# Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4

Martyna Maszota-Zieleniak, Adam Liwo, Sylvie Ricard-blum, Sergey A Samsonov

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Martyna Maszota-Zieleniak, Adam Liwo, Sylvie Ricard-blum, Sergey A Samsonov. Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4. Proteoglycan Research, 2023, 1 (3), pp.e10. 10.1002/pgr2.10. hal-05000576

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# Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4

Martyna Maszota-Zieleniak<fnref n="1" /> | Adam Liwo<fnref n="1" /> | Sylvie Ricard-Blum<fnref n="2" />
Sergey A. Samsonov<fnref n="1" />

## Correspondence

Sergey A. Samsonov, Faculty of Chemistry,
University of Gdansk, Gdansk 80-308, Poland.
Email: [sergey.samsonov@ug.edu.pl](mailto:sergey.samsonov@ug.edu.pl)

Sylvie Ricard-Blum, ICBMS, UMR 5246
University Lyon 1, CNRS, 69622 Villeurbanne
Cedex, France.
Email: [sylvie.ricard-blum@univ-lyon1.fr](mailto:sylvie.ricard-blum@univ-lyon1.fr)

## Funding information

PHC Polonium Program,
Grant/Award Number: 49131ZM; Institut
National du Cancer, Grant/Award Number:
PLBIO-258; Narodowe Centrum Nauki,
Grant/Award Number: UMO-2018/30/E/
ST4/00037

## Abstract

We have previously shown that the extracellular domains of the four syndecans are intrinsically disordered, and adopt a wide range of conformations. We report here the building of coarse-grained models of the extracellular domains of human syndecans 2 and 4 using small-angle X-ray scattering restraints. One, two or three heparan sulfate (HS) hexadecasaccharides, $(IdoA[2S]GlcNS[6S])_8$, were attached to three serine residues of the core proteins, resulting in eight variants for each syndecan that were used for all-atom molecular dynamics (MD) simulations (0.5–1 µs). Syndecan-4 had a larger conformational diversity than syndecan-2, and remained extended during MD simulations in absence of HS whereas syndecan-2 adopted more compact conformations. Their core proteins thus appeared to be structurally distinct. The HS chains also behave differently, the middle chain being more flexible in syndecan-4, and the third chain being able to interact with the core protein regions mediating cell adhesion. The cell adhesion sites on both core proteins were flexible, with or without HS chains, the NXIP motif of syndecan-2 being located in a particularly flexible region. In conclusion, the HS chains induce moderate changes in the conformational dynamics of both syndecans, depending on the number of HS chains and their location on the core protein, and on the core protein itself.

## KEYWORDS

glycosaminoglycans, heparan sulfate, intrinsically disordered proteins, models, molecular dynamics, syndecans

**Abbreviations:** CD, circular dichroism; CS, chondroitin sulfate; dp, degree of polymerization; DSSP, dictionary of secondary structure of proteins; GAG, glycosaminoglycan; H-bond, hydrogen bond; HP, heparin; HS, heparan sulfate; LIE, linear interaction energy; MD, molecular dynamics; MREMD, multiplexed replica exchange molecular dynamics simulation; NPT, constant-temperature, constant-pressure; NVT, constant-temperature, constant-volume; RMSD, root-mean-square deviation; SAXS, small-angle X-ray scattering.

Sylvie Ricard-Blum and Sergey A. Samsonov contributed equally to this study.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *Proteoglycan Research* published by Wiley Periodicals LLC.

<footnote n="1">¹Faculty of Chemistry, University of Gdansk, Gdansk, Poland</footnote>

<footnote n="2">²ICBMS, UMR 5246 University Lyon 1, CNRS, Villeurbanne, France</footnote>
## INTRODUCTION

Syndecans are transmembrane proteoglycans regulating cell adhesion and signaling, and involved in development and a variety of diseases.<sup>1-4</sup> Three heparan sulfate (HS) chains are covalently attached to the extracellular domain of the four mammalian syndecans, and chondroitin sulfate (CS) chains are also attached to syndecans 1 and 3.<sup>1</sup> We have recently shown using circular dichroism (CD), small-angle X-ray scattering (SAXS), and native mass spectrometry that the extracellular domains of the four human syndecans are intrinsically disordered, flexible and adopt a wide range of conformations including minor globular conformations and extended conformations.<sup>5</sup> These experiments were carried out with the protein moiety of the syndecans extracellular domains without their glycosaminoglycan (GAG) chains. However, it is important to determine if and how GAG chains influence the conformations of the syndecan extracellular domains to decipher the molecular mechanisms underlying the biological functions of syndecans. The few studies that have investigated the effects of GAGs on the structure of core proteins of aggrecan, decorin and biglycan have shown that they are GAG- and core protein-dependent.<sup>6,7</sup> The structural heterogeneity of the extracellular domains of syndecans makes their experimental characterization at the atomic level difficult, whereas computational approaches can describe the structural and dynamic properties of intrinsically disordered proteins. This prompted us to use molecular dynamics (MD) simulations to investigate the role of GAG chains on the flexibility, dynamics, and conformations of the extracellular domains of human syndecans 2 and 4. These syndecans were selected for this study because they have a shorter amino acid sequence than the extracellular domains of syndecans 1 and 3, and thus require shorter computational times.

We have first generated coarse-grained models of the extracellular domains of syndecans 2 and 4 based on the experimental SAXS data we collected.<sup>5</sup> The best models were then used for MD simulations in absence or presence of three HS chains to investigate their influence on the conformational diversity, flexibility, compactness and secondary structure of the core proteins of syndecans 2 and 4. Since the valency of HS chains is important for cell adhesion mediated by syndecan-4,<sup>8</sup> we have also explored the effect of the number and the location of HS chains by generating three single-chain and three two-chain variants for each syndecan (one HS chain and two HS chains attached to different serine residues on the core proteins respectively), and performed MD simulations for all these variants.

## METHODS

### Coarse-grained modeling of the extracellular domains of syndecans 2 and 4 with SAXS restraints

Molecular models of the extracellular domains of human syndecans 2 and 4 were generated with the UNRES coarse-grained approach

developed for proteins,<sup>9,10</sup> and the sequences used for SAXS experiments, including a N-terminal 6xHis tag and a C-terminal Flag tag (see below).<sup>5</sup> The serine residues serving as attachment sites of GAG chains are in bold and underlined.

Syndecan-2<sup>1</sup>GSSHHHHHHHS SGLVPRGSHM ESRAELTSDK DMYLDNSSIE EA$\S$GVYPIDD DDYASA$\S$G$G$ ADEDVESPEL TTSRPLPKIL LTSAAPKVET TTLNIQNKIP AQTKSPEETD KEKVHLSDSE RKMDPA EEDT NVYTEKHSDS LFKRTEDYKD DDDK<sup>154</sup>

Syndecan-4<sup>1</sup>GSSHHHHHHHS SGLVPRGSHM ESIRETEVID PQDILLE-GRYF $\S$GALPDDEDV VGPGQESDDF EL$\S$G$\S$GDLDD LEDSMIGPEV VHPLVPLDNH IPERAGSGSQ VPTEPKKLEE NEVIPKRISP VEESEDVSNK VSMSSTVQGS NIFERTEVLA GCPEHDYKDD DDK<sup>163</sup>

The multiplexed replica exchange MD simulations (MREMD)<sup>11</sup> were performed with restraints derived from SAXS data<sup>12</sup> at 12 temperatures (260-370 K) corresponding to 12 MREMD replicas. Each trajectory consisted of 2 × 10<sup>7</sup> MD steps, and the replicas were exchanged every 2 × 10<sup>4</sup> MD step. The relative free energy of each structure of the MREMD simulation was calculated using the Weighted Histogram Analysis Method (WHAM).<sup>13,14</sup> Then, structures corresponding to the lowest free energies were clustered,<sup>15,16</sup> and the averaged structures of each cluster were converted to all-atom models using the PULCHRA (PowerfUL CHain Restoration Algorithm),<sup>17</sup> and SCWRL (Side-Chains With a Rotamer Library)<sup>18</sup> programs for all-atom backbone and all-atom side-chain reconstruction respectively. Finally, the models were refined by MD simulations with the AMBER20 package,<sup>19</sup> the ff14SB force field<sup>20</sup> and harmonic restraints of 2 kcal/mol/$\AA^2$ applied to Cα atoms. The probability distribution (P[x]) for the distance of consecutive Cα atoms was calculated for the refined models. The models that best fitted the experimental distribution calculated from SAXS data were selected for further analyses.

### Attachment of HS chains to the models of syndecans 2 and 4

Three HS hexadecasaccharides (dp16, where dp stands for degree of polymerization) made up of the repeated trisulfated disaccharide IdoA(2S)-GlcNS(6S) were covalently attached to the selected all-atom models of the extracellular domains of syndecans 2 and 4 via a standard tetrasaccharide linker (GlcAβ(1-3)Galβ(1-3)Galβ(1-4)-Xylβ1).<sup>21</sup> The trisulfated disaccharide, more frequent in heparin (HP) than in HS, was selected because HP is a useful model compound for the sulfated NS domains of HS, which frequently interact with proteins, and both IdoA and IdoA2S residues exist in HS.<sup>22</sup> The complete glycan sequence attached to the core proteins was (IdoA(2S)-GlcNS(6S))<sub>8</sub>-GlcAβ(1-3)Galβ(1-3)Galβ(1-4)-Xylβ1. The molecular weight of the HS hexadecasaccharides was ~5 kDa, which corresponds to the shortest HS chains found in vivo (5-70 kDa<sup>23</sup>), and its size is consistent with the upper size of the sulfated NS domains of HS that ranges from dp6 to dp16.<sup>24</sup> The HS chains were termed GAG 1, GAG 2, and GAG 3 depending on the serine residue they were attached to, namely Ser41, Ser55, and Ser57 for human syndecan-2 (UniProtKB P34741) and Ser39,
Ser61, and Ser63 for human syndecan-4 (UniProtKB P31431). Each GAG chain was positioned in an arbitrary orientation relative to the core protein at the beginning of the simulation to avoid numerous initial contacts.

## MD simulations

The AMBER20 program was used to perform MD simulations.¹⁹ The GLYCAM06²⁵ and ff14SOnlysc²⁶ force fields were used for the GAG chains and the core proteins respectively. The initial structures were built in the xLeap module of AMBER20. The periodic boundary conditions were used with a TIP3P (Transferable Intermolecular Potential with three Points²⁷) octahedral water box containing approximately ~10⁵ water molecules and Na⁺ counterions for the neutralization of the system. Two steps of energy minimization were performed, first 500 cycles of steepest descent and 1000 conjugate gradient cycles with harmonic restraints of 10 kcal/mol/Å² on the solute (i.e., the syndecan core proteins), and then 3000 steepest descent and 3000 conjugate gradient cycles without restraints. Subsequently, the system was heated up to 300 K for 10 ps in the constant-temperature, constant-volume (NVT) ensemble followed by the simulations in the constant-temperature, constant-pressure (NPT) ensemble at the pressure of 10⁵ Pa until the density of the solvent converged. Finally, five independent MD production runs of 1 µs each were performed in NPT ensemble. The time integration step was 2 fs, and the cutoff for nonbonded interactions 8 Å. The SHAKE algorithm for the covalent bonds including hydrogen atoms²⁸ and the Particle Mesh Ewald procedure²⁹ were used for long-range electrostatic calculations. Each MD simulation in explicit solvent lasted about 90 days on GPU cards, mostly of Nvidia Tesla K40d architecture. Five repetitions of the MD simulations were carried out for the non-GAGosylated syndecans 2 and 4. The total MD duration differed for each trajectory (150–300 ns) depending on the time needed to reach convergence, which was assessed by the decay in root-mean-square deviation (RMSD) values. A series of MD simulations was performed with one or two HS hexadecasaccharides attached to different serine residues on the extracellular domains, which resulted in six additional variants for each syndecan, termed GAG 1, GAG 2, GAG 3, GAGs 1 + 2, GAGs 2 + 3, and GAGs 1 + 3 based on the location of the HS chain(s). Three 0.5-µs MD simulations were run for each variant.

