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Interactions That Influence the Binding of Synthetic Heparan Sulfate Based Disaccharides to Fibroblast Growth Factor‑2 Yi-Ching Li,† I-Hsin Ho,‡ Chiao-Chu Ku,§ Yong-Qing Zhong,‡ Yu-Peng Hu,‡ Zhi-Geng Chen,‡ Chun-Yen Chen,‡ Wei-Chen Lin,‡ Medel Manuel L. Zulueta,‡ Shang-Cheng Hung,*,‡ Min-Guan Lin,†,¶ Cheng-Chung Wang,§ and Chwan-Deng Hsiao*,† †

Institute of Molecular Biology, ‡Genomics Research Center, and §Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan ¶ Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan S Supporting Information *

ABSTRACT: Heparan sulfate (HS) is a linear sulfated polysaccharide that mediates protein activities at the cell−extracellular interface. Its interactions with proteins depend on the complex patterns of sulfonations and sugar residues. Previously, we synthesized all 48 potential disaccharides found in HS and used them for affinity screening and X-ray structural analysis with fibroblast growth factor-1 (FGF1). Herein, we evaluated the affinities of the same sugars against FGF2 and determined the crystal structures of FGF2 in complex with three disaccharides carrying N-sulfonated glucosamine and 2-O-sulfonated iduronic acid as basic backbones. The crystal structures show that water molecules mediate different interactions between the 3-Osulfonate group and Lys125. Moreover, the 6-O-sulfonate group forms intermolecular interactions with another FGF2 unit apart from the main binding site. These findings suggest that the water-mediated interactions and the intermolecular interactions influence the binding affinity of different disaccharides with FGF2, correlating with their respective dissociation constants in solution. ith 18 known members, the fibroblast growth factor (FGF) family plays a role in a variety of critical biological processes such as cell proliferation, survival, differentiation, cell migration, morphogenesis, and angiogenesis.1 FGFs bind to tyrosine kinase receptors and heparan sulfate (HS), a sugar component of proteoglycans widespread on cell surfaces and extracellular matrices. The interaction of FGFs with HS results in the formation and stabilization of oligomers. HS protects FGFs from inactivation and facilitates FGF receptor binding, leading to signaling and biological responses. Among the FGF family, FGF1 and FGF2 are most extensively investigated. Heparan sulfate is a highly sulfated linear polysaccharide with alternating 1 → 4-linked α-D-glucosamine (GlcN) and β-Dglucuronic acid (GlcA) or α-L-iduronic acid (IdoA). Its biosynthesis is a complex multistep process facilitated by membrane-bound enzymes in the endoplasmic reticulum and the Golgi apparatus.2,3 On assembly, the sugar backbone plainly involves N-acetyl D-glucosamine (GlcNAc) and GlcA disaccharide repeats. However, irregular modifications, such as Ndeacetylation, epimerization, and multiple N-sulfonations and O-sulfonations, transform the relatively simple structure into a complex polymer with 48 theoretical possibilities for the repeating disaccharide. Varying distributions of these disaccharide possibilities occur, but not all of them exist in Nature.3 Different combinations of these disaccharides complicate the detailed understanding of HS function. Commercial heparin, the structural analogue of HS mainly

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© XXXX American Chemical Society

obtained in mastocytes, is occasionally used as HS proxy in various biological studies.4 However, heparin is not homogeneous, and the data acquired from these studies do not fully clarify the HS motif responsible for function. Thus, chemical synthesis is often tapped to acquire well-defined materials for effective structure−activity relationship studies.5−12 Numerous HS-binding proteins recognize specific modification patterns along the sugar chain. Insights on the molecular details of these interactions have important biomedical significance. A classic example is the binding of antithrombin with a specific 3-O-sulfonated pentasaccharide motif in heparin, culminating in the inhibition of various proteins in the coagulation cascade.4 Conversely, a 3-O-sulfonated octasaccharide was found important in the attachment and entry of herpes simplex virus-1 to host cells.13 For FGF2, past studies showed that N-sulfonated GlcN (GlcNS) and 2-O-sulfonated IdoA (IdoA2S) are essential for HS interaction, while 6-Osulfonate groups are required for FGF2 receptor binding.14 Backed by NMR data, a tetrasaccharide containing the GlcNS and IdoA2S units was recently proposed as the minimal structural requirement for FGF2 binding.15 Previous FGF2 cocrystal structures utilized extended sugar sequences containing 6-O-sulfonated GlcNS and IdoA2S,16−18 precluding Received: February 7, 2014 Accepted: June 24, 2014

