Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM

Abstract

The hepatitis delta virus ribozyme is an efficient catalyst of RNA 2 ′ -O-transphosphorylation and has emerged as a key experimental system for identifying and characterizing fundamental features of RNA catalysis. Recent structural and biochemical data have led to a proposed mechanistic model whereby an active site Mg 2+ ion facilitates deprotonation of the O2 ′ nucleophile, and a protonated cytosine residue (C75) acts as an acid to donate a proton to the O5 ′ leaving group as noted in a previous study. This model assumes that the active site Mg 2+ ion forms an inner-sphere coordination with the O2 ′ nucleophile and a nonbridging oxygen of the scissile phosphate. These contacts, however, are not fully resolved in the crystal structure, and biochemical data are not able to unambiguously exclude other mechanistic models. In order to explore the feasibility of this model, we exhaustively mapped the free energy surfaces with different active site ion occupancies via quantum mechanical/ molecular mechanical (QM/MM) simulations. We further incorporate a three-dimensional reference interaction site model for the solvated ion atmosphere that allows these calculations to consider not only the rate associated with the chemical steps, but also the probability of observing the system in the presumed active state with the Mg 2+ ion bound. The QM/MM results predict that a pathway involving metal-assisted nucleophile activation is feasible based on the rate-controlling transition state barrier departing from the presumed metal-bound active state. However, QM/MM results for a similar pathway in the absence of Mg 2+ are not consistent with experimental data, suggesting that a structural model in which the crystallographically determined Mg 2+ is simply replaced with Na + is likely incorrect. It should be emphasized, however, that these results hinge upon the assumption of the validity of the presumed Mg 2+ -bound starting state, which has not yet been definitively verified experimentally, nor explored in depth computationally. Thus, further experimental and theoretical study is needed such that a consensus view of the catalytic mechanism emerges.

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

“…By comparing the pK a shifts with/without Mg 2+ in Table 3, we can estimate the net effect of Mg 2+ on the pK a of G12 to be (−1.2) − 3.7 = −4.9 pK a units, which is in qualitative agreement with our DFT result −7.0. In the hepatitis delta virus (HDV) ribozyme, which has a similar divalent metal ion requirement under physiological conditions, a pK a shift of similar magnitude (~−4 units) on the 2′-hydroxyl nucleophile induced by Mg 2+ was predicted by 3D-RISM calculations(95). Further, Amaro and co-workers have reported the Mg 2+ -induced pK a shift of lysine K82 in guanylyltransferase mRNA capping enzyme to be −4.2(94) which is very close to the result reported here using a similar TI approach.…”

Theory and Modeling of RNA Structure and Interactions with Metal Ions and Small Molecules

“…the model gives two peaks in the ion density distribution around a nucleic acid helix, a result observed in explicit MD simulation. Because the 3D-RISM model takes into account both the ion-ion and the ion-solvent correlations, it can also be used to investigate specific binding of ions (118). …”

Exploring RNA structure and dynamics through enhanced sampling simulations

“…A more robust alternative is provided by replicaexchange US simulations, used for instance in Refs. [32,41,59], at least if continuous trajectories are analyzed and transition events are detected as discussed above for T-REMD ( Figure 2). We notice that several works related to catalysis took advantage of the string method in order to sample http://creativecommons.org/licenses/by-nc-nd/4.0/ a reactive pathway [60,62,64,67].…”

“…Many of the works discussed here used simple geometric CVs such as distances between atoms or atom groups. Chemical reactions are typically accomplished by biasing a combination of distances, where some describe newly formed or expired contacts and others enforce related proton transfers [59,60,64,65,66,67,69]. Dihedral angles can be used to enforce the exploration of multiple rotamers [21,24,26,27,32,43].…”