The simulation data (i.e., coordinate trajectories and data files) were analyzed with the CPPTRAJ module of AMBER 20.³⁰ The free energy of interactions was calculated by the linear interaction energy (LIE) with a water dielectric constant of 80. The core proteins of syndecans and the HS chains were used as receptors and ligands respectively. The tetrasaccharide linker was not included as a part of the ligands in the calculation of LIE because LIE cannot be calculated for systems containing ligands covalently bound to receptors.

As implemented by default in the CPPTRAJ module, a distance cutoff of 4.0 Å for heavy atoms of the core protein and GAG chains, was applied to define contacts. The nature of the contacts (i.e., van

der Waals and electrostatic contacts) was not accounted for. The contacts corresponding to an occupancy ≥0.5 were considered for further analysis. The occupancy could be >1 when contacts were established between one amino acid residue and several GAG residues since the fractions were summed up per each amino acid residue and per GAG chain. Hydrogen bonds (H-bonds) were defined by the CPPTRAJ default values (i.e., distance donor-acceptor cutoff: 3.0 Å, angle cutoff: 135°). The fluctuations of the protein residues and the *radii* of gyration were calculated using the standard *atomicfluct* and *radgyr* commands of AMBER respectively.

## Determination of the secondary structure in the syndecan core protein models

The secondary structures were predicted using the Dictionary of Secondary Structure of Proteins DSSP based on backbone hydrogen bonds³¹ implemented in the CPPTRAJ module of AMBER 20.¹⁹

## Statistical analyses and visualization

Statistical analysis and graphical presentation of the data were performed with the R-package (RC Team, 2020). Each trajectory was visualized in VMD³² and Pymol (The PyMOL, 2019), which was also used to produce figures.

## RESULTS

### Coarse-grained modeling of the extracellular domains of syndecans 2 and 4 with SAXS restraints

Five models of the extracellular domains of syndecans 2 and 4 without their GAG chains were generated using the UNRES force field and 158 restraints on the distance distribution from SAXS data previously collected.⁵ The probability distribution (P[x]) for the distance of consecutive Ca atoms was calculated for the UNRES-generated models, and compared to the distribution calculated from experimental SAXS data to select the best models. Model 4 of the extracellular domain of syndecan-2 showed the best agreement with SAXS data. It contained two α-helices and six antiparallel β-strands, and had an elongated shape (Supporting Information: Figure S1) with a length of 175 Å, in agreement with the Dmax value of 185 Å determined by SAXS.⁵ The model of the syndecan-4 extracellular domain, which best fitted the SAXS data was model 1 (Supporting Information: Figure S2). It contained five α-helices and had a length of 175 Å, which was consistent with the Dmax value of 180 Å determined by SAXS.⁵ Models 4 and 1 of the extracellular domains of syndecans 2 and 4 respectively were then converted to all-atom models, and used for MD simulations without and with three HS chains. Only syndecan monomers were simulated with the UNRES dynamics and SAXS restraints.
## All-atom MD simulations of syndecans 2 and 4 with and without three GAG chains

All-atom MD simulations were performed on the selected models of the extracellular domains of syndecans 2 and 4 without and with three HS chains to investigate if the GAG chains induced changes in the core proteins. The RMSD values were calculated in the middle

(0.5 µs) and at the end (1 µs) of the MD simulations using the starting structures without HS chains as references. The simulated extracellular domains behave differently in some replicas, which confirmed their structural heterogeneity and their highly dynamic behavior (Figures 1 and 2). The non-GAGosylated extracellular domain of syndecan-2 exhibited a trend towards globular conformations during MD simulations (Figure 1). The RMSD values were quite large, up to

<figure><img src="image_2.png" /><figcaption>**FIGURE 1** Five molecular dynamics simulations (S1–S5) performed on the model 4 of the extracellular domain of human syndecan-2 without (left) and with three heparan sulfate (HS) hexadecasaccharides (right). Magenta: cartoon representation of the core protein, cyan: licorice representation of HS chains. The core protein and the glycosaminoglycan (GAG) chains were not drawn to scale.</figcaption></figure>
non-GAGosylated syndecan-4

GAGosylated syndecan-4

Model 1

<figure><img src="image_3.png" /><figcaption>**FIGURE 2** Five molecular dynamics simulations (S1–S5) performed on the model 1 of the extracellular domain of human syndecan-4 without (left) and with three heparan sulfate (HS) hexadecasaccharides (right). Magenta: cartoon representation of the core protein, cyan: licorice representation of HS chains. The core protein and the glycosaminoglycan (GAG) chains were not drawn to scale.</figcaption></figure>

25 Å for the non-GAGosylated form and 26.1 Å for its GAGosylated counterpart, 34% and 42% of the RMSD values were ≥20 Å in absence and presence of three attached HS chains, respectively (Supporting Information: Tables S1 and S2). This reflects a slight increase in the conformational space of the core protein of syndecan-2 sampled upon the attachment of the three HS chains. The largest changes in RSMD occurred during the second half of the simulations without HS chains (38% of the RSMD values were ≥20 Å) and the first half of the simulation with HS chains (49% of the RSMD values

were ≥20 Å). In contrast to syndecan-2, the extracellular domain of syndecan-4 remained mostly extended during the MD simulations (Figure 2). The RMSD values were larger than those calculated for syndecan-2, up to 38.3 Å for the non-GAGosylated form and 33.4 Å for the GAGosylated one. The percentages of RMSD values ≥ 20 Å were similar without and with HS chains (71% and 67% respectively) but the values ≥ 30 Å increased from 18% to 29% in the presence of the HS chains (Supporting Information: Tables S3 and S4). The three HS chains attached to the core protein of syndecan-4 appeared to
increase the heterogeneity of its most representative conformations, which might impact the accessibility of its biologically active motifs.

In most simulations of the GAGosylated syndecans 2 and 4, all three HS chains did not interact with the core proteins at the same time, and repelled each other due to their negative charges However, one HS chain adopted a compact conformation in the first simulation of syndecan-2, and in the first, second and fifth simulations of syndecan-4 (Figure 2). This might be due to the formation of intramolecular hydrogen bonds involving sulfate groups,³³ and to the presence of the counterions added for the simulations, which bind to the GAG chain and mask its charges.³⁴ The amino acid sequences located between the GAGosylation sites were not extended when HS chains were attached to the core proteins (Figures 1 and 2).

The flexibility of the core proteins of the non-GAGosylated and GAGosylated forms of syndecans 2 and 4 was evaluated by following the RMSD changes in the course of the five MD simulations. There were no significant differences between the non-GAGosylated and GAGosylated forms of syndecan-2 (Supporting Information: Figure S3). Two particularly flexible areas were observed in both forms of syndecan-2, one close to the attachment site of the first HS chain, and the other one close to the NXIP cell adhesion motif (N95-P98, UniProtKB numbering). The RMSD values were higher for the GAGosylated form of syndecan-4 than for the non-GAGosylated form, suggesting that the three HS chains might enhance the flexibility of the core protein of syndecan-4 (Supporting Information: Figure S3). The five MD trajectories were then aligned by the Cα atoms of the core proteins, and the flexibility of the GAG chains was evaluated by calculating the RMSD describing their movements with the reference to the core protein. The RMSD changes were more pronounced for the GAG 2 chain than for the two other GAG chains in syndecan-4, which reflected the higher flexibility of this HS chain attached to the serine residue 61 (Figure 3). In contrast, the three GAG chains attached to the core protein of syndecan-2 had a similar flexibility (Figure 3).

The radius of gyration (Rgyr) was calculated to evaluate the effect of the HS chains on the compactness of the extracellular domains of syndecans. It ranged from ~20 to 45 Å for the non-GAGosylated and GAGosylated forms of the extracellular domain of syndecan-2 (Supporting Information: Figure S4), compared to an average value of 42.90 Å determined by SAXS.⁵ The radius of gyration of the extracellular domain of syndecan-4 ranged from 25 to 55 Å for its non-GAGosylated form, compared to an average value of 41.53 Å determined by SAXS,⁵ and from 20 to 45 Å for its GAGosylated form (Supporting Information: Figure S4). These large variations were expected given that the syndecan extracellular domains are mostly disordered, unfolded, proteins. However, the amplitude of the fluctuations decreased during the MD simulations of both GAGosylated forms, suggesting that the presence of the three HS chains might increase their compactness (Supporting Information: Figure S4). The radius of gyration of the non-GAGosylated syndecan-4 was larger than that of the non-GAGosylated syndecan-2, which is consistent with the trend of

syndecan-2 to adopt globular conformations and of syndecan-4 to remain extended during the simulations as described above.

The fluctuations of protein residues showed that the presence of the three HS chains decreased the flexibility of the GAG attachment region in syndecan-2 (Figure 4A,B), and increased the flexibility of the C-terminus of the extracellular domain of syndecan-4 (Figure 4C,D). The flexibility of the biologically active motifs of either syndecans (i.e., the NXIP motif, the CD148 sequence, and the synstatin sequence) was not significantly affected by GAGosylation (Figure 4).

The extracellular domain of syndecan-4 had on average a seven-fold higher content in antiparallel β strands, a two-fold higher content in 3₁₀ helices and a 1.3–1.5-fold higher content in bends than the extracellular domain of syndecan-2 (Table 1). However, no significant differences in the total amount of secondary structures were observed between the non-GAGosylated and GAGosylated forms of both extracellular domains during the five simulations (Table 1, Supporting Information: Figures S5 and S6), indicating that the HS chains did not induce significant changes in the secondary structure content of both core proteins.

## All-atom MD simulations of the extracellular domains of syndecans 2 and 4 with one or two HS chains attached to various serine residues

Additional MD simulations were carried out with the extracellular domains of syndecans 2 and 4 bearing one or two HS chains attached to various serine residues to determine the possible influence of the number and location of the GAG chains on the core proteins. The percentages of the RMSD ≥ 20 Å ranged from 20% (GAG 2) to 47% (GAG 3) with an intermediate value (33%) for GAG 1 in syndecan-2 (Supporting Information: Table S5). The variant with the middle GAG chain had thus a reduced conformational heterogeneity. The variants bearing two GAG chains exhibited similar or lower percentages of RMSD values ≥ 20 Å (20% for GAG 1 + 2, 0% for GAG 1 + 3, and 27% for GAG 2 + 3) than the single-chain variants (Supporting Information: Table S5), whereas the presence of both GAG 1 and GAG 3 chains strongly reduced the conformational diversity of the syndecan-2 core protein compared to the other two-chain variants (Figure 5A,B). These results show that both the number and the location of the GAG chains matters, and support the importance of the number of GAG chains.

The percentage of RMSD values ≥ 20 Å for the MD simulations of syndecan-4 were higher than those calculated for syndecan-2. They varied from 53% to 55% for the single variants GAG 2 and GAG 3, and up to 87% for the GAG 1 variant (Supporting Information: Table S6). The RMSD values ≥ 20 Å represented 55%, 67%, and 87% of the RMSD value for the two-chain variants GAG 1 + 2, GAG 1 + 3, and GAG 2 + 3 respectively (Supporting Information: Table S6). The lowest structural diversity was thus observed for the single-variant GAG 2, and the two-chain variant GAG 1 + 2 of syndecan-4 (Figure 5C,D), although both RMSD values > 30 Å in single-chain variants were found in the GAG 2 variant (Supporting Information:
<figure><img src="image_4.png" /><figcaption>**FIGURE 3** The five molecular dynamics (MD) trajectories (S1-S5) were aligned by the Cα atoms of the core proteins, and the root-mean-square deviation (RMSD) of each trajectory relative to the first frame was calculated as a function of time for the three heparan sulfate (HS) chains attached to the extracellular domain of human syndecans 2 (left) and 4 (right) during the five MD simulations. (Pink: syndecan extracellular domain, blue: glycosaminoglycan [GAG] 1, red: GAG 2, green: GAG 3).</figcaption></figure>

Table S6). Single GAG chains attached to different serine residues on the core protein and combinations of two GAG chains modulated the conformational space of syndecans 2 and 4 but not to the same extent. The single-variant GAG 2 had the lowest heterogeneity

among the single variants of both syndecans, but the lowest diversity of two-chain variants was found for the GAG 1+3 variant for syndecan-2 and both GAG 1+2 and GAG 1+3 variants for syndecan-4. Both the number and the location of GAG chains
<figure><img src="image_5.png" /></figure>

**FIGURE 4** Fluctuations of the protein residues of the non-GAGosylated (A, C) and GAGosylated (B, D) extracellular domains of human syndecans 2 (A, B) and 4 (C, D) bearing three heparan sulfate (HS) chains during the five molecular dynamics (MD) simulations (simulation 1: dark blue, simulation 2: green, simulation 3: red, simulation 4: pink, simulation 5: black). The amino acid residues are numbered according to the sequences used in the MD simulation (see the Methods section). Solid black line: NXIP motif, dashed black line: CD148 binding site (syndecan-2) and synstatin (syndecan-4), solid red line: HS attachment region.

influence the structural diversity of syndecans 2 and 4, but in a core protein-dependent manner.

