Deciphering the Self-Cleavage Reaction Mechanism of Hairpin Ribozyme

Abstract

Hairpin ribozyme catalyzes the reversible self-cleavage of phosphodiester bonds which plays prominent roles in key biological processes involving RNAs. Despite impressive advances on ribozymatic self-cleavage, critical aspects of its molecular reaction mechanism remain controversially debated. Here, we generate and analyze the multidimensional free energy landscape that underlies the reaction using extensive QM/MM metadynamics simulations to investigate in detail the full selfcleavage mechanism. This allows us to answer several pertinent yet controversial questions concerning activation of the 2′-OH group, the mechanistic role of water molecules present in the active site, and the full reaction pathway including the structures of transition states and intermediates. Importantly, we find that a sufficiently unrestricted reaction subspace must be mapped using accelerated sampling methods in order to compute the underlying free energy landscape. It is shown that lower-dimensional sampling where the bond formation and cleavage steps are coupled does not allow the system to sufficiently explore the landscape. On the basis of a three-dimensional free energy surface spanned by flexible generalized coordinates, we find that 2′-OH is indirectly activated by adjacent G8 nucleobase in conjunction with stabilizing H-bonding involving water. This allows the proton of the 2′-OH group to directly migrate toward the 5′-leaving group via a nonbridging oxygen of the phosphodiester link. At variance with similar enzymatic processes where water wires connected to protonable side chains of the protein matrix act as transient proton shuttles, no such de/ reprotonation events of water molecules are found to be involved in this ribozymatic transesterification. Overall, our results support an acid-catalyzed reaction mechanism where A38 nucleobase directly acts as an acid whereas G8, in stark contrast, participates only indirectly via stabilizing the nascent nucleophile for subsequent attack. Moreover, we conclude that self-cleavage of hairpin ribozyme follows an A N + D N two-step associative pathway where the rate-determining step is the cleavage of the phosphodiester bond. These results provide a major advancement in our understanding of the unique catalytic mechanism of hairpin ribozyme which will fruitfully impact on the design of synthetic ribozymes.

Towards solvent regulated self-activation of N-terminal disulfide bond oxidoreductase-D

“…networks can be expected for this transfer. There has been considerable importance given for studying water networkassisted proton exchange in enzyme catalysis, 24,30,[79][80][81][82][83] which is inter-linked with intrinsic pK a for the side chain of the concerned residues. For example, the Grotthuss mechanism 84 of proton transfer (propagation via H 3 O + ) happens when the side chain of the terminal residues are acidic (pK a { 7) whereas proton-hole like (OH À ) propagation 85 ensues when the side chain residues are basic (pK a c 7).…”

“…[27][28][29] For example, the self-cleavage mechanism in hairpin ribosomes proceeds via proton transfer involving internal nucleophiles. 30 Moreover, Neves et al 19 reported nucleophilic disulfide bond rupture by the proton transfer mechanism in Protein Disulfide Isomerase (PDI), the mammalian counterpart of nDsbD. A similar reaction leading to the disulfide bond formation in nDsbD Red was suggested by Goulding and co-workers, 31 but the reaction mechanism remains undeciphered to date.…”

On the Adaptability of the Chemical Reaction of Hairpin Ribozyme to High Pressures

“…This profile has been computed from the minimum free energy pathway by using the corresponding three-dimensional free energy surface sampled at 10 kbar. Obviously, the chemical reaction at multi-kilobar pressure follows a strikingly different, yet much simpler pathway from what is known at 1 bar conditions. At such high pressures, the reaction occurs via a single-step concerted (A N D N ) mechanism, in stark contrast to the multistep associative (A N + D N ) mechanism at atmospheric pressure.…”

“…In what follows, we report the effects of multi-kilobar pressures exclusively on the covalent chemical reaction which underlies the self-cleavage process of HpRz. Building upon our previous work on this chemical reaction exclusively at ambient conditions and on our detailed investigations of rather global conformational and local solvation processes at ambient and also high pressures ,, (as summarized in Supporting Information section 5) together with pertinent computational studies of ribozymes, − we map the multidimensional free energy landscape of the chemical steps in HpRz self-cleavage using extensive ab initio QM/MM metadynamics simulations at high pressure conditions. Direct comparison to the reference scenario found at ambient conditions provides striking differences with considerable impact on the adaptability of HpRz to multi-kilobar pressure conditions.…”

“…Reaction free energy profiles of the chemical self-cleavage steps of HpRz at 10 kbar (a) vs 1 bar (b) pressures obtained from multidimensional QM/MM metadynamics sampling (see the text and Supporting Information; the 1 bar reference is adapted from ref ). R, TS, INT, and P represent the respective reactant, transition, intermediate, and product states according to the underlying distinctly different reaction mechanisms provided in Figure .…”