Determination of the active site protonation state of beta-secretase from molecular dynamics simulation and docking experiment: implications for structure-based inhibitor design

Memapsin 2 (BACE) is an aspartyl protease known as Beta-secretase that acts on the production of the beta-amyloid peptide in the human brain, a key event in the pathogenesis of Alzheimer's disease. Although it is expected that the net charge of the catalytic Asp diad would be -1 as in other kinds of aspartyl proteases, the exact protonation states of Asp32 and Asp228 have not been known without ambiguity. Two independent molecular dynamics (MD) simulations of BACE in complex with the potent inhibitor OM99-2 are carried out to determine the preferred protonation state of the Asp diad in the context that is consistent with the previous X-ray crystal structure. The results show that a strong hydrogen bond between the inhibitor hydroxyl group and Asp228 can be maintained only when Asp32 is neutral and Asp228 is ionized. The preference of this protonation state is further supported from the energetic and structural features found in the docking experiment of a novel potent inhibitor with the BACE active site. Thus, both MD and docking studies suggest that the role of hydrogen bond acceptor for the hydroxyl and piperazine groups of the inhibitors should be played by Asp228 instead of Asp32. This may be a key piece of information for the structure-based design/discovery of new inhibitor drugs.