The RMSD plots of the single- and two-chain variants showed a broader variation for syndecan-2 than for syndecan-4 (Supporting Information: Figure S7) as observed for the three-chain variants. The fluctuations of the core proteins of the single- and two-chain variants of syndecans 2 and 4 were similar to those of their three-chain variants (Figure 6), suggesting that neither the number nor the position of the HS chains did significantly affect the flexibility of the core proteins, although they did have an effect on their conformational diversity. The compactness of the single- and two-chain variants, assessed by the values of the radius of gyration, was higher for syndecan-2 than for syndecan-4 (Supporting Information: Figure S8), which was consistent with the data collected for the three-chain variants. There were no significant effects of the presence of one or two HS chains on the flexibility of the biologically active motifs (Figure 6) as previously observed for the three-chain variants.

The content in α-helix was lower in syndecan-2 single- and two-chain variants than in the three-chain variant and in absence of GAGs (Table 2). The content in antiparallel β strands decreased in the single-chain variant GAG 1 and in all the two-chain variants compared to the core protein without GAGs (Table 2), whereas the bend level increased in the single-chain variant GAG1 and in the two-chain variants GAG 1+2 and GAG 1+3 compared to the core protein without HS chain (Table 2). In contrast, no significant changes were observed in the single- and two-chain variants of syndecan-4 compared to that of the core protein without GAG chain, although

the content in antiparallel β strands was decreased in the two-chain variant GAG 1 + 3 (Table 2).

## Interactions between the three GAG chains and the core proteins of syndecans 2 and 4

We investigated the possible interactions between the core protein of syndecans 2 and 4 and their three covalently attached HS chains, which could potentially occur in in vitro experiments and within the shed syndecans released under a soluble form in the biological fluids, where they might serve as disease biomarkers.³⁵ GAG chains were considered as ligands and the extracellular domains of syndecans as receptors for the calculation of the LIE (Figure 7). The most favorable binding energies were found for the interactions of GAG 1 and GAG 3 chains with the core proteins of syndecans 2 and 4 (Table 3).

The fractions of native contacts between the GAG chains and the core proteins, defined as the contacts present in the initial frame of the MD simulation, were calculated and only the values above 0.5 were considered for further analysis. The fraction of native contacts ≥0.5 was higher in syndecan-2 than in syndecan-4 (44 and 28 respectively, Supporting Information: Tables S7 and S8). The GAG 3 chain was involved in 70% of the contacts established in syndecan-2, compared to 9% and 20% for the GAG 1 and GAG 2 chains respectively (Supporting Information: Table S7). The most frequent contact, observed in four out of the five simulations, was between Lys96 and the GAG 3 chain, and the strongest contact involved Arg142 and the GAG 3 chain (Supporting Information: Table S7).
<table><caption>**TABLE 1** Secondary structure content (%), assigned by the Dictionary of Secondary Structure of Proteins (DSSP) as implemented in the CPPTRAJ module of AMBER, of the non-GAGosylated and GAGosylated extracellular domains of syndecans 2 and 4 for the five molecular dynamics (MD) simulations.</caption><thead><tr><th>Syndecan</th><th>Simulation</th><th>Para</th><th>Anti</th><th>3-10</th><th>Alpha</th><th>Pi</th><th>Turn</th><th>Bend</th></tr></thead><tbody><tr><td rowspan="5">Non-GAGosylated syndecan-2</td><td>1</td><td>0.6</td><td>27.5</td><td>3.9</td><td>9.2</td><td>0</td><td>21.5</td><td>26.6</td></tr><tr><td>2</td><td>0.5</td><td>42.6</td><td>3.6</td><td>10.3</td><td>0</td><td>17.1</td><td>22.8</td></tr><tr><td>3</td><td>0.9</td><td>33.4</td><td>5.9</td><td>5.4</td><td>0</td><td>18.7</td><td>27.9</td></tr><tr><td>4</td><td>0.7</td><td>36.1</td><td>3.8</td><td>12.9</td><td>0.1</td><td>21.5</td><td>24.9</td></tr><tr><td>5</td><td>0.1</td><td>33.2</td><td>2.8</td><td>12.0</td><td>0</td><td>22.4</td><td>27.2</td></tr><tr><td rowspan="5">GAGosylated syndecan-2</td><td>1</td><td>0</td><td>26.7</td><td>5.2</td><td>2.0</td><td>0</td><td>18.2</td><td>32.9</td></tr><tr><td>2</td><td>0</td><td>28.5</td><td>3.7</td><td>5.1</td><td>0</td><td>17.7</td><td>30.4</td></tr><tr><td>3</td><td>0.6</td><td>34.9</td><td>2.8</td><td>10.2</td><td>0</td><td>18.0</td><td>26.6</td></tr><tr><td>4</td><td>1.4</td><td>27.6</td><td>5.0</td><td>2.4</td><td>0</td><td>21.2</td><td>31.5</td></tr><tr><td>5</td><td>0.3</td><td>27.4</td><td>4.6</td><td>8.0</td><td>0</td><td>18.0</td><td>24.3</td></tr><tr><td rowspan="5">Non-GAGosylated syndecan-4</td><td>1</td><td>0.1</td><td>4.7</td><td>11.0</td><td>12.4</td><td>0.1</td><td>33.0</td><td>40.8</td></tr><tr><td>2</td><td>1.9</td><td>5.5</td><td>9.2</td><td>3.8</td><td>0</td><td>33.5</td><td>38.3</td></tr><tr><td>3</td><td>0.4</td><td>3.0</td><td>8.6</td><td>10.4</td><td>0.2</td><td>29.9</td><td>33.9</td></tr><tr><td>4</td><td>0.7</td><td>1.0</td><td>13.7</td><td>9.6</td><td>0.1</td><td>35.9</td><td>38.1</td></tr><tr><td>5</td><td>2.8</td><td>4.0</td><td>9.2</td><td>8.9</td><td>0.2</td><td>31.7</td><td>37.3</td></tr><tr><td rowspan="5">GAGosylated syndecan-4</td><td>1</td><td>0.1</td><td>3.1</td><td>9.1</td><td>9.8</td><td>0.3</td><td>33.5</td><td>40.0</td></tr><tr><td>2</td><td>2.4</td><td>1.7</td><td>8.5</td><td>9.0</td><td>0.1</td><td>25.1</td><td>41.7</td></tr><tr><td>3</td><td>3.0</td><td>0.8</td><td>8.9</td><td>15.0</td><td>0.2</td><td>36.1</td><td>36.3</td></tr><tr><td>4</td><td>2.8</td><td>3.2</td><td>9.6</td><td>6.5</td><td>0</td><td>28.8</td><td>39.4</td></tr><tr><td>5</td><td>0.2</td><td>11.4</td><td>12.3</td><td>9.9</td><td>0.1</td><td>26.1</td><td>36.4</td></tr></tbody></table>

Note: Para, parallel beta-strand; anti, antiparallel beta-strand; 3-10, 3-10 helix; alpha, alpha-helix; Pi, pi-helix; Turn, turn; Bend, bend (region with high curvature).

The residue Lys96 is located within the NKIP cell adhesion motif (N95-P98), and the Arg142 residue is close to the N-terminus of the binding site of CD148 to syndecan-2, suggesting that intramolecular contacts between GAG chains and the core protein could interfere with syndecan-2 binding to cells and CD148. In syndecan-4 the GAG 1 and GAG 3 chains participated in 43% and 29% of the contacts, respectively, whereas the GAG 2 chain was involved in 18% of them. The most frequent contact, observed in four out of the five simulations of syndecan-4, was established between Ser61 and the GAG 2 chain (Supporting Information: Table S8). The GAG 2 chain was covalently attached to this serine residue in this variant, but although its contact with Ser61 might not be biologically relevant, it could formally be formed according to the 4-Å cut-off set up to define the contacts. The next most frequent and strongest contact was observed between Arg92 and the GAG 3 chain (Supporting Information: Table S8). The Arg92 residue being located within the synstatin motif (N87-M131) and close to the C-terminus of the NHIP (N87-P90) motif, its intramolecular contact with the GAG 3 chain could interfere with cell adhesion and the binding of synstatin to cell surface receptors. GAG 1, 2, and 3 chains predominantly established contacts with the N-terminal, the middle and the

C-terminal parts of the core proteins respectively (Supporting Information: Figure S9).

More H-bonds were established for syndecan-2 than for syndecan-4 during the five MD simulations (55 and 38 with an occupancy frequency >0.1 respectively, Supporting Information: Tables S9 and S10). The GAG 3 chain formed the highest number of H-bonds in syndecan-2 (59% compared to 16% and 25% for the GAG 1 and GAG 2 chains respectively, Supporting Information: Table S9). In contrast, the GAG 1 chain formed the highest number of H-bonds in syndecan-4 (50% compared to 10% and 40% for the GAG 2 and GAG 3 chains respectively, Supporting Information: Table S10). The most populated H-bond was observed between Arg142 and GAG 3 in syndecan-2, and between Arg92 and GAG 3 in syndecan-4 (Supporting Information: Tables S9 and S10).

DISCUSSION

We have generated coarse-grained models of the extracellular domains of syndecans 2 and 4 without GAG chains. A model of syndecan-4 bearing three HS chains has been generated several
<figure><img src="image_6.png" /><figcaption>**FIGURE 5** Root-mean-square deviation (RMSD) probability density plots of the three molecular dynamics (MD) simulations of 0.25 µs (A, C) and 0.5 µs (B, D) for the core proteins of the single-chain and two-chain variants of syndecans 2 (A, B) and 4 (C, D) (Pink: glycosaminoglycan [GAG] 1, blue: GAG 2, red: GAG 3, green: GAG 1 + 2, black: GAG 1 + 3, yellow: GAG 2 + 3).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**FIGURE 6** Fluctuations of the protein residues of the GAGosylated single-chain and two-chain variants of the extracellular domains of syndecans 2 (left panel) and 4 (right panel) for three molecular dynamics (MD) simulations (Pink: glycosaminoglycan [GAG] 1, blue: GAG 2, red: GAG 3, green: GAG 1 + 2, black: GAG 1 + 3, yellow: GAG 2 + 3). The amino acid residues are numbered according to the sequences used in the MD simulation (see the Methods section). Solid black line: NXIP motif, dashed black line: CD148 binding site (syndecan-2) and synstatin (syndecan-4), solid red line: heparan sulfate (HS) attachment region.</figcaption></figure>

years ago to investigate its behavior in a lipid bilayer submitted to various shear forces using MD simulations. However, no experimental structural data for the syndecan extracellular domains were available at that time, and the extracellular domain of syndecan-4 was represented as an elongated, unfolded, polypeptide.³⁶ Here, we used SAXS restraints to generate models consistent with experimental data. SAXS data were collected using tagged extracellular domains. Although a possible effect of the short N- and C-terminal tags (six and eight amino acid residues respectively) added to the recombinant extracellular domains of syndecans cannot be totally ruled out, it is unlikely that they significantly alter the dynamics of the highly disordered and highly dynamic core proteins of syndecans 2 and 4. Furthermore, we used the same SAXS-restrained models for the MD simulations of both syndecans without and with attached HS chains,

and we focused on the comparison of the data collected in presence and absence of HS chains, which greatly reduces a potential interference (if any) of the tags.

