The Interaction of Factor Xa and IXa with Non-Activated Antithrombin in Michaelis Complex: Insights from Enhanced-Sampling Molecular Dynamics Simulations.

The interaction between coagulation factors Xa and IXa and the activated state of their inhibitor, antithrombin (AT),have been investigated using X-ray diffraction studies. However, only mutagenesis data are available for non-activated AT. Our aim was to propose a model based on docking and advanced-sampling molecular dynamics simulations that can reveal the conformational behavior of the systems when AT is not binding a pentasaccharide. We built the initial structure for non-activated AT-FXa and AT-FIXa complexes using HADDOCK 2.4. The conformational behavior was studied using Gaussian accelerated molecular dynamics simulations. In addition to the docked complexes, two systems based on the X-ray structures were also simulated, with and without the ligand. The simulations revealed large variability in conformation for both factors. In the docking-based complex of AT-FIXa, conformations with stable Arg150-AT interactions can exist for longer time periods but the system also has a higher tendency for reaching states with very limited interaction with the "exosite" of AT. By comparing simulations with or without the pentasaccharide, we were able to gain insights into the effects of conformational activation on the Michaelis complexes. RMSF analysis and correlation calculations for the alpha-carbon atoms revealed important details of the allosteric mechanisms. Our simulations provide atomistic models for better understanding the conformational activation mechanism of AT against its target factors.