All-Atom Simulations Decrypt the Molecular Terms of RNA Catalysis in the Exon-Ligation Step of the Spliceosome

Borišek, Jure; Magistrato, Alessandra

doi:10.1021/acscatal.0c00390

Cited by 28 publications

(61 citation statements)

References 58 publications

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“…The QM atoms were treated using fully self-consistent Kohn–Sham density functional theory (DFT) using the BLYP functional , with the D3 dispersion correction included. Here, we favored BLYP functional as it allows for a convenient balance of accuracy and computational cost (given the extensive sampling required to carry out QM/MM metadynamics simulations to compute three-dimensional free energy surfaces with sufficient sampling statistics) and since it has been shown by others to perform well for several RNA systems. ,− Importantly, we additionally benchmarked for our specific QM description of hairpin ribozyme the performance of BLYP for all transition and intermediate states involved in the multistep self-cleavage reaction with respect to a linear-scaling variant, as explained in the Supporting Information, of the CCSD(T) method, the latter being often denoted as the “gold-standard in quantum chemistry”. Using such system-specific benchmark data, we find a mean absolute deviation (MAD) in the range of 2 ± 1 kcal/mol considering all states, thus suggesting a sufficient accuracy when using the BLYP functional to carry out the large-scale QM/MM metadynamics simulations which underly the current study (more validation details are provided in the Supporting Information).…”

Section: Methodsmentioning

confidence: 99%

“…As such, it plays a prominent role in many biological processes such as splicing of RNA, controlling of gene expression, and processing of tRNA to name but a few. − Chemically speaking, the mechanism of the self-cleavage reaction involves a nucleophilic attack of an endogenous, activated nucleophile (being the 2′-OH group of the same ribozyme) at the scissile phosphate and the subsequent breaking of the phosphodiester bond. This results in the formation of a 2′,3′-cyclic phosphate product and a 5′-OH group terminus, thus leading to site-specific scission of the RNA strand. ,, Unlike most other ribozymes, − the hairpin ribozyme does not require the presence of a divalent metal ion in the reaction site to be able to catalyze its self-cleavage; rather particular adjacent nucleobases, namely, A38 and G8, play an important role in the catalysis. ,,− The participation of nucleobases in lieu of metal ions is of significant importance as it allows the ribozyme to function in a standalone manner and, moreover, also increases the site-specificity much needed in gene-specific therapy and RNA processing. − Overall, major advances on understanding the self-cleavage reaction of hairpin ribozyme have been achieved over the decades, ,− ,− which are too numerous to be comprehensively referenced herein.…”

Section: Introductionmentioning

confidence: 99%

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Deciphering the Self-Cleavage Reaction Mechanism of Hairpin Ribozyme

Kumar

Marx

2020

J. Phys. Chem. B

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Deciphering the Self-Cleavage Reaction Mechanism of Hairpin Ribozyme

Kumar

Marx

2020

J. Phys. Chem. B

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“…One possible pathway is the transfer of the proton from the attacking group to the leaving group, mediated by the phosphate undergoing the substitution. 14,15 However, this scheme may not explain many of the experimental studies. 16,17,18 Figure 1: A) Superposition of Bh RNase H in crystallo reactant after 40ns (PDB 6DMV 19 , olive cartoon), HIV-1 QM/MM reactant model (gray cartoon, model based on PDB 1RTD 20 /1ZBL 6 )) and Hs RNase H (PDB 2QKK 21 , light cyan cartoon).…”

Section: Introductionmentioning

confidence: 99%

“…The identities of the general base deprotonating the nucleophilic water and the protonation of the leaving group leading to a stable product are still elusive. One possible pathway is the transfer of the proton from the attacking group to the leaving group mediated by the phosphate undergoing the substitution. , However, this scheme may not explain many of the experimental studies. − …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…Pre-messenger RNA (pre-mRNA) splicing, i.e., the removal of the intronic sequences from a nascent pre-mRNA transcript and the ligation of exons to generate a mature mRNA, is orchestrated by the spliceosome (SPL), a multi-megadalton protein/RNA machinery [1]. The SPL preserves genomic integrity by precisely recognizing/selecting specific sequences such as the 5 and the 3 splice sites (5 SS and 3 SS, respectively), at the exons/introns boundaries, and the branch point sequence (BPS), which contains the conserved branch point adenosine (BPA, the nucleophile of the first splicing step) [2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Investigating the Molecular Mechanism of H3B-8800: A Splicing Modulator Inducing Preferential Lethality in Spliceosome-Mutant Cancers

Spinello

Borišek

Malcovati

et al. 2021

IJMS

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The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre-mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B-8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B-8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome-mutant cancer cells remains elusive. Here, microsecond-long all-atom simulations addressed the binding/dissociation mechanism of H3B-8800 to wild type and K700E SF3B1-containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B-8800 binding. This supports the hypothesis that the selectivity of H3B-8800 towards mutant cancer cells is unrelated to its preferential targeting of K700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B-8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug-discovery efforts to specifically target mutant SF3b.

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All-Atom Simulations Decrypt the Molecular Terms of RNA Catalysis in the Exon-Ligation Step of the Spliceosome

Cited by 28 publications

References 58 publications

Deciphering the Self-Cleavage Reaction Mechanism of Hairpin Ribozyme

Deciphering the Self-Cleavage Reaction Mechanism of Hairpin Ribozyme

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

Investigating the Molecular Mechanism of H3B-8800: A Splicing Modulator Inducing Preferential Lethality in Spliceosome-Mutant Cancers

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