The models that best fitted to experimental SAXS data were used for MD simulations with and without HS chains. The simulations were carried out for 0.5 up to 1.0 µs for eight variants of each syndecan (no HS chain, one three-chain, three single-chain, and three two-chain variants) to determine if the number and position of GAG chains affect the conformational dynamics and flexibility of the core proteins. This is the first time that such computationally expensive µs-range all-atom MD simulations integrating SAXS-derived data have been performed for proteoglycans with GAG chains in explicit solvent (i.e., taking into account the movements and effects of the solvent molecules). This was challenging because the extracellular
<table><caption>**TABLE 2** Secondary structure content (%), assigned by the Dictionary of Secondary Structure of Proteins (DSSP) as implemented in the CPPTRAJ module of AMBER, of the GAGosylated extracellular domains of syndecans 2 and 4 with one to two heparan sulfate (HS) chains attached to various serine residues for the three molecular dynamics (MD) simulations.</caption><thead><tr><th>Syndecan</th><th>Simulation</th><th>Para</th><th>Anti</th><th>3-10</th><th>Alpha</th><th>Pi</th><th>Turn</th><th>Bend</th></tr></thead><tbody><tr><td rowspan="3">Syndecan-2 GAG 1</td><td>1</td><td>1.3</td><td>13.5</td><td>6.9</td><td>2.7</td><td>0.0</td><td>18.2</td><td>34.1</td></tr><tr><td>2</td><td>0.1</td><td>28.7</td><td>6.2</td><td>0.8</td><td>0.0</td><td>17.8</td><td>28.2</td></tr><tr><td>3</td><td>0.4</td><td>28.3</td><td>4.8</td><td>5.0</td><td>0.1</td><td>22.8</td><td>30.2</td></tr><tr><td rowspan="3">Syndecan-2 GAG 2</td><td>1</td><td>0.7</td><td>31.5</td><td>10.9</td><td>8.7</td><td>0.0</td><td>20.1</td><td>26.0</td></tr><tr><td>2</td><td>0.4</td><td>40.8</td><td>5.7</td><td>1.3</td><td>0.0</td><td>19.9</td><td>25.0</td></tr><tr><td>3</td><td>1.8</td><td>22.3</td><td>6.2</td><td>2.5</td><td>0.0</td><td>21.9</td><td>29.3</td></tr><tr><td rowspan="3">Syndecan-2 GAG 3</td><td>1</td><td>1.3</td><td>31.8</td><td>3.9</td><td>1.4</td><td>0.0</td><td>16.5</td><td>28.4</td></tr><tr><td>2</td><td>1.4</td><td>20.8</td><td>5.7</td><td>3.0</td><td>0.0</td><td>19.3</td><td>34.7</td></tr><tr><td>3</td><td>0.0</td><td>48.2</td><td>7.1</td><td>2.0</td><td>0.0</td><td>15.0</td><td>20.9</td></tr><tr><td rowspan="3">Syndecan-2 GAG 1 + GAG 2</td><td>1</td><td>0.1</td><td>22.6</td><td>5.7</td><td>3.7</td><td>0.0</td><td>17.8</td><td>32.7</td></tr><tr><td>2</td><td>0.1</td><td>23.5</td><td>2.7</td><td>1.8</td><td>0.0</td><td>12.9</td><td>35.2</td></tr><tr><td>3</td><td>0.2</td><td>30.3</td><td>4.4</td><td>0.8</td><td>0.0</td><td>13.2</td><td>30.1</td></tr><tr><td rowspan="3">Syndecan-2 GAG 1 + GAG 3</td><td>1</td><td>0.1</td><td>33.0</td><td>2.4</td><td>3.7</td><td>0.0</td><td>16.8</td><td>31.7</td></tr><tr><td>2</td><td>0.0</td><td>19.0</td><td>6.3</td><td>0.9</td><td>0.0</td><td>17.9</td><td>32.1</td></tr><tr><td>3</td><td>0.6</td><td>24.5</td><td>9.0</td><td>2.5</td><td>0.3</td><td>20.9</td><td>28.3</td></tr><tr><td rowspan="3">Syndecan-2 GAG 2 + GAG 3</td><td>1</td><td>2.6</td><td>24.5</td><td>4.2</td><td>3.6</td><td>0.0</td><td>17.8</td><td>27.2</td></tr><tr><td>2</td><td>0.1</td><td>23.5</td><td>6.3</td><td>3.8</td><td>0.0</td><td>20.4</td><td>29.6</td></tr><tr><td>3</td><td>2.7</td><td>25.7</td><td>2.9</td><td>2.3</td><td>0.0</td><td>17.8</td><td>31.4</td></tr><tr><td rowspan="3">Syndecan-4 GAG 1</td><td>1</td><td>2.9</td><td>3.9</td><td>5.2</td><td>14.1</td><td>0.0</td><td>25.5</td><td>40.2</td></tr><tr><td>2</td><td>1.2</td><td>5.2</td><td>10.3</td><td>7.6</td><td>0.1</td><td>30.3</td><td>34.7</td></tr><tr><td>3</td><td>1.7</td><td>2.3</td><td>13.9</td><td>6.4</td><td>0.0</td><td>28.0</td><td>37.2</td></tr><tr><td rowspan="3">Syndecan-4 GAG 2</td><td>1</td><td>1.6</td><td>4.9</td><td>11.3</td><td>7.4</td><td>0.0</td><td>30.4</td><td>36.1</td></tr><tr><td>2</td><td>0.2</td><td>8.6</td><td>9.4</td><td>9.9</td><td>0.2</td><td>27.8</td><td>39.9</td></tr><tr><td>3</td><td>1.3</td><td>1.0</td><td>11.5</td><td>5.3</td><td>0.0</td><td>30.6</td><td>43.0</td></tr><tr><td rowspan="3">Syndecan-4 GAG 3</td><td>1</td><td>0.1</td><td>2.1</td><td>6.8</td><td>8.1</td><td>0.1</td><td>33.3</td><td>39.1</td></tr><tr><td>2</td><td>1.3</td><td>1.9</td><td>8.9</td><td>12.0</td><td>0.1</td><td>25.5</td><td>43.4</td></tr><tr><td>3</td><td>0.8</td><td>6.8</td><td>9.7</td><td>8.8</td><td>0.1</td><td>26.4</td><td>40.8</td></tr><tr><td rowspan="3">Syndecan-4 GAG 1 + GAG 2</td><td>1</td><td>1.7</td><td>1.2</td><td>7.2</td><td>7.1</td><td>0.0</td><td>32.1</td><td>40.3</td></tr><tr><td>2</td><td>3.4</td><td>5.1</td><td>8.8</td><td>17.1</td><td>0.0</td><td>31.1</td><td>31.2</td></tr><tr><td>3</td><td>0.1</td><td>4.6</td><td>9.4</td><td>17.3</td><td>0.2</td><td>26.7</td><td>33.4</td></tr><tr><td rowspan="3">Syndecan-4 GAG 1 + GAG 3</td><td>1</td><td>2.0</td><td>1.1</td><td>4.8</td><td>7.7</td><td>0.0</td><td>28.5</td><td>45.0</td></tr><tr><td>2</td><td>0.7</td><td>0.8</td><td>10.0</td><td>7.9</td><td>0.0</td><td>35.3</td><td>39.0</td></tr><tr><td>3</td><td>0.9</td><td>1.8</td><td>11.2</td><td>22.2</td><td>0.1</td><td>33.0</td><td>32.3</td></tr><tr><td rowspan="3">Syndecan-4 GAG 2 + GAG 3</td><td>1</td><td>0.1</td><td>2.0</td><td>11.6</td><td>9.7</td><td>0.0</td><td>28.0</td><td>40.9</td></tr><tr><td>2</td><td>1.1</td><td>2.4</td><td>10.0</td><td>6.4</td><td>0.0</td><td>30.7</td><td>43.5</td></tr><tr><td>3</td><td>0.9</td><td>3.0</td><td>7.8</td><td>12.2</td><td>0.0</td><td>26.9</td><td>41.8</td></tr></tbody></table>

Note: Para, parallel beta-strand; anti, antiparallel beta-strand; 3-10, 3-10 helix; alpha, alpha-helix; Pi, pi-helix; Turn, turn; Bend, bend (region with high curvature).
<figure><img src="image_8.png" /><figcaption>**FIGURE 7** Linear interaction energy (LIE) calculated for the interactions between the core protein of the extracellular domains of syndecans 2 (A) and 4 (B) and the three glycosaminoglycan (GAG) chains (blue: GAG 1, red: GAG 2, green: GAG 3) for the five simulations.</figcaption></figure>

<table><caption>**TABLE 3** Mean values of the binding free energy (kcal/mol) calculated using the linear interaction energy (LIE) during the five simulations (S1-S5) for the binding of the core proteins of syndecans 2 and 4 to each glycosaminoglycan (GAG) chain.</caption><thead><tr><th>Molecular dynamics (MD) simulations</th><th>GAG 1</th><th>GAG 2</th><th>GAG 3</th></tr></thead><tbody><tr><td colspan="4">Binding free energy (kcal/mol) of GAGosylated syndecan-2</td></tr><tr><td>S1</td><td>-30.3</td><td>-16.3</td><td>-33.1</td></tr><tr><td>S2</td><td>-44.3</td><td>-9.0</td><td>-66.6</td></tr><tr><td>S3</td><td>-42.7</td><td>-15.4</td><td>-54.2</td></tr><tr><td>S4</td><td>-44.1</td><td>-0.2</td><td>-1.5</td></tr><tr><td>S5</td><td>-4.8</td><td>-8.1</td><td>-86.5</td></tr><tr><td colspan="4">Binding free energy (kcal/mol) of GAGosylated syndecan-4</td></tr><tr><td>S1</td><td>-44.4</td><td>-0.2</td><td>-28.1</td></tr><tr><td>S2</td><td>-27.6</td><td>-0.5</td><td>-73.5</td></tr><tr><td>S3</td><td>-44.9</td><td>-36.0</td><td>-1.5</td></tr><tr><td>S4</td><td>-7.8</td><td>0.0</td><td>-0.1</td></tr><tr><td>S5</td><td>-7.7</td><td>-0.1</td><td>-17.1</td></tr></tbody></table>

domains of syndecans 2 and 4 are intrinsically disordered,⁵ and the GAG chains are flexible. Iduronic acid residues provide flexibility to HS chains due to their conformational plasticity.³⁷ However, the conformations of iduronic acid residues are also regulated by the sulfation pattern of their flanking residues,³⁸,³⁹ and sulfation might thus also contribute to the flexibility of the HS oligosaccharide. The intrinsic flexibility of the disordered non-GAGosylated and GAGosylated forms of syndecans led to a significant heterogeneity in MD simulations, which prevented a detailed quantitative and statistically significant interpretation of the data despite the length of the performed MD simulations. All-atom MD simulations of intrinsically disordered proteins experience serious conceptual challenges indeed, due to the extremely long timescales of the simulations required to properly sample their broad conformational space, and to the lack of specialized force fields developed for intrinsically disordered proteins, in particular when posttranslational modifications are present as in the GAGosylated form of syndecans. Force field strategies have

been recently proposed for more accurate MD simulations of intrinsically disordered proteins, and could pave the way to future experiments.⁴⁰

The extracellular domain of syndecan-2 has a trend to adopt globular conformations during the MD simulations, whereas syndecan-4 remains mostly extended. This difference might not be due their disorder content that is similar (52% and 44% for syndecans 2 and 4, respectively) but it is consistent with the fact that the most extended conformations observed in electrospray ionization-mass spectrometry in native conditions were more abundant in syndecan-4 (85%) than in syndecan-2 (70%),⁵ which highlights the relevance of the computational models. In addition, syndecan-4 exhibits a larger conformational diversity than syndecan-2. The core proteins of these two closely related syndecans are thus structurally distinct, which might reflect different roles.

