Iffiffiffiffiffiffiffiffiffi uP u n u i {xCOM ? z i {yCOM ? z

Iffiffiffiffiffiffiffiffiffi uP u n u i {xCOM ? z i {yCOM ? z i {zCOM ? t Rg i 1 ??nprotein. It has been found empirically that about 500 HINT units correspond to 1 kcal/mol of binding free energy [38]. Water molecule placement was `focused’ in the pocket region, i.e., using the pre-computed cavity detection definition. The parameters for the water placement algorithm were set to ensure that the binding pocket was hydrated completely: the minimum ?water rotein distance was set to 3.0 A, the van der Waals bump scalar was set to 1.02, the minimum H2O 2O contact distance ?was set to 2.5 A, and the minimum HINT score for placement of a water was set at 21000. An analysis of the relevance of each water molecule in the cavity was performed using the Water Rank and Score Report function of HINT, where Rank is a parameter encoding the quantity and quality of hydrogen bonds a water molecule may make [40]. An additional derived parameter, Relevance, correlates with water conservation [41].Results Estimation of the Exposed Surface Area of Basic ResiduesThe MOLCAD functionality of SYBYL was used to generate a Fast Connolly surface for individual basic residues within the context of the HBS while taking into account neighboring residues; only the surface area that is exposed is included in the surface calculation. To generate a value for the maximal exposed surface area for each amino acid type, an analogous Connolly surface was generated for the central residue of a tripeptide Ala?X la with an ideal a-helical backbone conformation. The percent exposure value for each basic residue was calculated by dividing the HBS exposed surface area by its maximal exposed surface area. Although a number of crystal AKT inhibitor 2 site structures for thrombin and antithrombin have been available for several years, a thorough and quantitative exploration of their heparin binding order KS-176 regions has not been performed up until now. In fact, the previous descriptions of these sites have been quite qualitative, e.g., “the size of the thrombin-binding site can even be as small as mono- or disaccharide fragment” [42]. By application of a number of unique computational structure analysis tools the characteristics of these HBSs are here described.Surface Exposure of Basic Residues Present in the HBSThe binding site of GAGs on proteins is usually considered to be surface-exposed and readily accessible [30]. This implies that the basic residues of the HBSs are generally assumed to be fully exposed to the bulk solvent. However, are all basic side chains equally exposed? More importantly, does surface exposure of the HBS residues vary significantly amongst heparin-binding proteins (HBPs), especially between antithrombin and thrombin? The HBS of antithrombin consists of Lys11, Arg13, Arg46, Arg47, Lys114, Lys125, Arg129 and Arg132 residues, while in thrombin the basic residues are Arg93, Arg101, Arg126, Arg165, Arg233, Lys236 and Lys240. Of these, Lys114, Lys125 and Arg129 of antithrombin and Arg93, Arg101, Arg233, Lys236 and Lys240 of thrombin are important contributors to H/HS recognition [43], [44]. The exposed (water accessible) surface areas of each of these residues present in heparin co-crystal structures were calculated using the Fast Connolly surface ?generation algorithm. In this process, a sphere of 1.4 A, which simulates a water molecule, is rolled on the protein surface and the area of contact for each residue measured. A tripeptide Ala ?Ala, with X = Lys or Arg, was used as a control f.Iffiffiffiffiffiffiffiffiffi uP u n u i {xCOM ? z i {yCOM ? z i {zCOM ? t Rg i 1 ??nprotein. It has been found empirically that about 500 HINT units correspond to 1 kcal/mol of binding free energy [38]. Water molecule placement was `focused’ in the pocket region, i.e., using the pre-computed cavity detection definition. The parameters for the water placement algorithm were set to ensure that the binding pocket was hydrated completely: the minimum ?water rotein distance was set to 3.0 A, the van der Waals bump scalar was set to 1.02, the minimum H2O 2O contact distance ?was set to 2.5 A, and the minimum HINT score for placement of a water was set at 21000. An analysis of the relevance of each water molecule in the cavity was performed using the Water Rank and Score Report function of HINT, where Rank is a parameter encoding the quantity and quality of hydrogen bonds a water molecule may make [40]. An additional derived parameter, Relevance, correlates with water conservation [41].Results Estimation of the Exposed Surface Area of Basic ResiduesThe MOLCAD functionality of SYBYL was used to generate a Fast Connolly surface for individual basic residues within the context of the HBS while taking into account neighboring residues; only the surface area that is exposed is included in the surface calculation. To generate a value for the maximal exposed surface area for each amino acid type, an analogous Connolly surface was generated for the central residue of a tripeptide Ala?X la with an ideal a-helical backbone conformation. The percent exposure value for each basic residue was calculated by dividing the HBS exposed surface area by its maximal exposed surface area. Although a number of crystal structures for thrombin and antithrombin have been available for several years, a thorough and quantitative exploration of their heparin binding regions has not been performed up until now. In fact, the previous descriptions of these sites have been quite qualitative, e.g., “the size of the thrombin-binding site can even be as small as mono- or disaccharide fragment” [42]. By application of a number of unique computational structure analysis tools the characteristics of these HBSs are here described.Surface Exposure of Basic Residues Present in the HBSThe binding site of GAGs on proteins is usually considered to be surface-exposed and readily accessible [30]. This implies that the basic residues of the HBSs are generally assumed to be fully exposed to the bulk solvent. However, are all basic side chains equally exposed? More importantly, does surface exposure of the HBS residues vary significantly amongst heparin-binding proteins (HBPs), especially between antithrombin and thrombin? The HBS of antithrombin consists of Lys11, Arg13, Arg46, Arg47, Lys114, Lys125, Arg129 and Arg132 residues, while in thrombin the basic residues are Arg93, Arg101, Arg126, Arg165, Arg233, Lys236 and Lys240. Of these, Lys114, Lys125 and Arg129 of antithrombin and Arg93, Arg101, Arg233, Lys236 and Lys240 of thrombin are important contributors to H/HS recognition [43], [44]. The exposed (water accessible) surface areas of each of these residues present in heparin co-crystal structures were calculated using the Fast Connolly surface ?generation algorithm. In this process, a sphere of 1.4 A, which simulates a water molecule, is rolled on the protein surface and the area of contact for each residue measured. A tripeptide Ala ?Ala, with X = Lys or Arg, was used as a control f.