Structural specificity of heparin binding in the fibroblast growth factor family of proteins

Abstract

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) mediate a wide variety of complex biological processes by specifically binding proteins and modulating their biological activity. One of the best studied model systems for protein-HSGAG interactions is the fibroblast growth factor (FGF) family of molecules, and recent observations have demonstrated that the specificity of a given FGF ligand binding to its cognate receptor (FGFR) is mediated by distinct tissue-specific HSGAG sequences. Although it has been known that sulfate and carboxylate groups in the HSGAG chain play a key role by interacting with basic residues on the proteins, there is little understanding of how these ionic interactions provide the necessary specificity for protein binding. In this study, using all of the available crystal structures of different FGFs and FGF-HSGAG complexes, we show that in addition to the ionic interactions, optimal van der Waals contact between the HSGAG oligosaccharide and the protein is also very important in influencing the specificity of FGF-HSGAG interactions. Although the overall helical structure is maintained in the FGF-bound HSGAG compared with unbound HSGAG, we observe distinct changes in the backbone torsion angles of the oligosaccharide chain induced upon protein binding. These changes result in local deviations in the helical axis that provide optimal ionic and van der Waals contact with the protein. A specific conformation and topological arrangement of the HSGAG-binding loops of FGF, on the other hand, impose structural constraints that induce the local deviations in the HSGAG structure, thereby enabling maximum contact between HSGAG and the protein.

Figure 1

FGF–HSGAG structural complex. (A) Chemical structure of HSGAG oligosaccharide observed in all of the FGF–HSGAG cocrystal structures. The units are numbered from nonreducing to reducing end. The glycosidic torsion angles are p_ij (C_2j-C_1j-O_j-C_4j) and s_ij (C_1j-O_j-C_4j-C_3j), where i = 1 for an H-I linkage and 2 for an I-H linkage, and j is the monosaccharide number. (B) Orientation and chain direction of the HSGAG oligosaccharide relative to sheets S1, S2, and S3 (red) of a β-trefoil scaffold of FGF whose Cα trace is such that the pseudoaxis of threefold symmetry is roughly perpendicular to the plane of the paper. The orientation of the oligosaccharide (indicated by green circle) is at a slight angle to the plane of the paper between S1 and S3. The oligosaccharide interacts with the basic residues (blue) in the loop regions (in gray, numbered 1–3). The observed chain directions are such that either the nonreducing or reducing end is projecting out of the plane of the paper.

Figure 2

Kink in FGF-bound oligosaccharides. The direction of the chains shown in A and B is from nonreducing end to reducing end (left to right). (A) Superimposition of FGF-1-bound (green) and modeled unbound oligosaccharide (on the left) and FGF-2-bound (blue) and modeled unbound oligosaccharide (on the right). The ring atoms and the glycosidic oxygens (O1 and O4) of H3 (Fig. 1A) in bound and unbound oligosaccharide structures were superimposed. H3-I4-H5 (Fig. 1A) of FGF-1-bound oligosaccharide and H1-I2-H3 (Fig. 1A) of FGF-2-bound oligosaccharide constitute the trisaccharide-spanning kink. (B) Stepwise formation (top to bottom) of the kink and continuation of helical structure caused by changes in p_1j and s_1j (according to the boldface values in Table 1) of the oligosaccharide (blue) relative to the unbound oligosaccharide (red).

Figure 3

Distribution and orientation of sulfates in FGF-1- and FGF-2-bound oligosaccharides. (A) Designation of sulfates, where the chain direction is nonreducing end (left) to reducing end (right). (B–E) Projection of the sulfates down the helical axis represented on a helical wheel (Upper) and the axial distances between the sulfur atoms mapped on the helix axis (Lower). (B and D) Modeled unbound oligosaccharide structures with ring conformations corresponding to those observed in FGF-1 (25) and -2 (26) cocrystal structures, respectively. (C and E) Bound oligosaccharide structures obtained from the FGF-1 and -2 cocrystal structures, respectively. Note that in C the 2S2 is closer to the 6S2 (Upper) and there is a significant increase in the 6S1–NS2 axial distance and a decrease in the NS2–2S2 axial distance (Lower), compared with the unbound structure shown in B. Note that in E the 6S1 is closer to the NS2 (Upper), there is interchange of the 2S1 and NS1 (Upper), and there is a decrease in the NS2–2S2 axial distance and an increase in the 2S1–6S1 axial distance (Lower), compared with the unbound structure shown in D.

Figure 4

HSGAG-binding site in FGF-2 facilitates interaction with both oligosaccharide chain directions. The direction of the oligosaccharide chain (yellow) is fixed from nonreducing to reducing end (see arrow). The topological arrangement of the HSGAG-binding loops in FGF-2 (shown as a ribbon trace in gray) relative to oligosaccharide for both the chain directions, one of which is observed in FGF-2–HSGAG cocrystal structure as shown in A and the other in FGF-2–FGFR1–HSGAG cocrystal structure as shown in B. The critical amino acids are shown numbered according to their sequence in FGF-2 (K and R, colored in blue; Q and N, colored in purple). The kink in the oligosaccharide is induced in both modes of binding (toward the nonreducing end in A and the reducing end in B). The narrow pocket formed by loop2 and loop3, containing the critical K125 residue at the base of the pocket, causes the oligosaccharide chain to kink to provide optimal ionic and van der Waals contacts. K125, Q134, and N27 interact with the critical N-sulfate and 2-O-sulfate groups of the trisaccharide-spanning kink region in both of the chain directions. The other basic residues are distributed on the periphery of the binding site such that they can interact with the oligosaccharide chain in either direction.

Figure 5

Spatial distribution of basic amino acids in FGF. Shows Connolly surface representation of FGF-1 (purple), -2 (cyan), -4 (gray), -7 (green), and -9 (orange) with the basic residues shown in blue. Note that there is a significant difference in the spatial arrangement of the basic residues between the FGFs.