MD simulations confirm that the extracellular domains of both syndecans are highly flexible and dynamic as experimentally demonstrated,⁵ and show that the HS chains influence their conformational dynamics in a core protein-dependent manner. This is in agreement with reports showing that chondroitin sulfate, another GAG, has different effects depending on the core protein it is attached to. It could modulate the flexibility and size of aggrecan⁶ but has no significant effect on the structure or conformational stability of decorin.⁷ We also show that the effect of HS chains on the syndecan core proteins depends on the number of GAG chains attached to the core proteins, and on the serine residues they are attached to. The location of the GAG attachment site also matters for the GAG chain since the middle GAG chain attached to Ser61 in syndecan-4 is more flexible than the chains attached to Ser39 and Ser63. The roles of the number and location of GAG chains are further supported by the fact that the three single-chain variants and the three two-chain variants had different effects on the core proteins of syndecans. These findings are consistent with the importance of the valency of HS chains experimentally demonstrated for cell-matrix interactions mediated by syndecan-4.⁸

The NXIP cell adhesion motif of syndecan-2 is located in a highly flexible region, in contrast to the CD148 binding site (syndecan-2), and synstatin (syndecan-4). The particularly flexible region of syndecan-2 close to the NXIP site might be required for the function
of this syndecan. The conformational flexibility and dynamics of the biologically active motifs of both syndecans are not significantly affected by the HS chains. The fact that GAG 3 on either core protein may interact with the regions of the core proteins involved in adhesion raises the possibility that this could be an inhibitory process that may be overcome by HS interactions with other partners.

Beside the difficulty to get quantitative results mentioned above, other limitations of this study are that MD simulations were carried out with monomeric and not dimeric core proteins, and that the HS chains attached to the core proteins were very short compared to those found in vivo (~36–42 kDa⁸), and had all the same heparin-like sequences. Indeed, the sequences and size of HS chains may vary from one chain to another one on the same core protein, and in different cell types and tissues,⁴¹,⁴² and the sulfation density of HS chains of syndecans can vary when their number is reduced.⁸ The results reported here have been obtained with a unique, highly sulfated, HS sequence because MD simulations were time-consuming (each MD simulation lasted ~90 days of wall-clock-time on a GPU and the study included eight variants for each syndecan). Further MD simulations with longer HS chains, and various HS sequences mimicking those found in vivo (e.g., less sulfated sequences and GlcA-containing sequences) should be performed to determine if the conclusions drawn from this study can be extended to other HS sequences or if the effects of HS on the conformation of the extracellular domains of syndecans 2 and 4 are sequence- and/or size-dependent. Future studies should also focus on the tetrasaccharide linking the GAG chains to the serine residues of the core proteins, which behaves as a relatively rigid unit in MD simulations with conformational heterogeneity for rotation about bonds connecting the xylose and serine residues.²¹ The sugar residues of the linker can be phosphorylated, sulfated, and sialylated, and a noncanonical trisaccharide linker has been reported for bikunin.⁴³ It would be interesting to determine if the modifications and size of this linker affect the conformations of the core proteins of syndecans and/or the flexibility of the GAG chains, inasmuch as defective synthesis of the linker is associated with rare heritable connective tissue diseases known as linkeropathies.⁴⁴,⁴⁵

## CONCLUSION

We report here the first µ-range all-atom MD simulations in explicit solvent and integrating SAXS-derived data for proteoglycans with GAG chains. Although closely related, syndecans 2 and 4 appear to be structurally distinct during the MD simulations. The HS chains induce moderate changes in the conformational dynamics of both syndecans, depending on the number and location of the HS chains on the core protein and on the core protein itself. The cell adhesion sites on the core proteins are flexible, with or without HS chains, and the intramolecular contacts established by the GAG 3 chain in these regions might modulate their accessibility, and interfere with cell adhesion. The three HS chains attached to the core proteins are not

equivalent, the middle HS chain attached to Ser61 in syndecan-4 being more flexible than the two other HS chains.

## AUTHOR CONTRIBUTIONS

**Martyna Maszota-Zieleniak:** Investigation; formal analysis; writing—review and editing; writing—original draft; visualization. **Adam Liwo:** Conceptualization; writing—review and editing. **Sylvie Ricard-Blum:** Conceptualization; funding acquisition; writing—original draft; writing—review and editing; supervision. **Sergey A. Samsonov:** Conceptualization; writing—review and editing; writing—original draft; funding acquisition; formal analysis.

## ACKNOWLEDGMENTS

The computational resources were provided by the local cluster at the Faculty of Chemistry, University of Gdansk (Poland). We would like to thank John R. Couchman (University of Copenhagen, Denmark) for sharing with us his excellent expertise on syndecans, for stimulating discussions and support. This work was supported by the grant UMO-2018/30/E/ST4/00037 from the National Science Center of Poland (Narodowe Centrum Nauki) to Sergey A. Samsonov, a grant from the Institut National du Cancer PLBIO-258 to Sylvie Ricard-Blum, and partially supported by the "PHC POLONIUM" program (project number: 49131ZM), funded by the French Ministry for Europe and Foreign Affairs, the French Ministry for Higher Education and Research and the Polish NAWA to Sergey A. Samsonov and Sylvie Ricard-Blum.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## ETHICS STATEMENT

None.

## ORCID

Sylvie Ricard-Blum http://orcid.org/0000-0001-9263-1851

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** M. Maszota-Zieleniak, A. Liwo, S. Ricard-Blum, S. A. Samsonov, *Proteoglycan Research* **2023**, *1*, e10. https://doi.org/10.1002/pgr2.10
SUPPLEMENTARY FIGURES

Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4

Martyna Maszota-Zieleniak, Adam Liwo, Sylvie Ricard-Blum, Sergey A. Samsonov

<figure><img src="image_9.png" /><figcaption>**Figure S1.** The five models of the extracellular domain of human syndecan-2 without GAG chains generated with UNRES and SAXS restraints, and the probability distribution (P(x)) for the distance of consecutive Cα atoms calculated from experimental SAXS data (pink) and for UNRES-generated models (green). The topology diagram of model 4 selected for further analyses is displayed below the model (α-helix: red cylinder, β-strand: pink arrow, disordered sequence: blue). The amino acid residues were numbered according to the sequence used for the MD simulations (see the Methods section).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure S2.** The five models of the extracellular domain of human syndecan-4 without GAG chains generated with UNRES and SAXS restraints, and the probability distribution (P(x)) for the distance of consecutive Cα atoms calculated from experimental SAXS data (pink) and for UNRES-generated models (green). The topology diagram of model 1 selected for further analyses is displayed below the model (α-helix: red cylinder, β-strand: pink arrow, disordered sequence: blue). The amino acid residues were numbered according to the sequence used for the MD simulations (see the Methods section).</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Figure S3.** RMSD values calculated for the core proteins of non-GAGosylated (A, C) and GAGosylated (B, D) extracellular domains of syndecans 2 (A, B) and 4 (C, D) during the five MD simulations (simulation 1: dark blue, simulation 2: green, simulation 3: red, simulation 4: pink, simulation 5: black). The time displayed on the x-axis corresponds to the time required for RMSD convergence.</figcaption></figure>
<figure><img src="image_12.png" /></figure>

**Figure S4.** The radius of gyration (Rgyr) calculated for the non-GAGosylated (A, C) and GAGosylated (B, D) core protein of the extracellular domains of syndecans 2 (A, B) and 4 (C, D) for the five MD simulations (simulation 1: dark blue, simulation 2: green, simulation 3: red, simulation 4: pink, simulation 5: black). The time displayed on the x-axis corresponds to the time required for RMSD convergence.
<figure><img src="image_13.png" /><figcaption>**Figure S5.** Changes in the secondary structure, assigned by DSSP, of non-GAGosylated and GAGosylated (three heparan sulfate chains) extracellular domain of human syndecan-2 during the five simulations (S1-S5). The residues are numbered according to the sequence used in the MD simulations (see the Methods section in the main text). The time displayed on the x-axis corresponds to the time required for RMSD convergence. The amino acid residues were numbered according to the sequence used for the MD simulations (see the Methods section).</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure S6.** Change in the secondary structure, assigned by DSSP, of non-GAGosylated and GAGosylated (three heparan sulfate chains) extracellular domain of human syndecan-4 during the five simulations (S1-S5). The residues are numbered according to the sequence used in the MD simulations (see the Methods section in the main text). The time displayed on the x-axis corresponds to the time required for RMSD convergence. The amino acid residues were numbered according to the sequence used for the MD simulations (see the Methods section).</figcaption></figure>
<figure><img src="image_15.png" /></figure>

**Figure S7.** RMSD values as a function of time for the GAGosylated forms of the extracellular domains of syndecans 2 (A) and 4 (B) during three MD simulations (magenta: GAG 1, blue: GAG 2, red: GAG 3, green: GAG 1 + GAG 2, black: GAG 1 + GAG 3, yellow: GAG 2 + GAG 3).

<figure><img src="image_16.png" /></figure>

**Figure S8.** The radius of gyration ($R_{gyr}$) calculated for the core protein of GAGosylated forms of the extracellular domains of syndecans 2 (A) and 4 (B) in the three MD simulations (magenta: GAG 1, blue: GAG 2, red: GAG 3, green: GAG 1 + GAG 2, black: GAG 1 + GAG 3, yellow: GAG 2 + GAG 3).
<figure><img src="image_17.png" /><figcaption>**Figure S9.** Contact maps for the GAGosylated extracellular domains of syndecans 2 and 4 during the five MD simulations. The pairwise contacts were calculated for the core protein (y-axis) and each of the 3 GAG chains including the tetrasaccharide linker (x-axis) with a distance cut-off of 4 Å for all heavy atoms. The color code corresponds to the occupancy of the pairwise contacts. Values > 1 indicate that several atoms from the same protein or GAG residue establish contacts with another GAG or protein residue. The residues were numbered according to the sequence used in the MD simulations (see the Methods section).</figcaption></figure>
SUPPLEMENTARY TABLES

Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4

Martyna Maszota-Zieleniak, Adam Liwo, Sylvie Ricard-Blum, Sergey A. Samsonov

<table><caption>**Table S1.** RMSD between the conformations obtained at half (0.5 µs) and the end (1 µs) of each of the five MD simulations (S1-S5) for the non-GAGosylated extracellular domain of human syndecan-2.</caption><thead><tr><th rowspan="2">RMSD [Å]</th><th colspan="2">S1</th><th colspan="2">S2</th><th colspan="2">S3</th><th colspan="2">S4</th><th colspan="2">S5</th></tr><tr><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th></tr></thead><tbody><tr><td rowspan="2">S1</td><td>0.5 µs</td><td>X</td><td>10.9</td><td>19.4</td><td>20.6</td><td>21.4</td><td>15.2</td><td>16.6</td><td>16.5</td><td>15.6</td><td>14.8</td></tr><tr><td>1 µs</td><td>10.9</td><td>X</td><td>23.2</td><td>23.2</td><td>21.4</td><td>21.9</td><td>15.9</td><td>16.4</td><td>15.4</td><td>15.1</td></tr><tr><td rowspan="2">S2</td><td>0.5 µs</td><td>19.4</td><td>23.2</td><td>X</td><td>11.2</td><td>15.4</td><td>13.6</td><td>16.9</td><td>13.9</td><td>19.5</td><td>19.5</td></tr><tr><td>1 µs</td><td>20.6</td><td>23.2</td><td>11.2</td><td>X</td><td>14.8</td><td>12.0</td><td>18.5</td><td>23.8</td><td>21.8</td><td>22.1</td></tr><tr><td rowspan="2">S3</td><td>0.5 µs</td><td>21.4</td><td>21.4</td><td>15.4</td><td>14.8</td><td>X</td><td>14.5</td><td>23.2</td><td>21.2</td><td>20.5</td><td>21.2</td></tr><tr><td>1 µs</td><td>15.2</td><td>21.9</td><td>13.6</td><td>12.0</td><td>14.5</td><td>X</td><td>20.2</td><td>25.0</td><td>16.6</td><td>17.6</td></tr><tr><td rowspan="2">S4</td><td>0.5 µs</td><td>16.6</td><td>15.9</td><td>16.9</td><td>18.5</td><td>23.2</td><td>20.2</td><td>X</td><td>15.0</td><td>18.3</td><td>16.5</td></tr><tr><td>1 µs</td><td>16.5</td><td>16.4</td><td>13.9</td><td>23.8</td><td>21.2</td><td>25.0</td><td>15.0</td><td>X</td><td>15.8</td><td>16.2</td></tr><tr><td rowspan="2">S5</td><td>0.5 µs</td><td>15.6</td><td>15.4</td><td>19.5</td><td>21.8</td><td>20.5</td><td>16.6</td><td>18.3</td><td>15.8</td><td>X</td><td>3.0</td></tr><tr><td>1 µs</td><td>14.8</td><td>15.1</td><td>19.5</td><td>22.1</td><td>21.2</td><td>17.6</td><td>16.5</td><td>16.2</td><td>3.0</td><td>X</td></tr></tbody></table>

