Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 39

average oxygen-oxygen distance (3.36 ˚ ) than that observed in the X-ray
structure (around 4.3 ˚ ; Table 2.2 ). The reactant simulation with the
Mg 2 þ ion initially placed at the C-site (RT-C) shows that, although
Mg 2 þ does not move to the bridging position during simulation time
(12 ns), its distance to G10.1:N7 varies from around 2.0 to around 4.5 ˚ .
The distance between the A9 and scissile phosphates jumps to 6 ˚ at the
beginning of the simulation and returns to around 4 ˚ , resulting in an aver-
age of 5.06 ˚ ( Table 2.3 ).
In the deprotonated reactant state simulation (dRT-C), Mg 2 þ migrates
from the C-site to the bridging B-site position after about 2 ns while
maintaining coordination in the axial-equatorial mode. This results in a
shorter average in the A9 and scissile phosphate distance of 2.92 ˚
( Table 2.3 ). In all three reactant simulations with Mg 2 þ ion present, the dis-
tances between the A9 and scissile phosphates remain within 1.5 ˚ of the
crystallographic value of 4.3 ˚ .
In the RT-B simulation, the Mg 2 þ bridges the A9 and scissile phos-
phates, effectively tethering them at a distance of 3-4 ˚ ( Table 2.3 ),
whereas in the RT-C simulation the average distance is 5.06 ˚
( Table 2.3 ). In the deprotonated dRT-C simulation, the negative charge
facilitates the migration of the Mg 2 þ into a near-bridging position such
that results are quite similar to those of the RT-B simulation. It is also note-
worthy that the interaction of the implicated general acid and base
becomesstrongerwithMg 2 þ in a bridging position (either in the RT-B
simulation, of after migration in the dRT-C simulation). These results sug-
gest that in the reactant state the preferred binding mode of Mg 2 þ is at the
C-site, which is between A9 and N7 of G10.1 (through a water mole-
cule) 74,92,95 and that the negatively charged environment near the scissile
phosphate, formed after the initial pH-dependent general base reaction,
brings the Mg 2 þ into a bridging position between A9 and scissile phos-
phates leading to the transition state.
2.4.3 Mg 2þ binding in the transition state
In both ETS and LTS mimic simulations with Mg 2 þ at the bridging position
(ETS-B and LTS-B), the distance between the A9 and scissile phosphates is
around 4 ˚ and the Mg 2 þ coordination between the C1.1 and A9 phosphate
oxygens keeps an axial-axial position along the whole simulations
( Table 2.2 ). The distance between A9 and scissile phosphates in the crystal-
lographic structure is around 4.3 ˚ , which is well suited for Mg 2 þ bridging