Protein-ligand complexes for MD simulations were prepared as follows: 1) the crystal structure of C3b with Cp40 was prepared using the Protein Preparation Wizard as implemented in Maestro . Missing residues 76–77 were reconstructed and hydrogen atoms were added, assuming the physiological pH of 7.4. After protein-protein alignment, the binding site waters resolved at chains D and H of the C3c-compstatin complex were added to the model.
The first 24 ns of each simulation were considered an equilibration phase and thus were not included in the subsequent analyses. The binding affinities were calculated using the MMGB/SA analysis based on 50 frames sampled between 24 and 48 ns of the MD simulation. An in-house script was used to evaluate per residue intramolecular as well as intermolecular hydrogen bonding and hydrophobic interactions of compstatin and its analogue with the C3b.