<table><caption>**Table S2.** RMSD between the conformations obtained at half (0.5 µs) and the end (1 µs) of each of the five MD simulations (S1-S5) for the GAGosylated extracellular domain of human syndecan-2 bearing three heparan sulfate chains.</caption><thead><tr><th rowspan="2">RMSD [Å]</th><th colspan="2">S1</th><th colspan="2">S2</th><th colspan="2">S3</th><th colspan="2">S4</th><th colspan="2">S5</th></tr><tr><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th></tr></thead><tbody><tr><td rowspan="2">S1</td><td>0.5 µs</td><td>X</td><td>11.1</td><td>19.8</td><td>19.2</td><td>23.5</td><td>26.1</td><td>23.1</td><td>22.5</td><td>22.2</td><td>17.5</td></tr><tr><td>1 µs</td><td>11.1</td><td>X</td><td>21.5</td><td>20.4</td><td>24.1</td><td>20.3</td><td>22.0</td><td>21.1</td><td>24.7</td><td>19.6</td></tr><tr><td rowspan="2">S2</td><td>0.5 µs</td><td>19.8</td><td>21.5</td><td>X</td><td>9.2</td><td>20.3</td><td>14.7</td><td>20.3</td><td>20.0</td><td>18.5</td><td>11.3</td></tr><tr><td>1 µs</td><td>19.2</td><td>20.4</td><td>9.2</td><td>X</td><td>19.8</td><td>17.6</td><td>18.9</td><td>18.9</td><td>19.2</td><td>11.3</td></tr><tr><td rowspan="2">S3</td><td>0.5 µs</td><td>23.5</td><td>24.1</td><td>20.3</td><td>19.8</td><td>X</td><td>13.2</td><td>17.3</td><td>18.7</td><td>20.5</td><td>20.5</td></tr><tr><td>1 µs</td><td>26.1</td><td>20.3</td><td>14.7</td><td>17.6</td><td>13.2</td><td>X</td><td>22.4</td><td>19.0</td><td>18.3</td><td>17.3</td></tr><tr><td rowspan="2">S4</td><td>0.5 µs</td><td>23.1</td><td>22.0</td><td>20.3</td><td>18.9</td><td>17.3</td><td>22.4</td><td>X</td><td>4.2</td><td>19.6</td><td>20.0</td></tr><tr><td>1 µs</td><td>22.5</td><td>21.1</td><td>20.0</td><td>18.9</td><td>18.7</td><td>19.0</td><td>4.2</td><td>X</td><td>19.9</td><td>19.3</td></tr><tr><td rowspan="2">S5</td><td>0.5 µs</td><td>22.2</td><td>24.7</td><td>18.5</td><td>19.2</td><td>20.5</td><td>18.3</td><td>19.6</td><td>19.9</td><td>X</td><td>6.5</td></tr><tr><td>1 µs</td><td>17.5</td><td>19.6</td><td>11.3</td><td>11.3</td><td>20.5</td><td>17.3</td><td>20.0</td><td>19.3</td><td>6.5</td><td>X</td></tr></tbody></table>
<table><thead><tr><th rowspan="2">RMSD [Å]</th><th colspan="2">S1</th><th colspan="2">S2</th><th colspan="2">S3</th><th colspan="2">S4</th><th colspan="2">S5</th></tr><tr><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th></tr></thead><tbody><tr><td rowspan="2">S1</td><td>0.5 µs</td><td>X</td><td>14.8</td><td>18.6</td><td>16.5</td><td>27.0</td><td>23.0</td><td>25.8</td><td>26.3</td><td>21.1</td><td>20.7</td></tr><tr><td>1 µs</td><td>14.8</td><td>X</td><td>20.6</td><td>19.4</td><td>24.2</td><td>21.5</td><td>25.4</td><td>22.5</td><td>18.8</td><td>19.6</td></tr><tr><td rowspan="2">S2</td><td>0.5 µs</td><td>18.6</td><td>20.6</td><td>X</td><td>6.4</td><td>33.9</td><td>29.4</td><td>34.4</td><td>21.2</td><td>23.6</td><td>18.0</td></tr><tr><td>1 µs</td><td>16.5</td><td>19.4</td><td>6.4</td><td>X</td><td>31.2</td><td>25.9</td><td>31.0</td><td>24.4</td><td>24.1</td><td>17.7</td></tr><tr><td rowspan="2">S3</td><td>0.5 µs</td><td>27.0</td><td>24.2</td><td>33.9</td><td>31.2</td><td>X</td><td>15.0</td><td>16.9</td><td>35.8</td><td>25.3</td><td>30.3</td></tr><tr><td>1 µs</td><td>23.0</td><td>21.5</td><td>29.4</td><td>25.9</td><td>15.0</td><td>X</td><td>18.7</td><td>36.1</td><td>25.5</td><td>25.9</td></tr><tr><td rowspan="2">S4</td><td>0.5 µs</td><td>25.8</td><td>25.4</td><td>34.4</td><td>31.0</td><td>16.9</td><td>18.7</td><td>X</td><td>38.3</td><td>27.4</td><td>31.7</td></tr><tr><td>1 µs</td><td>26.3</td><td>22.5</td><td>21.2</td><td>24.4</td><td>35.8</td><td>36.1</td><td>38.3</td><td>X</td><td>21.9</td><td>22.6</td></tr><tr><td rowspan="2">S5</td><td>0.5 µs</td><td>21.1</td><td>18.8</td><td>23.6</td><td>24.1</td><td>25.3</td><td>25.5</td><td>27.4</td><td>21.9</td><td>X</td><td>16.1</td></tr><tr><td>1 µs</td><td>20.7</td><td>19.6</td><td>18.0</td><td>17.7</td><td>30.3</td><td>25.9</td><td>31.7</td><td>22.6</td><td>16.1</td><td>X</td></tr></tbody></table>

**Table S3:** RMSD between the conformations obtained at half (0.5 µs) and the end (1 µs) of each of the five MD simulations (S1-S5) for the non-GAGosylated extracellular domain of human syndecan-4.

<table><thead><tr><th rowspan="2">RMSD [Å]</th><th colspan="2">S1</th><th colspan="2">S2</th><th colspan="2">S3</th><th colspan="2">S4</th><th colspan="2">S5</th></tr><tr><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th><th>0.5 µs</th><th>1 µs</th></tr></thead><tbody><tr><td rowspan="2">S1</td><td>0.5 µs</td><td>X</td><td>18.2</td><td>26.0</td><td>26.1</td><td>19.7</td><td>19.5</td><td>19.6</td><td>25.8</td><td>19.7</td><td>25.9</td></tr><tr><td>1 µs</td><td>18.2</td><td>X</td><td>32.4</td><td>33.4</td><td>19.2</td><td>21.1</td><td>23.7</td><td>32.5</td><td>33.2</td><td>30.4</td></tr><tr><td rowspan="2">S2</td><td>0.5 µs</td><td>26.0</td><td>32.4</td><td>X</td><td>11.1</td><td>31.2</td><td>29.7</td><td>32.8</td><td>26.2</td><td>17.5</td><td>15.1</td></tr><tr><td>1 µs</td><td>26.1</td><td>33.4</td><td>11.1</td><td>X</td><td>31.7</td><td>30.6</td><td>32.1</td><td>27.4</td><td>18.6</td><td>17.8</td></tr><tr><td rowspan="2">S3</td><td>0.5 µs</td><td>19.7</td><td>19.2</td><td>31.2</td><td>31.7</td><td>X</td><td>6.9</td><td>19.2</td><td>24.5</td><td>28.1</td><td>30.7</td></tr><tr><td>1 µs</td><td>19.5</td><td>21.1</td><td>29.7</td><td>30.6</td><td>6.9</td><td>X</td><td>19.8</td><td>24.1</td><td>26.1</td><td>30.8</td></tr><tr><td rowspan="2">S4</td><td>0.5 µs</td><td>19.6</td><td>23.7</td><td>32.8</td><td>32.1</td><td>19.2</td><td>19.8</td><td>X</td><td>20.1</td><td>28.5</td><td>31.7</td></tr><tr><td>1 µs</td><td>25.8</td><td>32.5</td><td>26.2</td><td>27.4</td><td>24.5</td><td>24.1</td><td>20.1</td><td>X</td><td>22.7</td><td>28.4</td></tr><tr><td rowspan="2">S5</td><td>0.5 µs</td><td>19.7</td><td>33.2</td><td>17.5</td><td>18.6</td><td>28.1</td><td>26.1</td><td>28.5</td><td>22.7</td><td>X</td><td>18.0</td></tr><tr><td>1 µs</td><td>25.9</td><td>30.4</td><td>15.1</td><td>17.8</td><td>30.7</td><td>30.8</td><td>31.7</td><td>28.4</td><td>18.0</td><td>X</td></tr></tbody></table>

