Characterization of the Stromal Cell-derived Factor-1α-Heparin Complex

Abstract

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-1α (SDF-1α), 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-1α 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-1α 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- andN-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1α, 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. 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-1α (SDF-1α), 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-1α 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-1α 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- andN-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1α, 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. macrophage inflammatory peptides-1 glycosaminoglycan heparan sulfate stromal cell-derived factor-1α Tris-buffered saline monoclonal antibody degree of polymerization Chemokines are small structurally related chemo-attractant cytokines, characterized by conserved cysteine residues. Almost 40 chemokines have been identified to date which, based on the position of the first N-terminal cysteines, fall into four sub-families. Two of them have been well characterized, the CC group, which includes regulated on activation, normal T-cell expressed, and secreted, monocyte chemoattractant protein-1, and MIP-11 (macrophage inflammatory peptides-1), and the CXC group, the prototype of which is interleukin-8. The C chemokine (lymphotactine) and the CX 3C chemokine (fractalkine) sub-families have been identified more recently (1Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2083) Google Scholar, 2Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Greaves D.R. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1671) Google Scholar, 3Kelner M.P. Kennedy J. Bacon K.B. Kleyensteuber S. Largaespada D.A. Jenjins N.A. Copeland N.G. Bazan J.F. Moore K.W. Schall T.J. Zlotnik A. Science. 1994; 266: 1395-1399Crossref PubMed Scopus (624) Google Scholar, 4Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar). These proteins signal through G-protein-coupled seven transmembrane domain receptors (5Wells T.N.C. Power C.A. Proudfoot A.E.I. Trends Pharmacol. Sci. 1998; 19: 376-380Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and are primarily involved in immunosurveillance, activation, and recruitment of specific cell populations during disease (1Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2083) Google Scholar, 6Luster A.D. N. Engl. J. Med. 1998; 7: 436-445Crossref Scopus (3219) Google Scholar, 7Gale L.M. McColl S.R. BioEssays. 1999; 21: 17-28Crossref PubMed Scopus (113) Google Scholar, 8Melchers F. Rolink A.G. Schaniel C. Cell. 1999; 99: 351-354Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Most, if not all, chemokines bind to heparan sulfate (9Witt D.P. Lander A.D. 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PubMed Google Scholar) and have been shown to be involved in heparin the surface of the dimer for the of the electrostatic In the dimer, the by residues and are on the of the of them the of a Arg-41 and in the of the the and to be located to participate in the interaction. the is at the dimer and is more to play a role in in the interaction with heparin. It is that the N-terminal to the in the of the In the of the are for positively charged area on the accessible surface of the protein and also be involved in heparin The binding was characterized the GRID In this the interaction of a with a protein of structure is at and the an of energy The analysis of the GRID with a consisting of a charged a sulfate group, is in the most for binding sulfate is at the in the by four basic amino Lys-27 and Arg-41 of protein Lys-24 and Lys-43 also a binding site on the binding area the It is that a heparin to SDF-1α that SDF-1α on monosaccharide units A. A. A. M. M. M. F. F. J. 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Biol. 1999; Full Text Full Text PDF PubMed Scopus Google the and the are are the on a heparin of the and not in a analysis of the of and of SDF-1α to was with SDF-1α binding was in the of The the and the are are the on a heparin of the and not of that have been heparin to heparin to were for to with heparin for binding to shown in efficient competition was a fragment of monosaccharide units was a for oligosaccharides, the was and competition was of at investigate the also SDF-1α was with of heparitinase-resistant oligosaccharides, and the was used to the The fragment to bind to was an at a first in binding was with and and a was for more monosaccharide units which sulfate groups were in the binding with SDF-1α, a competition a of desulfated was in competition was and heparin, that and groups were involved in SDF-1α was a by not heparin to bind to heparin with heparin, that this position was In of the of SDF-1α the first N-terminal the of this amino acid in the this the of to the binding of SDF-1α to a that the first amino acids of SDF-1α A. A. A. M. M. M. F. F. