Assigning the protonation states of the key aspartates in β-Secretase using QM/MM X-ray structure refinement.

β-Secretase, a.k.a. β-APP cleaving enzyme (BACE), is an aspartyl protease that has been implicated as a key target in the pathogenesis of Alzheimer's disease (AD). The identification of the protonation states of the key aspartates in β-secretase is of great interest both in understanding the reaction mechanism and in guiding the design of drugs against AD. However, the resolutions of currently available crystal structures for BACE are not sufficient to determine the hydrogen atom locations. We have assigned the protonation states of the key aspartates using a novel method, QM/MM X-ray refinement. In our approach, an energy function is introduced to the refinement where the atoms in the active site are modeled by quantum mechanics (QM) and the other atoms are represented by molecular mechanics (MM). The gradients derived from the QM/MM energy function are combined with those from the X-ray target to refine the crystal structure of a complex containing BACE and an inhibitor. A total number of 8 protonation configurations of the aspartyl dyad were considered and QM/MM X-ray refinements were performed for all of them. The relative stability of the refined structures was scored by constructing the thermodynamic cycle using the energetics calculated by fully quantum mechanical self-consistent reaction field (QM/SCRF) calculations. While all 8 refined structures fit the observed electron density about equally well, we find the mono-protonated configurations to be strongly favored energetically, especially the configuration with the inner oxygen of Asp32 protonated and the hydroxyl of the inhibitor pointing towards Asp228. It was also found that these results depend on the constraints imposed by the X-ray data. We suggest that one of the strengths of this approach is that the resulting structures are a consensus of theoretical and experimental data and remark on the significance of our results in structure based drug design and mechanistic studies.