Endostatin (ES) is an endogenous angiogenesis inhibitor that has great capacity of solid tumors suppression. There are many hypotheses about its ligands and mechanisms of action. One possibility is that ES could form complexes, interacting with cell surface heparan sulfate (HS), since ES is easily purified using a heparin affinity column. Crystallographic analysis has shown that ES presents a dimeric organization. The relevance of this oligomerization for ES-HS interactions is still unknown.
    The first aim of this work was the construction and validation of the heparin topology parameters (bonds, angles, dihedrals, etc) for GROMOS96 force field, which is often used for proteins but lacks some sugar parameters. Besides, to understand the behavior of the ES-heparin complexes, we decided to analyze its stability using simulations, varying ES oligomerization state.
    In order to perform Molecular Dynamics (MD) with GROMACS software, the heparin topology was created in building blocks (saccharide units) using charges described by Verli et al. (2003) in both 1C4 and 2S0 states of the iduronate ring defined by dihedral angles. We identified low energies complexes with AUTODOCK. After that, using the complex structures of lowest docking energies we carried out 3ns MD simulations in an aqueous solution.
     Heparin dodecasaccharides was validated, resembling NMR angles and dihedrals with minor fluctuations. Docking analysis showed that dimers have better energies of interaction with heparin than monomers. Interestingly, all accepted heparin conformations for ES dimer belong to interface. Preliminary MD simulations on heparin-ES complexes demonstrated the stability of these complexes but with a non-specific interaction with arginine amino acid residues.
     In this work we provided sugar parameters in building blocks, which is important to any computational study of complexes involving heparin binding proteins. New models of ES-HS complexes, based on SAS sequences proposed by Kreuger et al. (2002), were suggested by docking analysis now interacting with ES dimers.
    At present, we are making less sulfated saccharides building blocks to construct a HS sequence that resembles the physiological ligand of ES, perhaps leading to more specific interactions in MD simulations than heparin.