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Structure in solution of fibronectin type III-domain 14 reveals its synergistic heparin binding site. Xueyin Zhong, Oliver Arnolds, Oktavian Krenczyk, Jana Gajewski, Stefanie Pütz, Christian Herrmann, and Raphael Stoll Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00771 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018
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Biochemistry
Structure in solution of fibronectin type III-domain 14 reveals its synergistic heparin binding site. Xueyin Zhong1, Oliver Arnolds1, Oktavian Krenczyk2, Jana Gajewski1, Stefanie Pütz1, Christian Herrmann2, Raphael Stoll1,* 1
Ruhr-University of Bochum, Faculty of Chemistry and Biochemistry, Biomolecular NMR, Bochum, 44780, Germany Ruhr-University of Bochum, Faculty of Chemistry and Biochemistry, Physical Chemistry, Bochum, 44780, Germany * corresponding author:
[email protected] KEYWORDS: Fibronectin type III-domain 14, Heparin, HIS Tag, NMR, ITC 2
Supporting Information Placeholder ABSTRACT: Fibronectin is a large multi-domain protein of the extracellular matrix that harbors two heparin binding sites, Hep-I and Hep-II, which support the heparin-dependent adhesion of melanoma and neuroblastoma cells.1,2,3 The stronger heparin/HS binding site on fibronectin, Hep-II, spans across fibronectin type III-domains 12 to 14. Previous site-directed mutagenesis, NMR chemical shift perturbation, and crystallographic structural studies all agree in that the main heparin binding site is located on the surface of fibronectin type III domain 13.4,5 However, the ‘synergy site’ for heparin binding located on fibronectin type III domain 14 still remained elusive since actual binding sites could not be identified. Using NMR spectroscopy and ITC, we show here that heparin is able to bind to a cationic ‘cradle’ of fibronectin type III-domain 14 formed by the PRARI sequence, which is involved in the integrin ɑ4β1 interaction,5,6 and to the flexible loop comprising residues KNNQKSE between the last two beta sheets D and E of FN14. Our data reveal that the individual FN14 domain binds to the sulphated sugars Dp8 and Reviparin with similar affinities as the individual domain FN13 that contains the Hep-II site.7 Noteworthy, by introducing the last beta strand of FN13 and the linker region between type III-domains 13 and 14, the perturbation of NMR chemical shifts by heparin is significantly reduced, especially at the PRARI site. This indicates that the Hep-II binding site of fibronectin is mainly located on FN13 and the synergistic binding site on FN14 only involves the KNNQKSE sequence.
Fibronectin (FN) is a large glycoprotein that mediates a wide range of molecular interactions within the extracellular matrix (ECM) important for cellular adhesion and migration.8 FN binds to integrins, collagen, syndecan, and fibrin.8 FN also contains two major heparin-binding domains, Hep-I and HepII, that interact with heparan sulfate proteoglycans (HSPG).9 Located at the N-terminus, Hep-I contains fibronectin type Idomains 1 to 5 and exhibits weaker affinity towards heparin. In contrast, Hep-II binds heparin much stronger and has been mapped to a region spanning FN type III-domains 12-14 near the C-terminus, promoting focal adhesion formation.10 A previous study using recombinant fragments that consisted of FN type III-domains 12 to 15 mapped the main heparin-binding sites to domain 13 and a second, synergistic site on the consecutive domain 14.4 The crystal structure of FN 12-15 that
contains the Hep-II site revealed a distinctive basic patch on FN13 and suggested a possible second heparin-binding site on FN14.5 However, a later nuclear magnetic resonance (NMR)based analysis of the interaction between FN13-14 and several sulfated sugar ligands did not show any significant contribution to the affinity of FN14 for heparin and a heparin-derived pentasaccharide.11 Indeed, no NMR chemical shift perturbations were observed for FN14, neither when analyzed as part of the tandem in FN type-III domain 13-14 form nor as an individual FN14 construct. This result has been challenged by two observations. Firstly, peptides derived from the FN14 domain were reported to weakly bind to heparin. Secondly, a sulfated heparin-derived Dp (disaccharides degree of polymerization) 14 sugar, which contains seven disaccharide units of L-iduronic acid and N-acetylglucosamine, was required for the maximal affinity between Hep-II domain and heparin to be observed.2,12 Here, we report the structure in solution of the individual 9.8 kDa FN type III-domain 14 and the characterization of its interaction with Reviparin, a low molecular weight heparin (LWH) with an average molecular weight of 4.2 kDa.7 Previously, binding of the individual FN14 to sulfated pentasaccharides could not be observed.11 In contrast, our individual FN14 domain construct clearly binds to Reviparin. Mapping of the heparin-binding site on FN14 by NMR chemical shift perturbation analysis locates the heparin binding site at the cationic ‘cradle’ including the integrin ɑ4β1 binding sequence, PRARI, and the flexible loop between the last two beta sheets. This is in concordance with a previous in silico prediction of heparin binding sites on FN14 for a Dp12 sulfated oligosaccharides.13 Further, isothermal titration calorimetry (ITC) data reveal that both FN13 and FN14 bind to Dp8 as well as Reviparin with very similar affinities. Notably, the binding affinity of FN14 towards heparin is decreased when a long linker preceding the FN14 domain is present. Size exclusion chromatography Similar to FN13, heparin provides multiple binding sites for FN14 and increases the apparent molecular weight observed in size-exclusion chromatograms (Fig.1). Heparin variants used in this work are listed in Table 1.
