Protonated Glu202 stabilizes the hydrogen bond network
To investigate the effects of the protonation states of Glu202, we have performed two sets of ~100 ns MD simulations, where the only difference is the protonation state of Glu202. Shown in Figure 2A is the RMSD traces of protonated Glu202 with respect to its crystal structure position. Clearly, the protonated Glu202 is pretty stable and stays at its initial crystal position throughout the entire simulation. However, as we can see from Figure 2B where Glu202 is deprotonated, the RMSD traces show that Glu202 is quite unstable, and moves away from its crystal structure position for most of the time during the simulation. To understand why the protonated Glu202 is much more stable than the deprotonated Glu202, we have compared the representative MD-simulated structures to the crystal structure. In Figure 2C, the carbon atoms of the representative structure and of the crystal structure are rendered with orange and green colors, respectively. We can see that the key hydrogen bond network in the simulated structure (orange carbons) is almost identical to that in the crystal structure (green carbons). There are four stable hydrogen bonds associate with the centered water molecule. This water molecule points its two hydrogen atoms toward Glu450 and Ser229, respectively. Thus, for the remaining two hydrogen bonds, the water oxygen needs to accept the hydrogen atoms from Gly448 and Glu202, respectively, evidently showing the Glu202 needs to be protonated. Clearly, if the Glu202 is deprotonated, then repulsion occurs between the water oxygen atom and the Glu202 carboxyl group, pushing the Glu202 away from its crystal structure position. As we can see from Figure 2D, where the carbon atoms of the representative MD-simulated structure are rendered with light blue color, the deprotonated Glu202 significantly deviates from its crystal structure position. The Glu450 also deviates largely from its crystal position. Importantly, the key water molecule is disappeared, suggesting the key hydrogen bond network is collapsed. This observation is consistent with what we discovered in the previous study, where the key hydrogen bond network in BChE eventually collapses without the support from Glu202.
Apparently, due to the multiple hydrogen bonds associate with the centered water molecule, the Glu202 needs to be protonated to join and stabilize the hydrogen bond network, whereas the deprotonated Glu202 can not join it, eventually resulting in the collapse of the key hydrogen bond network.