Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 40

coordination. However, in both ETS and LTS mimic simulations with
Mg 2 þ initially at the C-site position (ETS-C and ETS-C), the Mg 2 þ ion
migrates from the C-site to the bridging B-site position in less than 0.5 ns
and remains at the B-site (bridging position) for the remainder of the sim-
ulation. A similar situation is found in the deprotonated dRT-C simulation
(see above). The migration is also indicated by the broken coordination
between the Mg 2 þ ion and G10:N7 (details not shown here). This obser-
vation may again suggest that the B-site is the preferred position for
Mg 2 þ when an additional negative charge is accumulated at the scissile phos-
phate in formation of the transition state.
2.5. The big picture
In this section, we present a series of MD simulations of the eHHR in solu-
tion to study the Mg 2 þ -binding mode and conformational events at different
stages along the catalytic pathway. Our results suggest an HHR model
whereby the active site forms a region of local negative charge that requires
electrostatic stabilization to preserve its structural integrity, and that this sta-
bilization can be effected by divalent metal ion binding at the C-site or the
B-site in the ground (prereactive) state. A Mg 2 þ ion is observed to weakly
bind at the C-site position at solvent separation with G10.1, facilitating the
formation of near in-line attack conformations, particularly when in the
bridging position where there is increased interaction between the nucleo-
phile (C17:O2 0 ) and the implicated general base (G12).
Deprotonation of the nucleophile is correlated with the migration of the
Mg 2 þ from the C-site into a bridging position (B-site), and with the forma-
tion of the dianionic transition state, suggesting that the accumulation of
negative charge around the scissile phosphate center is sufficient to induce
a change in the binding mode of the Mg 2 þ . Once in the bridging position in
the transition state, the Mg 2 þ ion interacts with the O5 0 leaving group of
C1.1 and the 2 0 OH of G8, the implicated general acid catalyst. The
B-site Mg 2 þ ion can act both as a Lewis acid catalyst to stabilize directly
the accumulating charge on the O5 0 leaving group and can induce a p K a
shift on the 2 0 OH of G8 to facilitate general acid catalysis. Upon proton
transfer from G8:O2 0 to C1.1:O5 0 , the Mg 2 þ is poised to directly stabilize
the resulting 2 0 alkoxide (which could occur synchronously). Combined
QM/MM simulations on two points (LTS and ETS) along the reaction
coordinates suggest that the barrier for the general acid proton transfer step
may be sufficiently low so as to occur on the nanosecond timescale.