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Table 1. Heparin variants used in this study. The molecular weight of each sulfated oligosaccharide is given in parentheses in the first column. number of saccharides
number of sulfation sites per disaccharides
Dp8 (2.4 kDa)
8
3
Reviparin (4.2 kDa)
Ø 14
3
Figure 1. Size exclusion chromatography of FN14 in the absence (shown in black) and presence (shown in red) of Reviparin. FN14 (0.15 mM) was incubated with Reviparin (0.6 mM) before loaded on a SuperdexTM 75 column, previously equilibrated with phosphate-buffered saline (PBS) without any Reviparin. Size-exclusion chromatography clearly shows that, with an average of 14 saccharides, Reviparin is indeed able to bind three individual FN type III-domains 14 (Fig. 1). The black peak in Fig. 1 corresponds to the monomeric FN14 with apparent molecular weight (MW) of 9.8 kDa. The red peak is FN14 in the presence of Reviparin at a ratio of 1:4, which yields an apparent MW of 30.5 kDa (Table 2). Superdex 75 gel filtration
Mapping the binding sites by NMR spectroscopy In order to map the heparin binding site, 15N-enriched recombinant FN14 was purified from E coli and titrated with Reviparin in stoichiometric ratios ranging from 0.2 to 2.0 whilst observing NMR chemical shift perturbations in 2D 1H15 N heteronuclear single quantum coherence (HSQC) spectra (Fig. 2).
Figure 2. Overlay of 1H-15N HSQC spectra of fibronectin typeIII domain 14 in the presence (shown in red) and absence of Reviparin (shown in black). Both spectra are plotted at the same signal-to-noise level. The amino acid resonances that experience CSP twice the standard deviation are labeled. Assignments for FNA 14 and FN 13 have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under accession numbers BMRB27609 and 27610, respectively. Resonances of amino acids that experience significant CSP upon titrating FN14 with Reviparin are highlighted in Figs. 2 and 3. When projected onto the structure of FN14 in solution reported here (Figs. S1, S2, S3, Table S1, PDB ID 6HNF), they cluster at the interface between fibronectin type-III domains 13 and 14. (Figs. 3A, B) This includes the N-terminus, the PRARI sequence, and the in silico-predicted synergistic binding site KNNQKSE, which faces the same orientation as the heparin binding site on FN III domain 13 (Figs. 3B and 4.)13
number of polysaccharides
Vol (mL)
MW (kDa)
FN14
14.9
9.8
0
FN14-Reviparin
12.5
30.5
14
Table 2. Determination of molecular weight of individual FN III domain 14 with and without Reviparin by size exclusion chromatography. The approximate molecular weight of sulfated oligosaccharides is given in parentheses in the first column.