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dx.doi.org/10.1021/cb500298q | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

Letters

Table 1. Dissociation Constants of the Disaccharides That Bind to FGF1 and FGF2 Determined through ITC

a

Measured as association constant, the inverse of KD. bMeans of duplicate experiments with standard deviations indicated in parentheses. The full set of thermodynamic parameters is provided in Supplementary Table 1.

Table 2. X-ray Diffraction Data and Structure Refinement Statistics FGF2−S3I2

FGF2−S6I2

FGF2−S9I2

C2

P1

P1

84.4 58.1 32.4 90.0 109.1 90.0 30.0−1.5 171180 23559 7.3 (7.3) 99.8 (99.3) 45.4 (9.8) 6.2 (29.3)

30.4 33.1 35.6 65.5 72.6 77.4 30.0−1.8 40957 10466 3.9 (3.9) 94.2 (91.0) 36.2 (6.7) 4.6 (27.4)

30.4 33.0 35.6 65.5 71.8 77.0 30.0−1.6 57908 15020 3.9 (3.9) 95.4 (95.5) 46.3 (19.1) 3.4 (7.2)

21.2−1.5 16.33/18.54

24.3−1.8 16.91/22.01

19.6−1.6 17.10/21.50

1105 107

1046 95

1047 104

0.005 1.104

0.006 1.197

0.006 1.111

12.0 11.2 20.0

22.2 21.5 30.2

21.0 20.1 30.3

Data Collection space group unit cell parameters a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) resolution range (Å) no. of reflections collected no. of unique reflections redundancy of reflection completeness (%) overall I/σ(I) overall Rmerge (%)b overall

Refinement resolution range (Å) R-factorc/Rfree (%)d no. of atoms protein water RMSD bonds (Å2) angles (deg) average B-factor (Å2) all atoms protein water

a Values in the parentheses are for the highest resolution shell. bRmerge = Σ|I − ⟨I⟩|/ΣI, where I = observed intensity, and ⟨I⟩ = average intensity from multiple observations of symmetry related reflections. cR = P|Fo − Fc|/PFo, where Fo and Fc are observed and calculated structure factor amplitudes. d Rfree was calculated on the basis of 10% of the total number of reflections randomly omitted from the refinement.

B



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Figure 1. Heparan sulfate disaccharide interaction with FGF2. (a) Electrostatic potential mapped onto the molecular surface of FGF2 with a large patch of positive electrostatic potential distinguishing the HS binding site (S9I2 is shown in pink). (b) Crystal stereo image of the interactions between FGF2 and S9I2. S9I2 is shown in pink, and the surrounding FGF2 is shown in orange. Residues and water molecules mediating interactions are highlighted as atomic stick figures and red spheres. Atoms are color-coded by element (O, red; S, yellow; N, blue), and interactions are shown as dashed yellow lines. (c) Binding modes of the three disaccharides with Lys125 of FGF2. The distances of the interactions are shown as the number near the dashed lines. (d) Binding mode of S6I2 with Lys118 of FGF1.20

to those found binding with FGF1.20 However, in this case, S0I2 did not bind to FGF2 as it did with FGF1. S3I2 exhibited a dissociation constant (KD) of 1.73 μM, the highest affinity for FGF2 among the tested sugars. Previously, ITC measurements showed that FGF1 associated with these sugars exhibiting KD values ranging from 4.13 to 21.2 μM. Compared to FGF1− S6I2, the FGF2−S612 tandem (KD = 17.5 μM) displayed a slightly higher affinity. The affinity of FGF2 with S9I2 is about 2-fold higher than that with S6I2 but is about 5-fold lower than that with S3I2 despite being more anionic and holding the same 3-O-sulfonate group. The FGF2−S3I2 complex structure was determined at 1.5 Å by X-ray crystallography using the water-deleted and hexasaccharide-removed FGF2 (PDB code: 1BFC) as template for phase determination of FGF2 in the complex (Table 2). One FGF2 and one S3I2 were found in the asymmetric unit belonging to space group C2. The cocrystal structures of FGF2 with S6I2 and S9I2 were solved and refined at 1.8 and 1.6 Å, respectively. Despite both being in the distinct space group P1, one FGF2 molecule and one disaccharide are also present in the asymmetric unit. The three disaccharides are each bound to FGF2 in the same electropositive pocket of conserved sequence as observed in FGF1 (Figure 1, panel a). The primary

