Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 57

which happens during most of the simulation. During the periods of sim-
ulation where three Na þ ions bind to different ligand sites simulta-
neously, the in-line angle comes to a reaction-competent value (150 ).
Figure 2.8 illustrates the different Na þ binding patterns for cluster
A(definedin Table 2.6 , in-line conformation, Fig. 2.8 lower panels)
and cluster B (not in-line conformation, Fig. 2.8 upper panels) from
the dRT-Na simulation. The in-line cluster A clearly exhibits three
Na þ bridges that involve C17:O2 0 /C1.1:O2P, C1.1:O2P/G8:O2 0 ,and
C1.1:O2P/A9:O2P. For cluster B, on the other hand, the first two of
these bridges are absent with the third one being significantly less pro-
nounced. The above analysis suggests that the compensation of the neg-
ative charges of these three coordination sites, as well as the bridging
binding patterns of Na þ that bring them together, is necessary to keep
the in-line conformation in the deprotonated activated precursor state,
although the binding patterns are not as rigid as those of Mg 2 þ .
4.1.4 The HHR active site forms a local electronegative recruiting
pocket for cation occupation
In this section, we examine the preferential occupation of cations in the
HHR active site. The 3D density contour maps for the Na þ ion distribution
determined over the last 250 ns of simulation ( Fig. 2.9 ) show that the Na þ
ion density at a medium contour level (left panels, Fig. 2.9 ) is located near
the RNA's phosphate backbone, whereas at high contour level (right panels,
Fig. 2.9 ) the highest probability Na þ occupation sites were all concentrated
in the active site for both the reactant and activated precursor. No explicit
Na þ ions were initially placed in the active site, and Na þ ion exchange
events were observed to occur during simulations. This suggests that the
HHR folds to form a strong local electronegative pocket that attracts
Mg 2 þ or Na þ ions. A similar case has been observed in the tetraloop-
receptor complex analyzed by NMR, where the divalent ions were exper-
imentally found to be located at strong electronegative positions formed by
the RNA fold. 137 Together with the known divalent metal ion binding at
the C-site, these results provoke the speculation that perhaps the active sites
of some ribozymes such as the HHR have evolved to form electrostatic
cation-binding pockets that facilitate catalysis. In the case of the HHR, this
speculation is further supported by the simulated correlation of cation-
binding mode with the formation of active conformations discussed in detail
in the previous sections.