Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 49

ions. 130-132,134 Furthermore, the specific metal ion-binding modes in the
reactant state and the activated precursor state (with the nucleophile
C17:2 0 OH deprotonated) are also explored. Compared to the simulations
performed in Section 2 , here we significantly extended the simulation time
in order to further study the long-time dynamics of eHHR. The detailed
differences of simulation setup and protocols are discussed in Section 4.2 .
The following series of long-time simulations (300 ns each) were set up
( Table 2.3 ):
1. RT-C-Mg, the reactant state with Mg 2 þ at the C-site

RT-B-Mg, the reactant state with Mg 2 þ at the B-site 2.

dRT-C-Mg, the activated precursor with Mg 2 þ at the C-site 3.

dRT-B-Mg, the activated precursor with Mg 2 þ at the B-site

4.

RT-Na, the reactant state in the absence of Mg 2 þ (presence of only
Na þ )

5.

dRT-Na, the activated precursor in the absence of Mg 2 þ (presence of
only Na þ )
The dRT-C-Mg simulation of the activated precursor with Mg 2 þ initially
placed at the C-site quickly (less than 200 ps) leads to migration of the Mg 2 þ
ion into the B-site position, as was observed in a previously simulation study
( Section 2 and Ref. 75,76 ), and afterward exhibited nearly identical behavior
as if the Mg 2 þ ion was initially placed at the B-site (dRT-B-Mg). Therefore,
we only extended the dRT-B-Mg simulation to 300 ns and designated it
simply as dRT-Mg. All five simulations were carried out to 300 ns in a back-
ground of 0.14 M NaCl. The equilibration of each simulation was moni-
tored by the root-mean-square-deviation (RMSD) and reached steady
states after 30-50 ns. Hence, all analyses were performed over the last
250 ns of trajectories for each simulation.

6.

4.1.1 Threshold occupancy and specific coordination patterns of ions
are highly correlated with catalytically active conformations
To understand the relationship between the active in-line conformation and
the metal ion-binding patterns, we first examine the metal ion occupation
and coordination numbers in the active site. Four key coordination sites are
used for characterizing metal ion binding ( Fig. 2.6 ): G8:O2 0 , A9:O2P, C1.1:
O2P, and C17:O2 0 . The averages of the following three quantities were cal-
culated for both Mg 2 þ and Na þ ions: N is the number of ions with at least
one coordination to any one of the four key coordination sites, CN is the
total coordination number of ions with at least one coordination to any