investigations of other functional group combinations. Evidently, not only the number but also the positions of individual sulfonate groups determine the affinity of HS to FGFs. 19 Recently, all 48 potential disaccharides were synthesized and utilized for affinity screening with FGF1.20 Isothermal titration calorimetry (ITC) and X-ray analysis of the sugar−protein cocrystals showed that only four disaccharides, sharing the GlcNS−IdoA2S structure, bound FGF1. An additional 3-O-sulfonate group in GlcN improved affinity to FGF1, whereas the 6-O-sulfonate group did not participate in the encounter. In continuation of our efforts in defining the molecular details of FGF−HS interaction, we herein moved toward FGF2 by conducting ITC screening of the previously utilized 48 disaccharides to identify the protein binders and further initiating crystallographic analysis of the FGF2disaccharide system. In this paper, we adopted the naming scheme derived from the glycosaminoglycan structure code proposed by Lawrence et al.21 to simplify the disaccharide representation. Among the 48 disaccharides examined using ITC, only three sugars displayed substantial binding capability with FGF2 (Table 1, Supplementary Figure 1, Supplementary Table 1). These compounds share the GlcNS−IdoA2S structure, similar C



Letters

disaccharide binding site on FGF2 is formed by Asn27 and by the surface loop that consists of residues 119−135. The set of basic and polar residues in the binding site includes Asn27, Arg120, Lys125, Gln134, and Lys135 (Figure 1, panel b, Supplementary Table 2). These residues are the same or analogous to those observed in previous FGF2 complex structures with tetrasaccharide and hexasaccharide.17 The large patch of positive electrostatic potential surrounding the binding site corresponds to the area that interacts with the extended HS chain in the crystal structures with longer sugars. As anticipated from their structural similarities, the disaccharides made identical contacts with the protein, with the bulk of the interactions being sulfonate-mediated. Overall, the three disaccharides herein represent the shortest set of sugars based on HS that successfully bound FGF2 (Supplementary Figure 2). The 2-O-sulfonate of IdoA formed salt bridges with the side chain of Asn27, Lys125, and Q134 and hydrogen bonds with the main chain of Lys135 and Ala136. Analogous interactions occurred between the N-sulfonate group of GlcN and the side chains of Asn27 and Lys125 and the main chain of Arg120 (Figure 1, panel b). The 3-O-sulfonate group in S3I2 displayed electrostatic interaction with the side chain of Lys125 (Figure 1, panel c). Identical to that seen in FGF1, the 6-O-sulfonate and the carboxylate groups did not establish any contact with the main binding site. Moreover, we found one water molecule, conserved in both FGF2−S3I2 and FGF2−S9I2 complexes, bridging the N-sulfonate and the 3-O-sulfonate groups. An identical water-mediated interaction was observed between the N-sulfonate group and the 3-hydroxyl of GlcN in S6I2. This water molecule likely assists in stabilizing the disaccharides upon binding to FGF2. The direct interaction of the 3-O-sulfonate group with the Lys125 side chain (Figure 1, panel c) is surmised as the basis for the strong affinity of S3I2 with FGF2. While S9I2 adopted a similar orientation as S3I2, a water molecule was observed between the 3-O-sulfonate group and Lys125, indicating a water-mediated interaction. This behavior should account for the lower binding affinity of S9I2 compared to S3I2. In the FGF2−S6I2 complex, one water molecule, which bridges the 3hydroxyl of GlcN and Lys125, was also observed. The water molecule was located close to the position occupied by the oxygen atom of the 3-O-sulfonate group in S3I2, which may partially compensate for the function of this group. This watermediated interaction with S6I2 closely resembles that which involved the 3-O-sulfonate group in S9I2, resulting in comparable but still noticeably lower affinity. Comparison of the FGF1−S6I2 and FGF2−S6I2 cocrystal structures revealed a disparity in water molecules present in the sugar-binding regions (Figure 1, panels c and d). As already described, FGF2−S6I2 contains two tightly bound water molecules mediating interactions with the 3-hydroxyl of GlcN. However, no corresponding water molecule exists in the FGF1−S6I2 complex. These findings explain the slightly reduced affinity of S6I2 to FGF1 as compared to FGF2. An overlay of FGF2 in complex with the three disaccharides revealed no significant movement of the protein backbone and side chains in the binding site. Interestingly, differences were detected on S6I2 and S9I2, which are slanted at approximately 10° as compared to S3I2 (Figure 2, panel a). It should be noted that the 3-O-sulfonate of S3I2 is ion-paired to the side chain of Lys125 at a distance of 2.9 Å. However, analysis of the FGF2− S9I2 complex structure showed a significant slant of the bound