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Biochemistry
Figure 3. (A) NMR structural stereo ensemble of FN14 and mapping of its interaction with Reviparin. NMR backbone ensemble stereo representation of FN14 single domain in solution (this study). The atomic coordinates of human FN14 have been deposited at the Protein Data Bank (www.rcsb.org) under the PDB-ID accession number 6HNF. Residues that experience CSPs twice than the SD upon titration with Reviparin are highlighted in purple (KNNQKSE) and orange (PRARI). Located on FN14, the cationic ‘cradle’ of the heparin binding site is constituted by the KNNQKSE, PRARI sequences, and the flexible loop between the last two β-sheets D and E of FN14. (B) Surface representation of the FN12-14 Hep-II domain.5 The main heparin binding site located on FN13 is shown in blue and the synergistic heparin binding site KNNQKSE located on FN14 is shown in purple. The PRARI sequence is highlighted in orange.
Figure 4. The interaction between FN14 and Reviparin studied by NMR spectroscopy. Weighted CSPs plotted versus the amino acid sequence upon titration of FN14 with Reviparin. The following amino acids exhibit CSPs twice the standard deviation (∆δ ≥ 0.05): 4Asp, 5Ala, 27Arg, 28Ala, 30Ile, 31Thr, 33Tyr, 76Leu, 77Lys, 80Gln, 81Lys, 82Ser. Isothermal titration calorimetry (ITC) studies The interaction between Reviparin and the individual fibronectin type-III domains 13 and 14 was further investigated by isothermal titration calorimetry according to previously published protocols.11 This allowed for the extraction of thermodynamic parameters, such as binding enthalpy, and affinity, and stoichiometry, of the Reviparin-fibronectin type-III 13 and 14 complexes. Briefly, heparin placed in the ITC syringe was titrated into the ITC cell containing FN13 or 14 in 20 mM acetate buffer pH 4.5 and 150 mM NaCl. Based on the ITC data, Reviparin clearly binds to the individual FN14 domain. A single binding model was applied to fit the data yielding the values given in Table 3 (Fig. 5). For all protein constructs, the histidine tag was cleaved off using TEV protease as it influences heparin binding to fibronectin (Fig. S4, Table S2).
Figure 5. Calorimetric titration of fibronectin type-III domains 14 and 13 with Reviparin as well as Dp8. The upper panel shows the heat generation per each injection of 1.5 µl of heparin from the ITC syringe titrated into the ITC cell containing 200 µl of FN protein. The integration of each injection is shown in the lower panels. The best fit according to a single binding model is depicted as a red solid line. A: 0.5 mM Reviparin solution (syringe) titrated into a solution of 0.1 mM FN type-III domain 14 (cell), shown on the left; 1 mM Dp8 solution (syringe) titrated into a solution of 0.1 mM FN type-III domain 14 (cell), shown on the right. B: 0.45 mM Reviparin (syringe) solution titrated into a solution of 0.15 mM FN type-III domain 13 (cell), shown on the left; 1 mM Dp8 solution titrated into a solution of 0.1 mM FN type-III domain 13 (cell), shown on the right. Obviously, more than one FN14 molecule can bind to the sulfated oligosaccharides chain of Reviparin because an N value between 0.3 and 0.4 is obtained for all ITC-based titrations. This can be interpreted as binding of three FN14 domains to one sulfated oligosaccharide chain, which has also been observed for single FN1311. This is corroborated results obtained from size exclusion chromatography (Fig. 1, Table 2). The shorter oligosaccharide Dp8 is also able to interact with three FN13 or FN14 protein domains, albeit with a slightly lower affinity (Fig. 5, Table 3). Noteworthy, the individual FN14 domain binds both Reviparin and Dp8 with similar affinity as FN13 does. In order to rule out any error in determining protein concentration, a control ITC titration using the tandem fibronectin type-III domain 13-14 and Dp8 was carried under the same experimental conditions as described above. In concordance with previously published data, the tandem domain 13 to14 binds to LMW heparin (3K) in a 1:1 stoichiometric ratio (Fig. 6). However, the affinity for dp8 is lower than for the LMW heparin.11 With an average size of 14 saccharides, Reviparin is still able to bind three tandem FN13-14
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domains, preventing a detailed analysis by NMR spectroscopy due to excessive line broadening of resonances (Fig. 6, Table 3).
