds to Val50 in the I-AB Ca2+ -binding loop. Open table in a new tab SPR Analysis of Annexin A2-Heparin Interactions—SPR measurements were performed on a BIAcore 3000 (BIAcore AB, Uppsala, Sweden) operated using the version software. Streptavidin sensor chip was from BIAcore. There is an average of one unsubstituted amino group within each heparin chain (14Toida T. Yoshida H. Toyoda H. Koshiishi I. Imanari T. Hileman R.E. Fromm J.R. Linhardt R.J. Biochem. J. 1997; 322: 499-506Crossref PubMed Scopus (151) Google Scholar). This unsubstituted amino group was biotinylated as described previously (15Hernaiz M. Liu J. Rosenberg R.D. Linhardt R.J. Biochem. Biophys. Res. Commun. 2000; 276: 292-297Crossref PubMed Scopus (52) Google Scholar). The resulting biotinylated heparin was then immobilized on the streptavidin chip based on the manufacturer's protocol. The successful immobilization of heparin was confirmed by the observation of an ∼500 resonance unit increase in the sensor chip. For the measurement of protein interactions with heparin, HBS-P buffer (0.01 m HEPES, 0.15 m NaCl, 0.005% polysorbate 20 (v/v), pH 7.4, from BIAcore) with the addition of 5 mm CaCl2 was used as running buffer. Annexin A2 samples were diluted in HBS-P with Ca2+ and injected at a flow rate of 30 μl/min. At the end of each sample injection, the Ca2+-free buffer was flowed over the sensor surface to facilitate dissociation. After a 3-min dissociation time, the sensor surface was regenerated by injecting with 30 μl of 2 m NaCl in 100 mm acetate buffer, pH 4.5. The response was monitored as a function of time (sensorgram) at 25 °C. The resulting sensorgrams were used for kinetic parameter determination by globally fitting to a 1:1 Langmuir binding model using BIAEvaluation software 4.0.1 (BIAcore AB, Uppsala, Sweden). Annexin A2 Structure without Heparin Bound—The N-terminal 30 amino acids of the annexin A2 were not visible in the structure, leaving Asn31 as the first observed residue. Mass spectrometry run on the recovered crystal confirmed the absence, presumably due to proteolysis, of residues not The structure of the uncomplexed annexin A2 is to that of the human annexin A2 with Ca2+ bound A. H. Scholar) (Protein Data Bank entry main-chain r.m.s.d. of and overall r.m.s.d. of the annexin domain the from Asn31 to is into four each a domain containing five The molecular structure is that of a with the four in a Ca2+ are bound on the convex face of the protein Annexin this resolution structure, a is bound at the convex of domain IV the and electron density is observed for all sugar The structure of annexin A2 in the complex is r.m.s.d. of and overall r.m.s.d. of to that of uncomplexed annexin with in some This observation is with previous studies (7Kassam G. Manro A. Braat C.E. Louie P. Fitzpatrick S.L. Waisman D.M. J. Biol. Chem. 1997; 272: 15093-15100Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). of at the nonreducing end is in the residue of is in the The residue of the The residue D of GlcNpS6S, is in the same as that of residue In the residue at the nonreducing end of each heparin has been from to The modification a between and a of heparin the was I. Linhardt R.J. Chem. PubMed Scopus Google Scholar). The modification to the binding of residue to annexin A2. The binding interactions in and the atoms are in In the at the residue site and the interactions with either a or for as as the observed for Ca2+ ions are observed in the crystal structure of the tetrasaccharide-annexin A2 complex previously Ca2+-binding the binds at a Ca2+-binding site that has not been observed in other annexin A2 structures A. H. Scholar, A. S. J. A. P. V. J. J. Mol. Biol. PubMed Scopus Google Scholar). This novel Ca2+-binding site between the and where it in heparin binding. The three Ca2+ ions in the annexin A2 structure not in the annexin complex to the type type and type Ca2+-binding at the and the type type Ca2+-binding site at loop. In the absence of Ca2+, the a different and the chain in annexin A2 the site where is bound at the of the uncomplexed Two of the five Ca2+ ions observed in the complex participate directly or in binding heparin 2 and which is also in the of uncomplexed annexin does not directly with the interactions with the group of residue through a water In the novel Ca2+ ion with the of residues and of the and Table The shell of two oxygen atoms from the of residues and oxygen atoms from the chain and the and two water and are and are through two water as as which and through side-chain and main-chain oxygen between heparin oligosaccharides and protein atoms or Open table in a new tab In addition to the Ca2+ heparin with annexin A2 through between its oxygen atoms and the nitrogen atoms from the and including the N-terminal end of of domain residues of annexin A2 and Table The group of residue with the main-chain nitrogen of and the water the oxygen atoms of the and group with the side-chain nitrogen of residue its sole to the complex of an residue of an In are interactions between residue and the and For residue bonds between the and the oxygen and nitrogen atoms and the main-chain nitrogen atoms of and the sugar residue into Heparin an in solution B. M.J. D.B. Biochem. J. 1993; PubMed Scopus Google Scholar). The for the and are and and and B. M.J. 2000; PubMed Scopus Google Scholar). The observed in crystallographic or structures from by up to In the crystallographic tetrasaccharide-annexin A2 the as in B. M.J. 2000; PubMed Scopus Google Scholar) for the between residues and are and to the observed the between residues and D has of and which is from the common for this type of Annexin crystal structure of the annexin complex at the highest resolution all annexin crystal structures, is r.m.s.d. of to that of annexin density is observed for the first four sugar residues and for of the residue on the convex of domain IV of annexin A2 There is observed electron density for the residue. Comparison of the two complexes (annexin and annexin shows that the first three residues and of the are in the same and as the first three residues of the tetrasaccharide, the residue in the two structures 2 and Table In the annexin the group of residue D binds to annexin A2 through D in the annexin complex is in the it is by the between