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 H1901Nε and
LSubHα 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(H1901Nε-WatAO) and
d(LSubHα-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 H1901Nε and LSubHα 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).