Figure 3. Two key water molecules obtained by structural clustering analyses. (A) The representative structure of the most dominant cluster from md2 and md6 MD simulation trajectories; (B) The number distributions of water molecules within 5.00 Å of the reaction site H1901 and LSub respectively; (C) The distributions of the distances between the Cα atom of LSub and the O atom of the water molecules (Wat3466 and Wat4258) in md2 and md6 trajectories respectively. The light blue shaded area indicated the water molecule stayed within 5.00 Å of the Cα atom for a period of time.
Amazingly, two key water molecules were found in the representative structure of the most dominant cluster from md2 and md6 MD simulation trajectories (Figure 3A). One of the water molecules WatA stayed between the Nε atom of H1901 and the Hα atom of LSub. Because the distance d(H1901-WatAO) and d(LSub-WatAO) was small enough, two possible deprotonation paths were proposed, including direct deprotonation by H1901 and indirect deprotonation mediated by WatA. In order to confirm the feasibility of indirect deprotonation path, the total number of water molecules near the reaction site was counted (Figure 3B). The average numbers of water molecules within 5.00 Å of Nε and Hα atoms were 5.70 and 6.70 respectively, indicating there were abundant water molecules around the active site for indirect deprotonation. In addition, two water molecules Wat380 and Wat5671 were selected randomly from md2 and md6 trajectories to verify they could retain between H1901 and LSub for a certain time in 4 ns simulations (Figure S4).
In the opposite direction of the deprotonation site, the water molecule WatB was 3.98 Å away from the Cα atom of LSub, and formed hydrogen bonds (Hb1, Hb2 and Hb3) with the backbone of H1808 and the β-lactam ring of LSub. Owing to no other residue as the proton donor, the water molecule WatB was thought to provide the proton for the Cα atom from si face, finishing the L to D stereochemical inversion of the C-terminal Hpg. Similarly, two water molecules Wat3466 and Wat4258 were selected randomly. The distances between the Cα atom of LSub and the O atom of these water molecules and the populations of three important hydrogen bonds were analyzed (Figure 3C, Table S2). As these data shown, Wat3466 and Wat4258 could stayed within 5.00 Å of the Cα atom for a long time through these hydrogen bond interactions, getting ready for the subsequent re-protonation step. These similar structural characteristics concerning key water molecules were likewise observed in the pentapeptide covalently bound to NocTE system (Figure S5-S7, Table S3).