Rity of H/HS rotein interactions rely upon long-range and non-directional Coulombic interactions,which have a 1/r distance-dependence ?as compared to van der Waals forces with a 1/r3 to 1/r6 dependence. It is known that sulfate groups ( SO3? of H/HS can recognize arginines through the formation of directional, bidentate interactions [34], i.e., possessing both strong Coulombic and hydrogen bond components, and thus substantively enhancing binding energy. This implies that engineering specificity is possible through arginine ?sulfate interaction. Yet, even though thrombin has at least five arginine residues in its heparin-binding site (HBS), its interaction is non-specific. Beyond antithrombin /HS and thrombin /HS systems, no other Nobiletin protein /HS system has been studied extensively both in solution and in crystal form. Despite this limitation, understanding the differences in how antithrombin and thrombin recognize H/ HS is expected to provide a template for specificity features that can drive interactions of H/HS. Thus, we developed a simple structure analysis approach to explore the differences in HBSs of these proteins. Computation of solvent accessibilities and gyrational mobilities of arginines and lysines in the HBSs of the two proteins and analysis of their crystallographic thermal B-factors reveal sharp contrasts. Evaluating the distribution of positive charges in the two proteins reveals considerable asymmetry in antithrombin in contrast to substantial symmetry in thrombin. Cavity detection techniques suggest that although both HBSs are surface exposed, there are subtle differences between the two that allow H/HS to form a `hand-shake’ with antithrombin, while interacting only in a more transient `high-five’ with thrombin. Furthermore, there are differences in the solvation of these pockets that differentially affect the energetics of binding. Cumulatively, these differences in the binding sites result in major differences in recognition of H/HS sequences, which help explain specificity of binding. The work presents a foundation for understanding specificity at an atomic level and will be of value in the design of natural or synthetic H/HS sequences that target proteins in a specific manner.Tubastatin-A web Methods Computational Software/HardwareSYBYL-X 1.3 (Tripos International, St. Louis, MO) was used for molecular visualization and for in silico structural manipulation. Statistical analyses reported herein were also performed using SYBYL-X and implemented using SYBYL Programming Language (SPL). Molecular modeling was performed on Intel Xeonand AMD Opteron-based CentOS 5.5 Linux and Intel Xeonbased Mac OS-X 10.6 (Snow Leopard) MacPro graphical workstations.Antithrombin and Thrombin CoordinatesCrystal structures of antithrombin and thrombin co-crystallized with heparin or heparin-like fragments, obtained from the RCSB protein data bank (http://www.rcsb.org/pdb/), were used to analyze intra- and intermolecular interactions (Table 1). Coordinates of antithrombin and thrombin from 1TB6 [35] and the `A’ and `B chains of 1XMN [20] were extracted and used for cavity analysis and prediction of bound water studies. The unresolved heavy atoms of Lys240 in 1TB6 and Lys236 in 1XMN were added and assigned an extended conformation. Hydrogen atoms were added to each protein with SYBYL-X 1.3. The B-factors, which represent in part the thermal motion and potential disorder of atoms in an X-ray crystal structure, were analyzed for all side chain atoms in the struc.Rity of H/HS rotein interactions rely upon long-range and non-directional Coulombic interactions,which have a 1/r distance-dependence ?as compared to van der Waals forces with a 1/r3 to 1/r6 dependence. It is known that sulfate groups ( SO3? of H/HS can recognize arginines through the formation of directional, bidentate interactions [34], i.e., possessing both strong Coulombic and hydrogen bond components, and thus substantively enhancing binding energy. This implies that engineering specificity is possible through arginine ?sulfate interaction. Yet, even though thrombin has at least five arginine residues in its heparin-binding site (HBS), its interaction is non-specific. Beyond antithrombin /HS and thrombin /HS systems, no other protein /HS system has been studied extensively both in solution and in crystal form. Despite this limitation, understanding the differences in how antithrombin and thrombin recognize H/ HS is expected to provide a template for specificity features that can drive interactions of H/HS. Thus, we developed a simple structure analysis approach to explore the differences in HBSs of these proteins. Computation of solvent accessibilities and gyrational mobilities of arginines and lysines in the HBSs of the two proteins and analysis of their crystallographic thermal B-factors reveal sharp contrasts. Evaluating the distribution of positive charges in the two proteins reveals considerable asymmetry in antithrombin in contrast to substantial symmetry in thrombin. Cavity detection techniques suggest that although both HBSs are surface exposed, there are subtle differences between the two that allow H/HS to form a `hand-shake’ with antithrombin, while interacting only in a more transient `high-five’ with thrombin. Furthermore, there are differences in the solvation of these pockets that differentially affect the energetics of binding. Cumulatively, these differences in the binding sites result in major differences in recognition of H/HS sequences, which help explain specificity of binding. The work presents a foundation for understanding specificity at an atomic level and will be of value in the design of natural or synthetic H/HS sequences that target proteins in a specific manner.Methods Computational Software/HardwareSYBYL-X 1.3 (Tripos International, St. Louis, MO) was used for molecular visualization and for in silico structural manipulation. Statistical analyses reported herein were also performed using SYBYL-X and implemented using SYBYL Programming Language (SPL). Molecular modeling was performed on Intel Xeonand AMD Opteron-based CentOS 5.5 Linux and Intel Xeonbased Mac OS-X 10.6 (Snow Leopard) MacPro graphical workstations.Antithrombin and Thrombin CoordinatesCrystal structures of antithrombin and thrombin co-crystallized with heparin or heparin-like fragments, obtained from the RCSB protein data bank (http://www.rcsb.org/pdb/), were used to analyze intra- and intermolecular interactions (Table 1). Coordinates of antithrombin and thrombin from 1TB6 [35] and the `A’ and `B chains of 1XMN [20] were extracted and used for cavity analysis and prediction of bound water studies. The unresolved heavy atoms of Lys240 in 1TB6 and Lys236 in 1XMN were added and assigned an extended conformation. Hydrogen atoms were added to each protein with SYBYL-X 1.3. The B-factors, which represent in part the thermal motion and potential disorder of atoms in an X-ray crystal structure, were analyzed for all side chain atoms in the struc.