Figure 2. Notable details of the crystal structures. (a) Superposition of binding sites of FGF2 in complex with S3I2 (green), S6I2 (blue), and S9I2 (pink) showing a 45° rotation and 2.6 Å translation of the 3-Osulfonate of S9I2 compared with that of S3I2. (b) Close-up view of crystal packing observed in FGF2−S912.

disaccharide. Here, the 3-O-sulfonate of S9I2 exhibited a 45° rotation and a 2.6 Å translation compared to S3I2 as clearly defined by the electron density. The crystal packings of FGF2−S6I2 and FGF2−S9I2 complexes are distinct from that of FGF2−S3I2. Such difference probably resulted from the intermolecular interactions mediated by the 6-O-sulfonate of GlcN and the carboxylate of IdoA with another protein−sugar unit in the crystal. In both FGF2 complexes with S6I2 and S9I2, one water molecule bridged the 6-O-sulfonate of GlcN to the oxygen atom of Asp99 in another FGF2 unit. The same FGF2 molecule interacts with the carboxylate group of IdoA through Arg118 (Figure 2, panel b). S3I2, lacking the 6-O-sulfonate group, cannot mediate this unique assembly. Thus, the likely determinant for crystal packing is the presence or absence of the 6-O-sulfonate group. Although the overall complex structure of FGF2−S9I2 is generally comparable to that of FGF2−S3I2, the 6-O-sulfonate group forms intermolecular interactions with a separate FGF2, causing the 3-O-sulfonate of S9I2 to rotate away from Lys125. Consequently, a water molecule occupies the space between the 3-O-sulfonate group and Lys125 in FGF2−S9I2 complex. This ability for dual interaction of the 6-O-sulfonated disaccharides also accounts for the lower FGF2 binding affinity of S9I2 compared to S3I2. It is therefore evident that both S6I2 and S9I2 are capable of bridging two FGF2 molecules in trans-orientation22 in solution with the other FGF2 unit being less tightly bound. The present work showed that FGF2 possesses the means to associate directly or indirectly with the 3-O-sulfonate group if ever it is present in an HS sequence containing GlcNS and IdoA2S as core residues. Such interaction increases the affinity FGF2 to HS, which may be beneficial particularly in the initial stages of the encounter. Because the structural flexibility of a disaccharide differs from extended HS chains, it remains unclear whether the observed affinity shifts could be extrapolated to D



Letters

Structural Determination and Refinement. The water-deleted and hexasaccharide-removed FGF2 structure (PDB code: 1BFC) was used as a starting model for molecular replacement software AutoMR from PHENIX.27,28 Coot29 was used to adjust the FGF2-disaccharide models. Following initial model refinements by PHENIX, the unbiased electron density corresponding to the bound disaccharides were readily recognized in the resulting Fo−Fc map. COOT and PHENIX were used for subsequent rounds of manual model rebuilding and refinement. The data collection and refinement statistics are summarized in Table 2. The Ramachandran plots30 for these structures did not violate accepted backbone torsion angles.

