The two-step algorithm for identification of GBSs on proteins and elucidating their specificity.

<p>The process involves preparation of protein; identification of neutral hydrogen bond donors in the structure; calculation of 2DSE plots for the protein; and evaluation of ‘hot spots’ for deduction of specificity of GAG–protein interaction.</p

The structural basis for existence of hot spots in GBSs.

<p>Nature has designed specific GBSs by placing neutral hydrogen bond donors such as the ND2 and NE2 atoms of Asn and Gln respectively in close proximity to charged Arg or Lys residues, as seen in (a) antithrombin, (b) FGF2, (c) HS3ST3A1, (d) HS3ST1 and (e) HS2ST1. This close proximity maximizes the G_ES at these residues, thereby generating a specific GBS. Not all atoms are displayed, for the sake of visual clarity.</p

Specific proteins demonstrate unique, non-uniform distributions of electrostatic potential across neutral hydrogen-bond donors.

<p>Specific proteins such as antithrombin, FGF2, HS3ST1 and HS3ST3A1 demonstrate at least one location of electrostatic potential that deviates significantly from the mean. Nonspecific GAG-binding sites on proteins such as thrombin and serum albumin demonstrate a uniform, Gaussian distribution of the same, so no location is preferred significantly over another.</p

Polar residues present in various GBSs.

<p>These residues form direct interactions with GAGs, as evidenced by analysis of crystal structures.</p

<i>G</i>_<i>ES</i> at arginines and/or lysines does not identify the GBS on a protein.

<p><i>G</i>_<i>ES</i> for Arg/Lys residues are mapped using 2DSE plots for multiple GAG-binding proteins including <b>(a)</b> antithrombin, <b>(b)</b> thrombin, <b>(c)</b> FGF2, <b>(d)</b> HS3ST3A1, <b>(e)</b> HS3ST1 and <b>(f)</b> HS2ST1. The maps reveal that GAG-binding site Arg/Lys residues may not always possess high <i>G</i>_<i>ES</i> and not all Arg/Lys with high <i>G</i>_<i>ES</i> on a protein are part of the GAG-binding site.</p

Desolvation energy is critical for quantitative analysis of GAG–protein interaction.

<p>Neither ΔG_ES (<b>a</b> and <b>b</b>) nor ΔG_DS (<b>c</b> and <b>d</b>) alone explain the change in ΔG_OBS for antithrombin (<b>a</b> and <b>c</b>) and thrombin (<b>b</b> and <b>d</b>) mutants studied to date. Any enthalpic gain due to electrostatics is opposed by desolvation (R² = 0.99) in antithrombin <b>(e)</b> as well as in thrombin <b>(f)</b>, suggesting that desolvation is critical for quantitative analysis of GAG-protein interactions. In all cases, the correlation was found to be significant at α = 0.05.</p

2DSE plots for <i>G</i>_<i>ES</i> at neutral hydrogen bond donors.

<p>GAGs bind neutral hydrogen bond donors on the protein that possess significantly high G_ES. <b>(a)</b> Asn45 of antithrombin GAG-binding site possesses the highest G_ES within the structure. <b>(b)</b> In contrast, the nonspecific thrombin GAG-binding site demonstrates a diffused G_ES. Similarly, significantly high <i>G</i>_<i>ES</i> are observed at <b>(c)</b> Asn27 of the FGF2 GBS; Asn27Ala mutation affects GAG-binding (ΔΔG~1.1 kcal/mol) almost as much as K125A (ΔΔG~1.7 kcal/mol), which had the largest effect, <b>(d)</b> Asn255 of the HS3ST3A1 GAG-binding site; the N255A mutant is inactive, and <b>(e)</b> Gln163 of HS3ST1; Gln163Ala mutant loses ~65% activity. <b>(f)</b> Diffused G_ES of HS2ST1 may represent its ability to bind low-sulfated GAGs. However, Asn91 and 112 of the HS2ST1 GAG-binding site possess a potential higher than His106, mutation of which is already known to affect GAG-binding.</p

Electrostatic interactions and desolvation energies for AT-heparin pentasaccharide complexes reported in the literature.

<p>Electrostatic interactions and desolvation energies for AT-heparin pentasaccharide complexes reported in the literature.</p

Effect of electrostatic interactions on binding of various thrombin mutants with heparin.

<p>Effect of electrostatic interactions on binding of various thrombin mutants with heparin.</p

Designing Allosteric Inhibitors of Factor XIa. Lessons from the Interactions of Sulfated Pentagalloylglucopyranosides

We recently introduced sulfated pentagalloylglucopyranoside (SPGG) as an allosteric inhibitor of factor XIa (FXIa) (Al-Horani et al., J. Med Chem. 2013, 56, 867–878). To better understand the SPGG–FXIa interaction, we utilized eight SPGG variants and a range of biochemical techniques. The results reveal that SPGG’s sulfation level moderately affected FXIa inhibition potency and selectivity over thrombin and factor Xa. Variation in the anomeric configuration did not affect potency. Interestingly, zymogen factor XI bound SPGG with high affinity, suggesting its possible use as an antidote. Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa. Inhibition studies in the presence of heparin showed marginal competition with highly sulfated SPGG variants but robust competition with less sulfated variants. Resolution of energetic contributions revealed that nonionic forces contribute nearly 87% of binding energy suggesting a strong possibility of specific interaction. Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa. An SPGG molecule containing approximately 10 sulfate groups on positions 2 through 6 of the pentagalloylglucopyranosyl scaffold may be the optimal FXIa inhibitor for further preclinical studies