The Role of Conserved Residues in the DEDDh Motif: the Proton-Transfer Mechanism of HIV-1 RNase H

Abstract

RNase H is a prototypical example for two metal ion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present in important drug targets, such as the HIV-1 reverse transcriptase but also in many other nucleases such as Homo sapiens (Hs), Escherichia coli (Ec) RNases H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton transfer events are not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2'O atom of the sugar. Additionally, we also describe an alternative proton transfer mechanism via a conserved catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs and Ec RNase H, with the calculated pKa values of the DEDD residues and with crystallographic studies. The overall reaction barrier of ∼19 kcal mol -1 , encountered in the phosphate cleavage step, matches the slow experimental rate of ∼1-100 min -1 . Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of two-metal ion catalytic phosphate cleaving enzymes with a DDE motif.

QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1α RNase

“…They obtained an activation barrier energy of 15.5 kcal mol –1 for the rate-determining step, in agreement with experimental data. Using DFT (B3LYP) and CHARMM 27 force field, Dürr et al 76 conducted a series of extensive QM/MM calculations combined with Hamiltonian replica exchange to elucidate the RNA cleavage mechanism of HIV-1 “RNase H”. They found an overall reaction barrier of ∼19 kcal mol –1 , associated with the phosphate-cleavage step, which matches the experimental rate.…”

Catalytic Fields as a Tool to Analyze Enzyme Reaction Mechanism Variants and Reaction Steps

“…Information about conserved residues has sometimes been helpful in the search for enzyme reaction mechanisms; , however, to our knowledge, there has not yet been an attempt in the literature to verify alternative enzyme reaction mechanism variants by the systematic matching of all conserved amino acids with CFs.…”

“…This indicates the significant flexibility of charged side chains already reported in other related studies, 30−32 which will also be extensively explored in this work. Information about conserved residues has sometimes been helpful in the search for enzyme reaction mechanisms; 38,39 however, to our knowledge, there has not yet been an attempt in the literature to verify alternative enzyme reaction mechanism variants by the systematic matching of all conserved amino acids with CFs.…”

Accelerating ab initio QM/MM Molecular Dynamics Simulations with Multiple Time Step Integration and a Recalibrated Semi-empirical QM/MM Hamiltonian

“…However, due to their high computational cost, the routine application of the ai-QM/MM potentials is often limited to minimum energy pathway (MEP) calculations, where only O(10 2 ) QM/MM energy/force calculations are required to optimize each point along a discretized reaction pathway. Less common is the direct employment of ai-QM/MM models in the minimum free energy a) Electronic mail: jpu@iupui.edu b) Electronic mail: kwangho.nam@uta.edu c) Electronic mail: yihan.shao@ou.edu pathway (MFEP) simulations, [21][22][23][24][25][26][27][28][29][30] which typically involve O (10 4 ) QM/MM energy/force calculations to sample each point/window along the free energy pathway and thus require hundreds of thousands (if not millions) of computer core-hours. Consequently, direct QM/MM MFEP simulation often resort to less-accurate semiempirical QM/MM (se-QM/MM) Hamiltonians based on AM1, 31 PM3, 32 PM6, 33 or tight-binding DFT [34][35][36][37] methods.…”