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). SDF-1α was with a of of and the were with The of SDF-1α was with The showed that to SDF-1α the binding to a of its in the complex with the to the It is that an to the in the SDF-1α dimer to with the interaction and the a also show that the binding of the of was by the the with a this a heparin was in the positively charged at the SDF-1α dimer interface. The of heparin was the with a that has been to in C. J. PubMed Scopus Google Scholar). of and the polysaccharide was together with the of the acid in the of docking In the polysaccharide the and 13 to were to the the residues. to the of in the docking was not to of and the that is more to in this in shape not the ribbon shape of the The of the binding was and the potential are in the a energy of to the electrostatic the heparin and the positively charged amino In the energetically binding the heparin on the and acid residues The energy the was not the of sulfate in the creation of a the a potential energy the and be that this binding with the with the of the crevice, was the for the the of are to the where the are by and the same the The energy is into the protein and the and and the interaction energy in a are to the where the are by and the same the The energy is into the protein and the and and the interaction energy In the to the energy binding the heparin consisting of to have the size for binding the the but with the protein The positively charged amino acids involved in with the sulfate and groups of heparin were Lys-1, Lys-24, Lys-27, and Arg-41 of and of B and Lys-43 of A. of the electrostatic were not for and B of the dimer, the of the polysaccharide to the of the The of amino and were also involved in with the heparin study of the interaction that Lys-24, Lys-27, and to a and Arg-41 the major role in binding the polysaccharide fragment of them at with the residues. in the heparin fitted with the shape of the crevice, the with the N-terminal of the SDF-1α During the energy minimization process, heparin lost the perfect ribbon shape with a that been to in C. 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Science. 1994; 266: 1395-1399Crossref PubMed Scopus (624) Google Scholar, 4Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, T.N.C. Power C.A. Proudfoot A.E.I. Trends Pharmacol. Sci. 1998; 19: 376-380Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 6Luster A.D. N. Engl. J. Med. 1998; 7: 436-445Crossref Scopus (3219) Google Scholar, 7Gale L.M. McColl S.R. BioEssays. 1999; 21: 17-28Crossref PubMed Scopus (113) Google Scholar) are the in a The (1Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2083) Google Scholar, 2Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Greaves D.R. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1671) Google Scholar, 3Kelner M.P. Kennedy J. Bacon K.B. Kleyensteuber S. Largaespada D.A. Jenjins N.A. Copeland N.G. Bazan J.F. Moore K.W. Schall T.J. Zlotnik A. Science. 1994; 266: 1395-1399Crossref PubMed Scopus (624) Google Scholar, 4Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, T.N.C. Power C.A. Proudfoot A.E.I. Trends Pharmacol. Sci. 1998; 19: 376-380Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 6Luster A.D. N. Engl. J. Med. 1998; 7: 436-445Crossref Scopus (3219) Google Scholar, 7Gale L.M. McColl S.R. BioEssays. 1999; 21: 17-28Crossref PubMed Scopus (113) Google Scholar) are the SDF-1α, and structurally heparin were used to the on the heparin-binding consensus sequence by and A.D. PubMed Google Scholar), a first of basic amino acids residues has been shown to be involved in heparin The study that this Lys-24 and Lys-27 are the of of residues the heparin binding of also identified other amino acids that play a role in heparin These residues and Lys-43) are located in a but of them (Arg-41 and Lys-43) together with Lys-24 and Lys-27 to a basic electrostatic calculations show that are to a on the SDF-1α residues in a into a domain to a heparin-binding have been in other W. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and W. C. J. Immunol. 1999; Google Scholar). It is not well of the four SDF-1α amino acids that the basic surface of them and are essential for the other are in the of basic residues for heparin binding have been for in which, of the basic residues that are clustered into a basic was found to be critical a role and and the and not to participate in the interaction W. C. J. Immunol. 1999; Google Scholar). It is that in of the Lys-24 and Lys-27, the critical more with heparin Arg-41 and but more structural will be required to the heparin-binding amino acids are located to the in the SDF-1α with the GRID showed that the most energetically domain for binding groups is located at the and the binding site to be by the of the has been that has for heparin to bind the at the of this chemokine binding to heparin to D. D. J. 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Finally, also that and are involved in with heparin contribute to the interaction. this has not been is that have been found in other in which and are in the interaction M. J. T.J. J. PubMed Scopus Google Scholar). of was also to on the heparin structure required for binding to show that a to was required to with the and in this to the to the heparin-binding site on the SDF-1α dimer, the N-terminal residues. of 2-O- groups in heparin the binding to that groups contribute to the of has In the binding of heparin to required of sulfate and a sequence of monosaccharide units D. D. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of units for SDF-1α binding to be are with for SDF-1α with the of sequence together with the study us to of the are to F. for the and the of and to M. for the of oligosaccharides, and to J. for the of desulfated