N
Ka [M-1]
ΔH [cal/mol]
FN14 Reviparin
0.39 0.01
±
5.70·104 ± 2.91·103
-9600 ± 300
FN14 Dp8
0.43 0.06
±
1.3·104 1·103
±
-6500 ± 1100
FN13 Reviparin
0.30 0.02
±
2.5·104 2 ·103
±
-1.5·104 ± 1200
FN13 Dp8
0.44 0.03
±
2.3·104 2·103
±
-5100± 400
FN1314 Reviparin
0.35 0.01
±
8.4·104 6·103
±
-8000 ± 200
FN1314 Dp8
1.00 0.02
±
2.2·104 1 ·103
±
-3200 ± 100
Table 3. Summary of ITC data on the interaction between various fibronectin type-III domains (individual FN13 and FN14 as well as tandem FN13-14) and sulfated oligosaccharides (Reviparin and Dp8).
Figure 6. Calorimetric titration of FN type-III domain 13-14 (0.1 mM) with Reviparin (0.5 mM) and Dp8 (1 mM) in 20 mM acetate buffer containing 150 mM NaCl at pH 4.5. The upper panel shows the heat generation per each injection of 1.5 µl of heparin from the ITC syringe titrated into the ITC cell containing 200 µl of protein. The integration of each injection is shown in the lower panel. The best fit according to a single binding model is depicted as a red solid line.
Elongation of the FN14 N-terminus influences heparin binding The location of the mapped heparin binding site on the protein surface of the individual FN14 domain immediately suggests that heparin binding would be interrupted by the presence of the linker sequence located between FN13 and FN14 (Fig. 3). A previous NMR study could not detect binding of pentasaccharides to FN14 neither as an individual nor as part of a tandem FN13-14 construct.11 Presumably, the longer N-terminus of the single FN14 construct used in this study prevented the formation of an FN14heparin complex. In order to prove this theory, we cloned a single domain FN14 construct with an N-terminal elongation that contains the last beta strand of FN13. Applying the same experimental conditions, this N-terminal elongated version of FN14 was titrated with increasing stoichiometric amounts of Reviparin ranging from 0.2 to a twofold molar excess and the weighted CSP was plotted versus the FN14 amino acid sequence (Fig. 7). The N-terminal elongation of FN14 domain significantly reduces the chemical shift perturbation in 2D 1H-15N HSQC spectra of FN14 induced by heparin (Fig. 7). This holds especially true for PRARI-site of FN14, for which CSP is almost completely abolished (Fig. 4, 7). In sharp contrast, however, the KNNQKSE-site still remains accessible for Reviparin and may still contribute heparin-binding of full-length fibronectin. This corroborates previous biochemical studies that identified FN14 to be also required for full heparin-binding capacity of FN131,14. Notably, the histidine tag can contribute significantly to the binding of Reviparin at pH 4.5, as judged by ITC analysis and should thus be removed prior to interaction studies (Fig. S4, Table S2). As the interaction between heparin and proteins, such as fibronectin, is mainly electrostatic and as thus based on Coulomb forces, the cluster of six positively charged histidine side chains at the N-termini of FN13 and FN14 are apparently indeed able to bind to Reviparin (Fig. S4, Table S2).
Figure 7. Elongated FN14 titrated with a twofold stoichiometric excess of Reviparin. Weighted CSPs are plotted versus the amino acid sequence. In conclusion, by using NMR and ITC, we could identify a heparin binding site on single domain FN14 formed by the PRARI sequence, which is important for its interaction with integrin ɑ4β1, and the flexible loop comprising residues KNNQKSE between the last two beta sheets D and E of FN14. Surprisingly, individual FN14 binds to the sulfated sugars Dp8 and Reviparin with similar affinities as the individual domain FN13. However, in the pres-
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Biochemistry ence of the linker region between type III-domains 13 and 14, the interaction between FN14 and heparin is significantly reduced, especially at the PRARI site. This indicates that the Hep-II binding site of fibronectin is mainly located on FN13 and the synergistic binding site on FN14 only involves the KNNQKSE sequence. Nonetheless, the FN14 KNNQKSE-site, which is in a cis-position to the heparin binding site on FN13 still remains accessible for heparin or any other sulfated oligosaccharides. Hence, FN14 might be able to contribute to heparin-binding of full-length fibronectin in the extracellular matrix that contains sulfated sugars of sufficient length to bridge the heparin binding sites in FN13 and FN14. Therefore, the extended interaction between FN13, FN14, and heparin might indeed contribute to scaffolding multiprotein-heparin complexes. As the KNNQKSE sequence is located opposite to the PRARI sequence element, (synergistic) binding of both heparin and integrin ɑ4β1 to fibronectin might indeed be possible to form a ternary complex under specific physiological conditions.5
ABBREVIATIONS Hep-I and Hep-II, heparin binding domain 1 and 2; FN13, Fibronectin type III domain 13; FN14, Fibronectin type III domain 14; Dp8: disaccharides degree of polymerization 8; SD, standard deviation.