longer chains. Nevertheless, the associations revealed by the FGF2-disaccharide cocrystals could guide the design of FGF2 inhibitors. HS oligomers form helices wherein the repeating disaccharides orient at opposite sides of one another in a sequence.3 This arrangement allows for attachment of separate FGF2 molecules at adjacent HS binding sites containing the sugars structures represented by the three disaccharides identified here and ultimately provides the FGF2 dimer required for receptor activation. Conversely, the weak interactions formed by Asp99 and Arg118 of FGF2 and the carboxylate and 6-O-sulfonate groups of HS may assist in the translocation of FGF2 from one HS binding site to another.23 In conclusion, we generated and evaluated the individual cocrystals of FGF2 and three disaccharides identified through ITC from a set of 48 synthetic HS-based disaccharides. Comparisons between the cocrystal structures revealed differences in the interaction potential of the 3-O-sulfonate and 6-Osulfonate groups that correlated with the KD values calculated from ITC. The affinities of S6I2 and S9I2 to FGF2 were also explained by the ability of the sugars in bridging two FGF2 units and the water-mediated interactions involved therein. The presence of the 6-O-sulfonate group, in these cases, remarkably reduces the contribution of the 3-O-sulfonate group in enhancing the binding affinity. Such structural binding details can have implications in future designs of materials intended to bind FGF2.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Coordinates and structure factors of the FGF2−S3I2, FGF2− S6I2, and FGF2−S9I2 structures have been deposited in the PDB under accession numbers 4OEE, 4OEF, and 4OEG, respectively.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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METHODS

ACKNOWLEDGMENTS We are grateful for the access to the synchrotron radiation beamlines at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan. This work was supported by research grants from Academia Sinica and the National Science Council (NSC101-2311-B-001-023-MY3 for C.-D.H. and NSC 100-2113-M-001-019-MY3 and NSC 101-2628-M-001-006MY3 for S.-C.H.), Taiwan.

Protein Expression and Purification. The recombinant human double mutant FGF2 gene has Cys69 and Cys87 mutated to serine residues for higher yield.24 This gene was cloned in pET-32a expression vector and transformed into the BL21 (RIPL) strains, which were cultured in LB medium supplemented with 100 μg mL−1 ampicillin. When light absorption reaches 0.6 at 550 nm, protein expression was induced with 0.6 mM isopropyl 1-thio-β-D-galactopyranoside at 22 °C overnight. The cells collected by centrifugation were then disintegrated using a French press, and the fusion protein was subsequently purified in a nickel affinity column. Dialysis against a solution containing 50 mM Tris-HCl buffer (pH 7.8), 50 mM NaCl, 2 mM CaCl2, and 1 mM dithiothreitol and cleavage by the catalytic subunit of human recombinant enterokinase at 20 °C for 42 h supplied the FGF2 fragment, which was purified in a heparin affinity column. The FGF2 fractions were further passed through a HisTrap FF column to obtain the pure protein. The purified FGF2 was dialyzed against 20 mM Tris (pH 7.6) with 100 mM NaCl and concentrated to levels suitable for ITC and cocrystallizations. The concentration of FGF2 was determined spectrophotometrically at 280 nm. ITC Measurement. All ITC experiments were carried out at 25 °C using Tris buffer (20 mM Tris, 100 mM NaCl, pH 7.6) as solvent. The FGF2 solution (50−80 μM) was placed in the calorimeter cell and titrated with the sugar solution (3 mM, 2 μL injections with 3 min spacing). To account for the heat of the dilution, the buffer without the protein was also titrated with the sugar solution, and the generated data were subtracted from the results of the protein titration. The titration isotherms were fitted to an equation modeling the interaction (One Sites) to generate the fitting parameters. Crystallization and X-ray Data Collection. FGF2 (10 mg mL−1) was separately mixed with S3I2, S6I2, and S9I2 at a 1:2 molar ratio in 20 mM Tris-HCl buffer containing 100 mM NaCl at pH 7.6. The hanging-drop vapor diffusion method25 was used to set up crystallization trials. All cocrystals were obtained at RT by mixing equal amounts of protein complex solution and a reservoir solution consisting of 40% poly(ethylene glycol) 600 and 100 mM Na2HPO4/ citric acid (pH 4.2). X-ray diffraction data sets were collected on beamline BL13C1 and BL15A1 (National Synchrotron Radiation Research Center, Taiwan) and processed by using HKL2000.26

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