Or 100 surface exposure. Table 2 lists the relative exposure of individual basic residues present in the antithrombin pentasaccharide binding site (PBS) and thrombin exosite II. Figure 1 shows the values for antithrombin and thrombin mapped onto surfaces generated from 1TB6 and 1XMN, respectively. The surface exposure of the basic residues in the HBS of thrombin ranges from 66 to 85 , except for Arg101, which is 35 . In contrast, antithrombin’s residues show a surface exposure range of 39 to 76 , except for Arg13, which displays 91 . Interestingly, only four of eight basic residues in antithrombin are predominantly surface exposed (exposure .2/3rd of fully exposed), while for thrombin, the proportion rises to five out of seven. This simple analysis shows a fundamental difference SQ 34676 between two apparently highly surface-exposed binding sites.Identification of Binding Pockets and Conserved Water MoleculesBinding pockets on the surface of antithrombin and thrombin were detected using the vectorial identification of cavity extents (VICE) algorithm [37] implemented in a local version of HINT [38] as a module within SYBYL. The VICE algorithm was used to Enzastaurin site search for pockets within the HBSs of thrombin and antithrombin (PDB ID = 1TB6). For antithrombin, the HBS was defined to ?include amino acid residues within 10 A of the Nf (NZ) atom of ?Lys125, while for thrombin it was 15 A from the Nf atom of ?Lys236. The grid resolution was set at 0.5 A and the minimum ?closed contour value was set to be 60 A3. The default cavity definition was set to 0.45 and the contour value was set to 0.4. All other variables were kept at their default values. To investigate the extent of hydration, we used the binding site hydration algorithm of HINT [39]. In this approach, a grid-based algorithm combined with the HINT scoring function is used to identify the most probable locations of water molecules in the binding site. The HINT scoring function is atom-based and empirically parameterized and takes the form of equation 6. bij ai Si aj Sj Tij rij zRij ??In this equation, `bij’ is the interaction score between atoms i and j, `a’ is the hydrophobic atom constant, `S’ is the solvent-accessible surface area using a standard H2O probe, `Tij’ is a logic function that has a value of 1 or 21 depending on the nature of the interacting atoms (attractive or repulsive, respectively), `rij’ is a function of the distance between atoms i and j (e2r) and `Rij’ is an implementation of the Lennard ones potential [38]. This formulation implicitly takes into account the entropic component of the free energy of binding of a small molecule, e.g., H2O, with aSpecificity of Heparan Sulfate InteractionsTable 2. Exposed surface area (SA) and radius of gyration (Rg) of arginine and lysine residues in the pentasaccharide binding site of antithrombin and exosite II of thrombin.*?Number of Observations{ Exposed SA ?S.D. (A2) Exposure{ ?Rg (A2)Amino Acid/Protein Antithrombin Lys11 Arg13 Arg46 Arg47 Lys114 Lys125 Arg129 Arg132 Thrombin Arg93 Arg101 Arg126 Arg165 Arg233 Lys236 LysH-bond Partners10 10 9 13 13 10 129261 13263 10663 5663 7862 5963 69647261 9162 7363 3962 6262 4762 47642.19 3.92 3.08 0.32 0.75 1.87 0.63 3.– Asp14 — Ser112, Thr115 Phe122 Asn45 Thr44, Glu414 –11 11 10 11 11 710562 5163 11762 10263 9562 108637361 3562 8062 7062 6662 85622.52 0.77 3.10 0.52 2.20 3.29 1.– Asp100 — Met180 Asp178, Asn179 — Gln*Exposed Surface Area was calculated using the Connolly surface area analysis,.Or 100 surface exposure. Table 2 lists the relative exposure of individual basic residues present in the antithrombin pentasaccharide binding site (PBS) and thrombin exosite II. Figure 1 shows the values for antithrombin and thrombin mapped onto surfaces generated from 1TB6 and 1XMN, respectively. The surface exposure of the basic residues in the HBS of thrombin ranges from 66 to 85 , except for Arg101, which is 35 . In contrast, antithrombin’s residues show a surface exposure range of 39 to 76 , except for Arg13, which displays 91 . Interestingly, only four of eight basic residues in antithrombin are predominantly surface exposed (exposure .2/3rd of fully exposed), while for thrombin, the proportion rises to five out of seven. This simple analysis shows a fundamental difference between two apparently highly surface-exposed binding sites.Identification of Binding Pockets and Conserved Water MoleculesBinding pockets on the surface of antithrombin and thrombin were detected using the vectorial identification of cavity extents (VICE) algorithm [37] implemented in a local version of HINT [38] as a module within SYBYL. The VICE algorithm was used to search for pockets within the HBSs of thrombin and antithrombin (PDB ID = 1TB6). For antithrombin, the HBS was defined to ?include amino acid residues within 10 A of the Nf (NZ) atom of ?Lys125, while for thrombin it was 15 A from the Nf atom of ?Lys236. The grid resolution was set at 0.5 A and the minimum ?closed contour value was set to be 60 A3. The default cavity definition was set to 0.45 and the contour value was set to 0.4. All other variables were kept at their default values. To investigate the extent of hydration, we used the binding site hydration algorithm of HINT [39]. In this approach, a grid-based algorithm combined with the HINT scoring function is used to identify the most probable locations of water molecules in the binding site. The HINT scoring function is atom-based and empirically parameterized and takes the form of equation 6. bij ai Si aj Sj Tij rij zRij ??In this equation, `bij’ is the interaction score between atoms i and j, `a’ is the hydrophobic atom constant, `S’ is the solvent-accessible surface area using a standard H2O probe, `Tij’ is a logic function that has a value of 1 or 21 depending on the nature of the interacting atoms (attractive or repulsive, respectively), `rij’ is a function of the distance between atoms i and j (e2r) and `Rij’ is an implementation of the Lennard ones potential [38]. This formulation implicitly takes into account the entropic component of the free energy of binding of a small molecule, e.g., H2O, with aSpecificity of Heparan Sulfate InteractionsTable 2. Exposed surface area (SA) and radius of gyration (Rg) of arginine and lysine residues in the pentasaccharide binding site of antithrombin and exosite II of thrombin.*?Number of Observations{ Exposed SA ?S.D. (A2) Exposure{ ?Rg (A2)Amino Acid/Protein Antithrombin Lys11 Arg13 Arg46 Arg47 Lys114 Lys125 Arg129 Arg132 Thrombin Arg93 Arg101 Arg126 Arg165 Arg233 Lys236 LysH-bond Partners10 10 9 13 13 10 129261 13263 10663 5663 7862 5963 69647261 9162 7363 3962 6262 4762 47642.19 3.92 3.08 0.32 0.75 1.87 0.63 3.– Asp14 — Ser112, Thr115 Phe122 Asn45 Thr44, Glu414 –11 11 10 11 11 710562 5163 11762 10263 9562 108637361 3562 8062 7062 6662 85622.52 0.77 3.10 0.52 2.20 3.29 1.– Asp100 — Met180 Asp178, Asn179 — Gln*Exposed Surface Area was calculated using the Connolly surface area analysis,.
