Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 51

coordination site, and NB is the number of ions which coordinate to at least
two of the four coordination sites.
The active conformation can be examined by the distribution of the
nucleophile in-line attack angle and distance. Their distributions from all
simulations are shown in Fig. 2.7 and manifest two k-mean clusters 136 : clus-
ter A (light gray) containing structures in the near in-line attack conforma-
tions, and cluster B (dark gray) containing structures not in the near in-line
attack conformations, with the exception of the dRT-Mg simulation where
only cluster A is found.
The metal ion occupation and coordination numbers were then ana-
lyzed for each cluster and the results are listed in Table 2.6 .Duringallsim-
ulations, when present in the active site, one Mg 2 þ ion remained stably
bound at full occupancy (NMg 2 þ

1.00). In the reactant state simulations
(RT-C-Mg and RT-B-Mg), there is no significant population of Na þ ions
in the active site (NNa þ > 0.1), which suggests that the Mg 2 þ ion is suf-
ficient to neutralize the local charge of the A9 and scissile phosphates.
In the RT-C-Mg (Mg 2 þ at the C-site) simulation, the Mg 2 þ ion directly
coordinates only A9:O2P of the RNA (CNMg 2 þ

¼

¼ 1.00), and thus is not

involved in a bridge (NBMg 2 þ 0.00). Cluster A (in-line conformation)

represents approximately 21% of the sampled data over the last 250ns of
simulation, whereas cluster B (not in-line conformation) represents the
remaining 79%.
The RT-B-Mg (Mg 2 þ at the B-site) simulation, on the other hand, is
dramatically different. In this simulation, the Mg 2 þ ion directly coordi-
nates both A9:O2P and the scissile phosphate C1.1:O2P of the RNA
(CNMg 2 þ

¼

2.00)asastablebridge(NBMg 2 þ 1.00). Cluster A, con-

taining a high degree of in-line near attack conformations, represents
the vast majority of the sampled data (over 99%), whereas cluster B is
observed less than 1% of the time. This suggests that a bridging Mg 2 þ
ion contributes to stabilization of catalytically active conformations in
the reactant simulations.
This feature is even more pronounced in the dRT-Mg simulations (acti-
vated precursor state with Mg 2 þ ). There is only a single cluster with in-line
conformation (

¼ ¼

155). For dRT-Mg, an additional negative charge in the
active site arises from deprotonation of the nucleophile, and a single Na þ ion
is observed in the active site (NNa þ

y ¼

¼ 0.97) that makes direct coordination

to only one RNA ligand (CNNa þ ¼ 1.01) with essentially no bridging

interactions.