REFERENCES (1) Barkalow, F. J. B., and Schwarzbauer, J. E. (1991) Localization of the major heparin-binding site in fibronectin. J. Biol. Chem. 266, 7812–7818. (2) McCarthy, J. B., Chelberg, M. K., Mickelson, D. J., and Furcht, L. T. (1988) Localization and Chemical Synthesis of Fibronectin Peptides with Melanoma Adhesion and Heparin Binding Activities. Biochemistry 27, 1380–1388. (3) Drake, S. L., Varnum, J., Mayo, K. H., Letourneau, P. C., Furcht, L. T., and McCarthy, J. B. (1993) Structural features of fibronectin synthetic peptide FN-C/HII, responsible for cell adhesion, neurite extension, and heparan sulfate binding. J. Biol. Chem. 268, 15859–15867.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website and contains a description of amino acid sequences of FN constructs used in this study, additional ITC data, NMR spectroscopic techniques, structure calculation protocols, and statistics of the FN14 NMR structure ensemble in PDF file format.
(4) Ingham, K. C., Brew, S. A., Migliorini, M. M., and Busby, T. F. (1993) Binding of Heparin by Type III Domains and Peptides from the Carboxy Terminal Hep-2 Region of Fibronectin. Biochemistry 32, 12548–12553. (5) Sharma, A., Askar, J. A., Humphries, M. J., Jones, E. Y., and Stuart, D. I. (1999) Crystal structure of a heparin-and integrinbinding segment of human fibronectin. EMBO J. 18, 1468–1479.
AUTHOR INFORMATION Corresponding Author Raphael Stoll, Ruhr-University of Bochum, Faculty of Chemistry and Biochemistry, Biomolecular NMR, Bochum, 44780, Germany, phone: +492343225466, fax: +492343205466, email:
[email protected] Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.
(6) Mould, A. P., and Humphries, M. J. (1991) Identification of a novel recognition sequence for the integrin alpha4beta1 in the COOH-terminal heparin-binding domain of fibronectin. EMBO J. 10, 4089–4095. (7) Breddin, H. K. (2002) Reviparin sodium - a new low molecular weight heparin. Expert Opin. Pharmacother. 3, 173– 182. (8) Pankov, R., and Yamada, K. M. (2002) Fibronectin at a glance. J. Cell Sci. 115, 3861–3863. (9) Woods, A., and Couchman, J. R. (1994) Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component. Mol. Biol. Cell 5, 183–92.
Funding Sources German Science Foundation (DFG) and Ruhr University of Bochum (RUB) Research SchoolPlus.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We are grateful to the DFG (INST 213/757-1 FUGG) and the RUB Research SchoolPlus for generous financial support.
(10) Woods, A., McCarthy, J. B., Furcht, L. T., and Couchman, J. R. (1993) A synthetic peptide from the COOH-terminal heparinbinding domain of fibronectin promotes focal adhesion formation. Mol. Biol. Cell 4, 605–613. (11) Sachchidanand, Lequin, O., Staunton, D., Mulloy, B., Forster, M. J., Yoshida, K., and Campbell, I. D. (2002) Mapping the heparin-binding site on the 13-14 F3 fragment of fibronectin. J. Biol. Chem. 277, 50629–50635. (12) Lyon, M., Rushton, G., Askari, J. A., Humphries, M. J., and Gallagher, J. T. (2000) Elucidation of the structural features of heparan sulfate important for interaction with the Hep-2 domain of fibronectin. J. Biol. Chem. 275, 4599–4606. (13) Carpentier, M., Denys, A., Allain, F., and Vergoten, G. (2014) Molecular docking of heparin oligosaccharides with Hep-
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II heparin-binding domain of fibronectin reveals an interplay between the different positions of sulfate groups. Glycoconj. J. 31, 161–169. (14) Ingham, K. C., Brew, S. a, and Atha, D. H. (1990) Interaction of heparin with fibronectin and isolated fibronectin domains. Biochem. J. 272, 605–11.
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Biochemistry Graphic entry for the Table of Contents (TOC):
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