*3.4. GAG Structures in Solution*

The database contains the structure of heparin as established by NMR in solution (PDB entry 1HPN, 1XT3) and analogue (2ERM). Other structures have been reported for the solution structures of four different heparin oligosaccharides, determined by a combination of analytical ultracentrifugation, synchrotron X-ray solution scattering that gave the radii of gyration and maximum length extension [30,83] (PDB code 3IRI, 3IRJ, 3IRK, and 3IRL). Constrained molecular modeling of randomized heparin conformers resulted in 9–15 best-fit structures for each degree of polymerization (dp) DP18, DP24, DP30, and DP36 that indicated flexibility and the presence of short linear segments in mildly bent structures. All the conformations of the experimental conformations are somewhat scattered. They are all located in the lowest energy region of the corresponding Φ and Ψ maps (see Figure 9). The idopyranose residues experienced some changes, either <sup>1</sup>C<sup>4</sup> or <sup>2</sup>S0, without any influence on the Φ and Ψ maps. This establishes a model of heparin in solution as a semi-rigid object. *Biomolecules* **2020**, *10*, x FOR PEER REVIEW 12 of 17 any influence on the and maps. This establishes a model of heparin in solution as a semi-rigid object.

**Figure 9.** 3D representation of heparin in a helical conformation (left panel) and in disordered **Figure 9.** 3D representation of heparin in a helical conformation (left panel) and in disordered conformation. The distribution of the Φ and Ψ angles are reported on the two corresponding potential energy surfaces (drawn with SweetUnityMol [82]).

conformation. The distribution of the and angles are reported on the two corresponding potential energy surfaces (drawn with SweetUnityMol [82]). Such a computational protocol was used to model the disordered features of hyaluronic acid Such a computational protocol was used to model the disordered features of hyaluronic acid [81] and chondroitin sulfate [84]. As with heparin, the semi-rigid behavior and the stiffness of these GAGs polysaccharides could be established.

### [81] and chondroitin sulfate [84]. As with heparin, the semi-rigid behavior and the stiffness of these **4. Conclusions**

GAGs polysaccharides could be established. **4. Conclusions**  The aim of the article was to integrate three-dimensional data of GAGs, GAGs oligosaccharides as complexed with proteins. The sources of data are multiple: X-ray fiber diffraction, solution NMR, small angle X-ray scattering for GAGs, and X-ray biomolecular crystallography for protein-GAGs and protein-GAG mimetics complexes. A series of descriptors were selected to guide the search. They include cross-references to PDB, UniProtKB, MatrixDB, and GlyTouCan. GAG-DB opens the possibility of deciphering the full potential of GAGs as bioactive fragments or a structurally The aim of the article was to integrate three-dimensional data of GAGs, GAGs oligosaccharides as complexed with proteins. The sources of data are multiple: X-ray fiber diffraction, solution NMR, small angle X-ray scattering for GAGs, and X-ray biomolecular crystallography for protein-GAGs and protein-GAG mimetics complexes. A series of descriptors were selected to guide the search. They include cross-references to PDB, UniProtKB, MatrixDB, and GlyTouCan. GAG-DB opens the possibility of deciphering the full potential of GAGs as bioactive fragments or a structurally important multivalent scaffold for interaction synergy at assembling proteins within quaternary structures. The inspection of the many features of the database supports the reporting of robust facts/knowledge

amount and the quality of the 3D structures of GAG-protein complexes are amenable to comparison between the observed and the calculated 3D descriptors. Such a rich set of experimental information provides a solid basis for validating and improving computational strategies. We could confirm previously described features such as the lack of counterion effect in the interaction between GAGs; the definition of the preferred amino acids bringing the electrostatic neutrality of the interaction; and the lack of influence of sulfate groups on the glycosidic torsion angles. All the observed conformations fell within the low energy basins, thereby comforting the suitability of the computational protocol to model GAGs conformation in a disordered state. An emerging picture is the description of these polysaccharide chains as propagating linearly in a preferred direction, with and the determination of what remains to be investigated or discovered. The amount and the quality of the 3D structures of GAG-protein complexes are amenable to comparison between the observed and the calculated 3D descriptors. Such a rich set of experimental information provides a solid basis for validating and improving computational strategies. We could confirm previously described features such as the lack of counterion effect in the interaction between GAGs; the definition of the preferred amino acids bringing the electrostatic neutrality of the interaction; and the lack of influence of sulfate groups on the glycosidic torsion angles. All the observed conformations fell within the low energy basins, thereby comforting the suitability of the computational protocol to model GAGs conformation in a disordered state. An emerging picture is the description of these polysaccharide chains as propagating linearly in a preferred direction, with extended fragments separated by kinks. The semi-rigid character of the chains involves microarchitectural domains. They contain preformed conformation for optimal binding to protein targets. The separation of such domains, at a long enough distance, offers the possibility of multivalent binding to create further spatial arrangements that can induce the formation of functional assemblies of proteins.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/10/12/1660/s1, Figure S1: Detailed of the analysis of GAG binding in crystal structure of a ternary FGF1-FGFR2-Heparin complex (PDB 2GD4) available from different souces; PDBe; Swiss-Model; PLIP, LIGPOLT. Figure S2: Quaternary organisation in the crystal Structure of the Antithrombin-S195A Factor Xa-Pentasaccharide Complex (PDB 1EO0) computed and displayed by PISA.

**Author Contributions:** Methodology, S.P., O.M., and S.R.B.; software, F.B. and S.P.; data curation, S.P. and S.R.B.; writing—original draft preparation, S.P., O.M., and S.R.B; writing—review and editing, all; funding acquisition, A.I., S.P., S.R.B., and F.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research profited from Campus Rhodanien Co-Funds (http://campusrhodanien. unige-cofunds.ch. Support from ANR PIA Glyco@Alps [ANR-15-IDEX-02]; Alliance), and Labex Arcane/CBH-EUR-GS [ANR-17-EURE-0003] is acknowledged. Innogly COST Action covered open access charge. O.M thanks for partial financial support from the government assignment for FRC Kazan Scientific Center of Russian Academy of Science.

**Conflicts of Interest:** The authors declare no conflict of interest.