**Table S4.** RMSD between the conformations obtained at half (0.5 µs) and the end (1 µs) of each of the five MD simulations (S1-S5) for the GAGosylated extracellular domain of human syndecan-4 bearing three heparan sulfate chains.
<table><caption>**Table S5.** RMSD (Å) at half (0.25 µs) and the end (0.5 µs) of 3 MD simulations for the core protein of syndecan-2 with one or two HS chains attached to various serine residues.</caption><thead><tr><th rowspan="2">Extracellular<br>domain of<br>syndecan-2</th><th rowspan="2"></th><th rowspan="2"></th><th colspan="2">S1</th><th colspan="2">S2</th><th colspan="2">S3</th></tr><tr><th>0.25 µs</th><th>0.5 µs</th><th>0.25 µs</th><th>0.5 µs</th><th>0.25 µs</th><th>0.5 µs</th></tr></thead><tbody><tr><td rowspan="6">GAG1</td><td rowspan="2">S1</td><td>0.25 µs</td><td>X</td><td>11.5</td><td>23.2</td><td>16.3</td><td>23</td><td>20.6</td></tr><tr><td>0.5 µs</td><td>11.5</td><td>X</td><td>21.2</td><td>15.2</td><td>22.2</td><td>19.1</td></tr><tr><td rowspan="2">S2</td><td>0.25 µs</td><td>23.2</td><td>21.2</td><td>X</td><td>17.8</td><td>16.0</td><td>17.9</td></tr><tr><td>0.5 µs</td><td>16.3</td><td>15.2</td><td>17.8</td><td>X</td><td>17.2</td><td>19.3</td></tr><tr><td rowspan="2">S3</td><td>0.25 µs</td><td>23.0</td><td>22.2</td><td>16.0</td><td>17.2</td><td>X</td><td>10.5</td></tr><tr><td>0.5 µs</td><td>20.6</td><td>19.1</td><td>17.9</td><td>19.3</td><td>10.5</td><td>X</td></tr><tr><td rowspan="6">GAG 2</td><td rowspan="2">S1</td><td>0.25 µs</td><td>X</td><td>12.5</td><td>22.3</td><td>19.2</td><td>13.8</td><td>13.8</td></tr><tr><td>0.5 µs</td><td>12.5</td><td>X</td><td>22.6</td><td>20.3</td><td>18.3</td><td>14.8</td></tr><tr><td rowspan="2">S2</td><td>0.25 µs</td><td>22.3</td><td>22.6</td><td>X</td><td>9.1</td><td>14.8</td><td>17</td></tr><tr><td>0.5 µs</td><td>19.2</td><td>20.3</td><td>9.1</td><td>X</td><td>18.8</td><td>19.2</td></tr><tr><td rowspan="2">S3</td><td>0.25 µs</td><td>13.8</td><td>18.3</td><td>14.8</td><td>18.8</td><td>X</td><td>12.5</td></tr><tr><td>0.5 µs</td><td>13.8</td><td>14.8</td><td>17</td><td>19.2</td><td>12.5</td><td>X</td></tr><tr><td rowspan="6">GAG 3</td><td rowspan="2">S1</td><td>0.25 µs</td><td>X</td><td>5.8</td><td>21.1</td><td>18.2</td><td>22.9</td><td>21.9</td></tr><tr><td>0.5 µs</td><td>5.8</td><td>X</td><td>19.8</td><td>18.9</td><td>22.8</td><td>22.6</td></tr><tr><td rowspan="2">S2</td><td>0.25 µs</td><td>21.1</td><td>19.8</td><td>X</td><td>14.2</td><td>25.4</td><td>25.1</td></tr><tr><td>0.5 µs</td><td>18.2</td><td>18.9</td><td>14.2</td><td>X</td><td>18.6</td><td>19.0</td></tr><tr><td rowspan="2">S3</td><td>0.25 µs</td><td>22.9</td><td>22.8</td><td>25.4</td><td>18.6</td><td>X</td><td>6.0</td></tr><tr><td>0.5 µs</td><td>21.9</td><td>22.6</td><td>25.1</td><td>19.0</td><td>6.0</td><td>X</td></tr><tr><td rowspan="6">GAG 1 + GAG 2</td><td rowspan="2">S1</td><td>0.25 µs</td><td>X</td><td>6.4</td><td>19.8</td><td>22.6</td><td>12.5</td><td>18.1</td></tr><tr><td>0.5 µs</td><td>6.4</td><td>X</td><td>18.9</td><td>23.2</td><td>14.3</td><td>19.0</td></tr><tr><td rowspan="2">S2</td><td>0.25 µs</td><td>19.8</td><td>18.9</td><td>X</td><td>12.1</td><td>16.2</td><td>18.7</td></tr><tr><td>0.5 µs</td><td>22.6</td><td>23.2</td><td>12.1</td><td>X</td><td>18.2</td><td>20.6</td></tr><tr><td rowspan="2">S3</td><td>0.25 µs</td><td>12.5</td><td>14.3</td><td>16.2</td><td>18.2</td><td>X</td><td>15.3</td></tr><tr><td>0.5 µs</td><td>18.1</td><td>19.0</td><td>18.7</td><td>20.6</td><td>15.3</td><td>X</td></tr><tr><td rowspan="6">GAG 1 + GAG 3</td><td rowspan="2">S1</td><td>0.25 µs</td><td>X</td><td>3.8</td><td>18.4</td><td>15.0</td><td>14.3</td><td>17.0</td></tr><tr><td>0.5 µs</td><td>3.8</td><td>X</td><td>17.3</td><td>16.2</td><td>13.6</td><td>16.9</td></tr><tr><td rowspan="2">S2</td><td>0.25 µs</td><td>18.4</td><td>17.3</td><td>X</td><td>11.7</td><td>15.5</td><td>14.8</td></tr><tr><td>0.5 µs</td><td>15.0</td><td>16.2</td><td>11.7</td><td>X</td><td>15.6</td><td>15.2</td></tr><tr><td rowspan="2">S3</td><td>0.25 µs</td><td>14.3</td><td>13.6</td><td>15.5</td><td>15.6</td><td>X</td><td>3.9</td></tr><tr><td>0.5 µs</td><td>17.0</td><td>16.9</td><td>14.8</td><td>15.2</td><td>3.9</td><td>X</td></tr><tr><td rowspan="6">GAG 2 + GAG 3</td><td rowspan="2">S1</td><td>0.25 µs</td><td>X</td><td>9.9</td><td>19.2</td><td>19.5</td><td>19.0</td><td>18.4</td></tr><tr><td>0.5 µs</td><td>9.9</td><td>X</td><td>13.3</td><td>15.1</td><td>21.1</td><td>20.9</td></tr><tr><td rowspan="2">S2</td><td>0.25 µs</td><td>19.2</td><td>13.3</td><td>X</td><td>7.3</td><td>15.3</td><td>19.2</td></tr><tr><td>0.5 µs</td><td>19.5</td><td>15.1</td><td>7.3</td><td>X</td><td>16.3</td><td>18.9</td></tr><tr><td rowspan="2">S3</td><td>0.25 µs</td><td>19.0</td><td>21.1</td><td>15.3</td><td>16.3</td><td>X</td><td>8.6</td></tr><tr><td>0.5 µs</td><td>18.4</td><td>20.9</td><td>19.2</td><td>18.9</td><td>8.6</td><td>X</td></tr></tbody></table>
<table><caption>**Table S6. RMSD (Å) at half (0.25 µs) and the end (0.5 µs) of 3 MD simulations for the core protein of syndecan-4 with one or two HS chains attached to various serine residues.**</caption><thead><tr><th rowspan="2">Extracellular domain of syndecan-4</th><th rowspan="2"></th><th rowspan="2"></th><th colspan="2">S 1</th><th colspan="2">S 2</th><th colspan="2">S 3</th></tr><tr><th>0.25 µs</th><th>0.5 µs</th><th>0.25 µs</th><th>0.5 µs</th><th>0.25 µs</th><th>0.5 µs</th></tr></thead><tbody><tr><td rowspan="6">GAG 1</td><td rowspan="2">S 1</td><td>0.25 µs</td><td>X</td><td>18.3</td><td>27.7</td><td>19.9</td><td>21.4</td><td>25.5</td></tr><tr><td>0.5 µs</td><td>18.3</td><td>X</td><td>34.5</td><td>24.2</td><td>22.1</td><td>22.6</td></tr><tr><td rowspan="2">S 2</td><td>0.25 µs</td><td>27.7</td><td>34.5</td><td>X</td><td>26.3</td><td>29.5</td><td>22.6</td></tr><tr><td>0.5 µs</td><td>19.9</td><td>24.2</td><td>26.3</td><td>X</td><td>29.5</td><td>26.1</td></tr><tr><td rowspan="2">S 3</td><td>0.25 µs</td><td>21.4</td><td>22.1</td><td>29.5</td><td>29.5</td><td>X</td><td>20.4</td></tr><tr><td>0.5 µs</td><td>25.5</td><td>22.6</td><td>22.6</td><td>26.1</td><td>20.4</td><td>X</td></tr><tr><td rowspan="6">GAG 2</td><td rowspan="2">S 1</td><td>0.25 µs</td><td>X</td><td>15.3</td><td>20.8</td><td>21.5</td><td>20.2</td><td>21.4</td></tr><tr><td>0.5 µs</td><td>15.3</td><td>X</td><td>21.2</td><td>22.1</td><td>16.8</td><td>19.0</td></tr><tr><td rowspan="2">S 2</td><td>0.25 µs</td><td>20.8</td><td>21.2</td><td>X</td><td>9.7</td><td>16.8</td><td>22.5</td></tr><tr><td>0.5 µs</td><td>21.5</td><td>22.1</td><td>9.7</td><td>X</td><td>18.1</td><td>22.7</td></tr><tr><td rowspan="2">S 3</td><td>0.25 µs</td><td>20.2</td><td>16.8</td><td>16.8</td><td>18.1</td><td>X</td><td>17.5</td></tr><tr><td>0.5 µs</td><td>21.4</td><td>19.0</td><td>22.5</td><td>22.7</td><td>17.5</td><td>X</td></tr><tr><td rowspan="6">GAG 3</td><td rowspan="2">S 1</td><td>0.25 µs</td><td>X</td><td>16.4</td><td>16.1</td><td>25.8</td><td>25.6</td><td>28.6</td></tr><tr><td>0.5 µs</td><td>16.4</td><td>X</td><td>17.9</td><td>19.9</td><td>21.1</td><td>18.2</td></tr><tr><td rowspan="2">S 2</td><td>0.25 µs</td><td>16.1</td><td>17.9</td><td>X</td><td>20.4</td><td>20.3</td><td>27.3</td></tr><tr><td>0.5 µs</td><td>25.8</td><td>19.9</td><td>20.4</td><td>X</td><td>19.0</td><td>19.1</td></tr><tr><td rowspan="2">S 3</td><td>0.25 µs</td><td>25.6</td><td>21.1</td><td>20.3</td><td>19.0</td><td>X</td><td>20.9</td></tr><tr><td>0.5 µs</td><td>28.6</td><td>18.2</td><td>27.3</td><td>19.1</td><td>20.9</td><td>X</td></tr><tr><td rowspan="6">GAG 1 + GAG 2</td><td rowspan="2">S 1</td><td>0.25 µs</td><td>X</td><td>21</td><td>27.1</td><td>23.7</td><td>28.8</td><td>28.9</td></tr><tr><td>0.5 µs</td><td>21.0</td><td>X</td><td>21.9</td><td>20.5</td><td>22.6</td><td>24.2</td></tr><tr><td rowspan="2">S 2</td><td>0.25 µs</td><td>27.1</td><td>21.9</td><td>X</td><td>5.7</td><td>18.2</td><td>16</td></tr><tr><td>0.5 µs</td><td>23.7</td><td>20.5</td><td>5.7</td><td>X</td><td>19.9</td><td>17.9</td></tr><tr><td rowspan="2">S 3</td><td>0.25 µs</td><td>28.8</td><td>22.6</td><td>18.2</td><td>19.9</td><td>X</td><td>12.2</td></tr><tr><td>0.5 µs</td><td>28.9</td><td>24.2</td><td>16.0</td><td>17.9</td><td>12.2</td><td>X</td></tr><tr><td rowspan="6">GAG 1 + GAG 3</td><td rowspan="2">S 1</td><td>0.25 µs</td><td>X</td><td>8.2</td><td>26.9</td><td>21.4</td><td>19.6</td><td>20.3</td></tr><tr><td>0.5 µs</td><td>8.2</td><td>X</td><td>26.0</td><td>23.9</td><td>18.4</td><td>20.0</td></tr><tr><td rowspan="2">S 2</td><td>0.25 µs</td><td>26.9</td><td>26.0</td><td>X</td><td>21.2</td><td>23.9</td><td>25.7</td></tr><tr><td>0.5 µs</td><td>21.4</td><td>23.9</td><td>21.2</td><td>X</td><td>19.8</td><td>21.0</td></tr><tr><td rowspan="2">S 3</td><td>0.25 µs</td><td>19.6</td><td>18.4</td><td>23.9</td><td>19.8</td><td>X</td><td>5.9</td></tr><tr><td>0.5 µs</td><td>20.3</td><td>20.0</td><td>25.7</td><td>21.0</td><td>5.9</td><td>X</td></tr><tr><td rowspan="6">GAG 2 + GAG 3</td><td rowspan="2">S 1</td><td>0.25 µs</td><td>X</td><td>4.8</td><td>25.7</td><td>26.2</td><td>20.8</td><td>27.3</td></tr><tr><td>0.5 µs</td><td>4.8</td><td>X</td><td>27.4</td><td>28.2</td><td>22.3</td><td>27.3</td></tr><tr><td rowspan="2">S 2</td><td>0.25 µs</td><td>25.7</td><td>27.4</td><td>X</td><td>9.2</td><td>20.7</td><td>24.9</td></tr><tr><td>0.5 µs</td><td>26.2</td><td>28.2</td><td>9.2</td><td>X</td><td>21.3</td><td>24.9</td></tr><tr><td rowspan="2">S 3</td><td>0.25 µs</td><td>20.8</td><td>22.3</td><td>20.7</td><td>21.3</td><td>X</td><td>21.4</td></tr><tr><td>0.5 µs</td><td>27.3</td><td>27.3</td><td>24.9</td><td>24.9</td><td>21.4</td><td>X</td></tr></tbody></table>