residues and In the bonds or are between the oxygen atoms of residue D and main-chain and side-chain nitrogen atoms of from the loop. this of residue the of the are and to the observed for the B. M.J. 2000; PubMed Scopus Google Scholar). residue D of the binds to the residue D of the with the of the shows binding to protein atoms or bound Ca2+ the interactions are through water The annexin complex is r.m.s.d. of to that of annexin its the the same interactions the first five sugar and the electron density is for the for the first four In of complexes, residues the first five not with the protein and clear electron of its to the the complex is not of with crystals of the annexin complexes are grown pH the binding properties of annexin A2 to full-length heparin at this pH were using measurements were made at pH and 7.4, with or without Ca2+ added The that in the absence of Ca2+, annexin A2 binding to the heparin chip at either pH 7.4 or pH not in the presence of 5 mm Ca2+, binding is observed at pH Table shows the data calculated for the binding between full-length heparin and annexin A2. The binding affinity is at pH at pH 7.4 of and 17 kinetic measurements of annexin A2 interactions with heparin polysaccharide in the presence of 5 mm × × × × × Open table in a new tab of heparin-binding proteins have been to of a Ca2+-dependent binding to heparin as follows: and and annexin a of annexin A2 I. Linhardt R.J. Chem. PubMed Scopus Google Scholar). this annexin A5 (8Capila I. Hernaiz M.J. Mo Y.D. Mealy T.R. Campos B. Dedman J.R. Linhardt R.J. Seaton B.A. Structure (Camb.). 2001; 9: 57-64Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and annexin A2 have been as crystallographic complexes with Ca2+ and heparin The crystallographic data that Ca2+-dependent heparin binding from the Ca2+-dependent protein conformational that a heparin-binding site complex are in the annexin The is by the of the through of from various crystallographic and studies indicates that Ca2+ within annexin type type the In all four of the annexin A2 crystal structures, the are by of the presence or absence of heparin oligosaccharide. the by a binding surface for residue A. In the of upon complex in which is a The of a Ca2+ site as a for a Ca2+ site that as of a complex with another has been previously for annexin A5 Dedman J.R. Seaton B.A. Nat. Biol. 1995; PubMed Scopus Google Scholar). In the annexin A2 the ion is similarly in the and complexes, through five protein oxygen ligands and two water on the other with the the ion is through two oxygen ligands from the protein, two from residues and and two water The two Ca2+ are through interactions with two water and Ca2+ have been observed in other proteins e.g. Sci. 2000; 9: PubMed Scopus Google Scholar). The crystal structure of annexin A5 in complex with Ca2+ and heparin (8Capila I. Hernaiz M.J. Mo Y.D. Mealy T.R. Campos B. Dedman J.R. Linhardt R.J. Seaton B.A. Structure (Camb.). 2001; 9: 57-64Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) shows a Ca2+-dependent of binding that is to the observed in annexin A2. In the annexin A5 the does not the Ca2+ binds to protein atoms in a I-AB Ca2+-binding loop. The annexin complex on the concave surface of that protein is not observed in the annexin A2 crystal at this may in annexin A2 as a of heterotetramer which the concave protein The of heparin binding in all three annexin A2 complexes common that may important for heparin polysaccharide by the In all complexes, the nonreducing end of the binds at the with a physiological site for the nonreducing end of the heparin or the heparan chain to a IdoAp2S, the of Δ4UAp2S, bind and either or the The crystallographic data that either the site the nonreducing end or that the modification on residue binding affinity at the in the crystallographic In despite in the first three sugar residues bind with at the same site on the the residue in the has different from that of the residue D in the The conformational difference may by its of the and the resulting with the nitrogen of the group within the residue D sugar and Table the heparin the oxygen of residue D is not in the forms the to residue with the In of the the residue is clear in electron of and and subsequent residues are not observed at of the three complex structures that the first residues the nonreducing are for heparin and binding and that the sugar residues in a heparin polysaccharide are to the in the The work a structural basis for previously reported to the binding in solution of heparin to (7Kassam G. Manro A. Braat C.E. Louie P. Fitzpatrick S.L. Waisman D.M. J. Biol. Chem. 1997; 272: 15093-15100Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). the previous solution that the binding interaction shows Ca2+ dependence and a of The crystallographic data are with all three oligosaccharides bind to annexin A2 the same four sugar residues and binding for a in the The SPR data that binding of A2 to the chip in the absence of In the binding for A2 and at despite different measurement for A2 and for are 17 and respectively. The SPR kinetic data a 1:1 one heparin polysaccharide one annexin A2 with the crystallographic data that a site on the The combined data from the two studies that the heparin-binding site in the crystallographic complexes the site of heparin interaction in annexin A2 and The two studies are also in conformational to heparin binding by annexin A2 or The crystallographic and data in that heparin binding significant conformational change in annexin A2 it is that a conformational in that observed in (7Kassam G. Manro A. Braat C.E. Louie P. Fitzpatrick S.L. Waisman D.M. J. Biol. Chem. 1997; 272: 15093-15100Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This observation that heparin in some with the p11 the complex to a structural in The data also that a conformational change in This proposed that may a of in domain IV the concave surface of the annexin A2 in with p11 In this that annexin A2 significant Ca2+-dependent heparin-binding properties either as a protein or as a component of the properties from a heparin-binding site by two Ca2+-binding at the convex of domain IV that binds residues from the nonreducing The of this site provide a structural basis for Ca2+-dependent of heparin by annexin A2.