The effects of Glu202 protonation states on AChE-ACh complex
Now that it is clear the protonation states of Glu202 play important role in stabilizing the key hydrogen bond network and consequently stabilizing the catalytic triad in apo AChE. Whether the same effects could be observed when AChE binds a substrate remains uncertain. To answer this question, we have performed ~100 ns MD simulations with ACh binding at the active site. As we can see from Figure 4A, the RMSD trace of the protonated Glu202 vibrates around small values, indicating a stable Glu202 that stays firmly at its crystal structure position, whereas the RMSD trace of the deprotonated Glu202 vibrates around large values, showing a Glu202 that moves away from its crystal structure position throughout the entire simulation. Similar behaviors are observed for the catalytic His447. In Figure 4B, we can see that His447 also stays firmly at its crystal position when Glu202 is protonated, and dramatically moves away when Glu202 is deprotonated.
Shown in Figure 5 are the comparisons between the representative MD-simulated structures and the crystal structure. In Figure 5A, the key hydrogen bond network is almost identical to that in the crystal structure. The lengths of four hydrogen bonds are 1.8±0.1 Å, 1.9±0.2 Å, 1.8±0.1 Å, and 2.4±0.3 Å, respectively, implying a very stable hydrogen bond network throughout the entire simulation where Glu202 is protonated. As expected, Figure 5B shows that the hydrogen bond network is collapsed when Glu202 is deprotonated, illustrating that the deprotonated Glu202 causes the collapse of the key hydrogen bond network no matter there is ACh binding at the active site or not. As for the catalytic His447, the representative MD-simulated structure, which is given in Figure 5C, clearly shows that the His447 is very close to that in the crystal structure. The distances of the two hydrogen bonds in the catalytic triad are 1.9±0.2 Å, and 1.7±0.1 Å, respectively, indicating a very stable catalytic triad with Glu202 being protonated. However, if Glu202 is deprotonated, as shown in Figure 5D, the catalytic His447 deviates significantly away from its crystal structure position, and the hydrogen bond between His447 and Ser203, which is critical in maintaining the catalytic triad, is lost due to the shift of His447. Shown in Figure 5E and F are the binding modes of ACh with Glu202 being protonated and deprotonated, respectively. In both structures, the averaged distances between the carbonyl oxygen of ACh and the oxyanion hole are no more than 3.2 Å, and their standard deviations are very small, indicating that ACh binds tightly with the oxyanion hole regardless of the protonation state of Glu202. Also, the distances between the carbonyl carbon of ACh and the hydroxyl oxygen of Ser203 are 3.4±0.4 Å with Glu202 being protonated and 3.2±0.2 Å with Glu202 being deprotonated, suggesting the Ser203 is at an appropriate position for the nucleophilic attack no matter what the protonation state of Glu202 is. Apparently, the protonation states of Glu202 barely alter the binding mode of ACh. Actually, if Glu202 is deprotonated, ACh binds slightly tighter with the oxyanion hole and also slightly closer to the nucleophilic hydroxyl group of Ser203. However, as clearly shown in Figure 5F, the catalytic His447 significantly moves away from Ser203, and thus lost its role in assisting the nucleophilic attack on ACh performed by Ser203. As a result, the catalytic hydrolysis of ACh hardly starts and the catalytic efficiency should be remarkably decreased if Glu202 is deprotonated.