Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 54

On the other side, the simulations without an active site Mg 2 þ ion are
considerably different than those with the divalent ion present. In the
reactant RT-Na simulation, cluster A is dominant (87% of the time)
and shows a high degree of in-line conformations ( 153). Most of

cluster A population contains a single Na þ ion in the active site
(NNa þ

y ¼

1.15), less frequently two Na þ ions, and the average number
of bridging Na þ ions is 0.88. On the other hand, cluster B has a slightly
higher active site Na þ occupation (NNa þ

¼

¼ 1.38), but a lower average

number of bridging ions (0.66).
In the activated precursor dRT-Na simulation, the average Na þ occu-
pancy (NNa þ ) increases to approximately 3 and 2.5 for clusters A and B,
respectively. Cluster B (not in-line conformation) is the dominate popula-
tion occurring 76% of the time. Cluster A (in-line conformation) occurs
24% of the time.
A striking feature that distinguishes cluster A from B is that it exhibits a
very high degree of bridging ion character in addition to higher Na þ occu-
pancy. For cluster A, the average number of these ions that coordinate at
least two RNA ligands (NBNa þ ) is 2.68, while the number is only 1.36
for cluster B. These results suggest that the bridging coordination patterns
are highly correlated with formation of in-line conformations for both cases
with and without Mg 2 þ ions. Besides the above ion occupation and coor-
dination number analysis, we further look into the specific binding patterns
for both cases with or without the Mg 2 þ ion.

4.1.2 A bridging Mg 2þ ion maintains rigid coordination patterns
that stabilize in-line attack conformations
In this section, we compare the effect of different Mg 2 þ -binding modes
in both the neutral reactant and activated (deprotonated 2 0 OH) precursor
states on the active site structure and fluctuations. Table 2.7 lists the aver-
ages of key in-line indexes, the A9/scissile phosphate-phosphate distance,
and Mg 2 þ coordination distances for the RT-C-Mg, RT-B-Mg, and
dRT-Mg simulations. Figure 2.6 shows a general schematic view of
the active site metal ion coordination from the simulations. The distances
and standard deviations in Table 2.7 indicate that the Mg 2 þ ion retains
rigid coordination with the phosphate oxygens over the course of the
simulation, being directly coordinated to A9:O2P in all simulations. In
the RT-C-Mg simulation, the Mg 2 þ ion coordinates G10.1:N7 indi-
rectly through one of four inner-sphere water molecules. However, this