<table><caption>**Table S7.** Contacts (C, fraction > 0.5) between the core protein of syndecan-2 and the GAG chains during the five MD simulations. Amino acid (AA) residues were numbered according to the UniProtKB entry P34741, and heparan sulfate (HS) chains according to the serine residue they are attached to as described in the Methods section.</caption><thead><tr><th colspan="3">S1</th><th colspan="3">S2</th><th colspan="3">S3</th><th colspan="3">S4</th><th colspan="3">S5</th></tr><tr><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>26</td><td>2</td><td>0.73</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>27</td><td>2</td><td>0.53</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>41</td><td>1</td><td>0.53</td><td></td><td></td><td></td><td>41</td><td>1</td><td>0.60</td><td></td><td></td><td></td><td>41</td><td>1</td><td>0.58</td></tr><tr><td></td><td></td><td></td><td>43</td><td>1</td><td>0.52</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>50</td><td>2</td><td>0.76</td><td></td><td></td><td></td><td>50</td><td>3</td><td>1.14</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>55</td><td>2</td><td>0.92</td><td></td><td></td><td>55</td><td>2</td><td>0.83</td><td></td></tr><tr><td>57</td><td>3</td><td>0.88</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>57</td><td>3</td><td>0.94</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>70</td><td>3</td><td>0.75</td><td></td><td></td><td></td><td>70</td><td>3</td><td>0.65</td></tr><tr><td></td><td></td><td></td><td>71</td><td>3</td><td>0.73</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>72</td><td>3</td><td>1.06</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>76</td><td>3</td><td>0.86</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>85</td><td>3</td><td>1.20</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>89</td><td>3</td><td>0.81</td><td></td><td></td><td></td><td>89</td><td>3</td><td>0.53</td></tr><tr><td>90</td><td>3</td><td>0.88</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>92</td><td>3</td><td>0.89</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>96</td><td>3</td><td>1.45</td><td>96</td><td>3</td><td>0.63</td><td>96</td><td>3</td><td>1.43</td><td></td><td></td><td></td><td>96</td><td>3</td><td>0.79</td></tr><tr><td></td><td></td><td></td><td>100</td><td>3</td><td>0.85</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>101</td><td>3</td><td>0.70</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>102</td><td>3</td><td>1.32</td><td></td><td></td><td></td><td></td><td></td><td></td><td>102</td><td>3</td><td>1.38</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>103</td><td>3</td><td>0.85</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>109</td><td>3</td><td>0.60</td><td></td><td></td><td></td><td>109</td><td>3</td><td>0.72</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>111</td><td>3</td><td>0.99</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>115</td><td>3</td><td>0.54</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>120</td><td>3</td><td>0.57</td><td></td><td></td><td></td><td></td><td></td><td></td><td>120</td><td>3</td><td>1.06</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>126</td><td>3</td><td>0.54</td></tr><tr><td></td><td></td><td></td><td>142</td><td>3</td><td>3.18</td><td>142</td><td>2</td><td>1.10</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>143</td><td>3</td><td>0.62</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>148</td><td>2</td><td>0.88</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>152</td><td>2</td><td>0.91</td><td></td><td></td><td></td><td>152</td><td>2</td><td>0.69</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>**Table S8.** Contacts (C, fraction > 0.5) between the core protein of syndecan-4 and the GAG chains during the five MD simulations. Amino acid (AA) residues were numbered according to the UniProtKB entry P31431, and heparan sulfate (HS) chains according to the serine residue they are attached to as described in the Methods section.</caption><thead><tr><th colspan="3">S1</th><th colspan="3">S2</th><th colspan="3">S3</th><th colspan="3">S4</th><th colspan="3">S5</th></tr><tr><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th><th>AA</th><th>HS</th><th>C</th></tr></thead><tbody><tr><td>22</td><td>1</td><td>0.67</td><td></td><td></td><td></td><td></td><td></td><td></td><td>22</td><td>1</td><td>0.55</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>39</td><td>1</td><td>0.62</td><td>39</td><td>1</td><td>0.55</td><td>39</td><td>1</td><td>0.67</td><td>39</td><td>1</td><td>0.70</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>45</td><td>1</td><td>0.65</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>57</td><td>2</td><td>0.50</td><td></td><td></td><td></td></tr><tr><td>61</td><td>2</td><td>0.60</td><td>61</td><td>2</td><td>0.91</td><td>61</td><td>2</td><td>0.87</td><td>61</td><td>2</td><td>0.81</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>63</td><td>3</td><td>0.53</td><td></td><td></td><td></td><td>63</td><td>3</td><td>0.51</td><td></td><td></td><td></td></tr><tr><td>92</td><td>3</td><td>2.90</td><td>92</td><td>3</td><td>2.45</td><td>92</td><td>1</td><td>1.49</td><td></td><td></td><td></td><td>92</td><td>3</td><td>2.21</td></tr><tr><td></td><td></td><td></td><td>95</td><td>3</td><td>0.71</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>97</td><td>3</td><td>0.62</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>104</td><td>1</td><td>0.74</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>105</td><td>1</td><td>0.78</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>114</td><td>1</td><td>0.55</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>108</td><td>3</td><td>0.51</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>115</td><td>3</td><td>2.05</td><td>115</td><td>1</td><td>2.69</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>128</td><td>3</td><td>0.90</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>156</td><td>3</td><td>0.60</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>**Table S9.** Hydrogen bonds (A: acceptor, D: donor) with an occupancy (Occ) > 0.1 established by the core protein of human syndecan-2 and the three heparan sulfate (HS) chains during the five MD simulations. When H-bonds were established between one amino acid residue and several GAG residues, the occupancy could be higher than 1 since the fractions were summed up per each residue and per GAG chain. The occupancy is ranked from the highest (top) to the lowest (bottom) values. Amino acid (AA) residues were numbered according to the UniProtKB entry P34741.</caption><thead><tr><th colspan="3">Simulation 1</th><th colspan="3">Simulation 2</th><th colspan="3">Simulation 3</th><th colspan="3">Simulation 4</th><th colspan="3">Simulation 5</th></tr><tr><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th></tr></thead><tbody><tr><td>HS 3</td><td>90</td><td>0.46</td><td>HS 3</td><td>142</td><td>1.83</td><td>HS 2</td><td>142</td><td>0.88</td><td>113</td><td>HS 1</td><td>0.59</td><td>50</td><td>HS 3</td><td>0.83</td></tr><tr><td>HS 2</td><td>72</td><td>0.36</td><td>HS 3</td><td>101</td><td>0.59</td><td>HS 3</td><td>70</td><td>0.67</td><td>19</td><td>HS 1</td><td>0.44</td><td>HS 3</td><td>70</td><td>0.56</td></tr><tr><td>105</td><td>HS 3</td><td>0.35</td><td>43</td><td>HS 1</td><td>0.41</td><td>26</td><td>HS 2</td><td>0.46</td><td>43</td><td>HS 1</td><td>0.35</td><td>66</td><td>HS 3</td><td>0.34</td></tr><tr><td>92</td><td>HS 3</td><td>0.29</td><td>HS 3</td><td>100</td><td>0.31</td><td>HS 3</td><td>36</td><td>0.42</td><td>HS 2</td><td>53</td><td>0.23</td><td>HS 3</td><td>89</td><td>0.34</td></tr><tr><td>HS 3</td><td>90</td><td>0.22</td><td>HS 1</td><td>42</td><td>0.25</td><td>148</td><td>HS 2</td><td>0.36</td><td>HS 1</td><td>114</td><td>0.23</td><td>HS 3</td><td>120</td><td>0.32</td></tr><tr><td>62</td><td>HS 2</td><td>0.19</td><td>71</td><td>HS 3</td><td>0.14</td><td>HS 2</td><td>148</td><td>0.32</td><td>59</td><td>HS 2</td><td>0.26</td><td>HS 3</td><td>56</td><td>0.29</td></tr><tr><td>60</td><td>HS 3</td><td>0.19</td><td>115</td><td>HS 3</td><td>0.13</td><td>68</td><td>HS 3</td><td>0.31</td><td></td><td></td><td></td><td>HS 3</td><td>90</td><td>0.28</td></tr><tr><td>33</td><td>HS 1</td><td>0.16</td><td>HS 1</td><td>72</td><td>0.12</td><td>50</td><td>HS 2</td><td>0.29</td><td></td><td></td><td></td><td>HS 3</td><td>72</td><td>0.24</td></tr><tr><td>151</td><td>HS 2</td><td>0.14</td><td>34</td><td>HS 1</td><td>0.12</td><td>HS 3</td><td>90</td><td>0.20</td><td></td><td></td><td></td><td>54</td><td>HS 3</td><td>0.23</td></tr><tr><td>HS 3</td><td>92</td><td>0.13</td><td>HS 3</td><td>117</td><td>0.11</td><td>27</td><td>HS 2</td><td>0.19</td><td></td><td></td><td></td><td>HS 3</td><td>57</td><td>0.16</td></tr><tr><td></td><td></td><td></td><td>HS 3</td><td>115</td><td>0.11</td><td>24</td><td>HS 2</td><td>0.19</td><td></td><td></td><td></td><td>HS 3</td><td>103</td><td>0.14</td></tr><tr><td></td><td></td><td></td><td>71</td><td>HS 3</td><td>0.11</td><td>52</td><td>HS 2</td><td>0.16</td><td></td><td></td><td></td><td>89</td><td>HS 3</td><td>0.12</td></tr><tr><td></td><td></td><td></td><td>HS 2</td><td>53</td><td>0.10</td><td>119</td><td>HS 3</td><td>0.15</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>52</td><td>HS 3</td><td>0.14</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th colspan="3">Simulation 1</th><th colspan="3">Simulation 2</th><th colspan="3">Simulation 3</th><th colspan="3">Simulation 4</th><th colspan="3">Simulation 5</th></tr><tr><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th><th>A</th><th>D</th><th>Occ</th></tr></thead><tbody><tr><td>HS 3</td><td>92</td><td>1.38</td><td>HS 3</td><td>92</td><td>0.75</td><td>HS 1</td><td>115</td><td>1.15</td><td>57</td><td>HS 2</td><td>0.38</td><td>HS 3</td><td>92</td><td>0.97</td></tr><tr><td>108</td><td>HS 3</td><td>0.31</td><td>HS 3</td><td>115</td><td>0.34</td><td>45</td><td>HS 1</td><td>0.32</td><td>HS 1</td><td>22</td><td>0.22</td><td>47</td><td>HS 1</td><td>0.35</td></tr><tr><td>93</td><td>HS 3</td><td>0.24</td><td>157</td><td>HS 3</td><td>0.18</td><td>127</td><td>HS 1</td><td>0.20</td><td>HS 1</td><td>39</td><td>0.11</td><td>91</td><td>HS 3</td><td>0.23</td></tr><tr><td>HS 1</td><td>22</td><td>0.23</td><td>96</td><td>HS 3</td><td>0.14</td><td>124</td><td>HS 1</td><td>0.17</td><td></td><td></td><td></td><td>36</td><td>HS 1</td><td>0.22</td></tr><tr><td>138</td><td>HS 1</td><td>0.23</td><td>95</td><td>HS 3</td><td>0.14</td><td>46</td><td>HS 1</td><td>0.16</td><td></td><td></td><td></td><td>HS 1</td><td>39</td><td>0.14</td></tr><tr><td>58</td><td>HS 2</td><td>0.23</td><td>HS 1</td><td>20</td><td>0.12</td><td>44</td><td>HS 1</td><td>0.13</td><td></td><td></td><td></td><td>37</td><td>HS 1</td><td>0.11</td></tr><tr><td>136</td><td>HS 1</td><td>0.22</td><td>127</td><td>HS 3</td><td>0.11</td><td>116</td><td>HS 1</td><td>0.11</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>133</td><td>HS 1</td><td>0.22</td><td></td><td></td><td></td><td>65</td><td>HS 3</td><td>0.10</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>88</td><td>HS 3</td><td>0.19</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>67</td><td>HS 2</td><td>0.15</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>107</td><td>HS 3</td><td>0.14</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>68</td><td>HS 2</td><td>0.13</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>132</td><td>HS 1</td><td>0.12</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HS 3</td><td>91</td><td>0.11</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Table S10.** Hydrogen bonds (A: acceptor, D: donor) with an occupancy (Occ) > 0.1 established by the core protein of human syndecan-4 and the three heparan sulfate (HS) chains during the five MD simulations. When H-bonds were established between one amino acid residue and several GAG residues, the occupancy could be higher than 1 since the fractions were summed up per each residue and per GAG chain. The occupancy is ranked from the largest (top) to the lowest (bottom) values. Amino acid (AA) residues are numbered according to the UniProtKB entry P31431.