PB0535 - Structural basis of activated factor X binding to antithrombin not activated by heparinoids, insights from enhanced sampling molecular dynamics simulations

Background: The activity of antithrombin, a critical regulator of the coagulation process, is significantly enhanced by high affinity heparin pentasaccharide against factor Xa and IXa. An X-ray diffraction structure has been published for pentasaccharide-activated antithrombin complexed with an inactivated factor Xa mutant. However, the currently available information about the binding of the non-activated serpin to FXa is limited to mutagenesis data.

Aims: Our aim was to build model systems using protein docking and enhanced sampling molecular dynamics simulations that can describe the binding of factor Xa to antithrombin in its non-activated state.

Methods: We have performed molecular docking using the available structures of the two proteins using HADDOCK (the GLA domain was removed from factor Xa). The highest-ranked structure was then used in Gaussian Accelerated Molecular Dynamics simulations for further refinement of the structure and to evaluate its stability. Additionally, we started simulations based on the available X-ray structure using the same method, both for systems containing and not containing the pentasaccharide.

Results: Based on the molecular docking and enhanced sampling molecular dynamics simulations, we propose a model structure in which factor Xa can interact with non-activated antithrombin in a conformation with a “closed” hinge region and no heparin pentasaccharide bound. The simulations show differences in the interaction with the “exosite” of antithrombin compared to the available X-ray structure for the complex with the activated form. The proposed complex conformation is in good agreement with the available mutagenesis data. From the simulations based on the X-ray structure with the pentasaccharide deleted, we have investigated the changes in conformation compared to the ligand-bound form. These changes provide information on how the pentasaccharide affects the interaction with FXa allosterically.

Conclusion(s): Our results provide a deeper insight into the mechanisms how pentasaccharide ligands activate antithrombin against its target factors.