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. 2023 Dec 20;14(1):8482.
doi: 10.1038/s41467-023-44174-2.

Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome

Jana Aupič  1 , Jure Borišek  2 , Sebastian M Fica  3 , Wojciech P Galej  4 , Alessandra Magistrato  5
Affiliations

Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome

Jana Aupič et al. Nat Commun. .

Abstract

Cleavage and formation of phosphodiester bonds in nucleic acids is accomplished by large cellular machineries composed of both protein and RNA. Long thought to rely on a two-metal-ion mechanism for catalysis, structure comparisons revealed many contain highly spatially conserved second-shell monovalent cations, whose precise function remains elusive. A recent high-resolution structure of the spliceosome, essential for pre-mRNA splicing in eukaryotes, revealed a potassium ion in the active site. Here, we employ biased quantum mechanics/ molecular mechanics molecular dynamics to elucidate the function of this monovalent ion in splicing. We discover that the K+ ion regulates the kinetics and thermodynamics of the first splicing step by rigidifying the active site and stabilizing the substrate in the pre- and post-catalytic state via formation of key hydrogen bonds. Our work supports a direct role for the K+ ion during catalysis and provides a mechanistic hypothesis likely shared by other nucleic acid processing enzymes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Scheme of the spliceosome complex pre-organized for the first transesterification reaction.
a Cryo-EM structure of the yeast C complex spliceosome (PDBID: 7B9V) used in this study. Relevant splicing factors (Yju2, Cwc25), small nuclear RNAs (U2, U5, and U6), and pre-mRNA are shown in the new cartoon representation. The protein core is shown as a white surface. b Close up view of the active site containing two Mg2+ ions (M1 and M2) and K+ ion (K1) along with their coordinating residues. c Branching is initiated by the attack of the branch point adenosine (BPA) in the intron sequence on the 5’-splice site, leading to the formation of the intron lariat-3’-exon intermediate and free 5’-exon.
Fig. 2
Fig. 2. Branching reaction follows a two-step associative mechanism.
a Close-up view of the reaction site in the reactant state. The reaction proceeds by the nucleophilic 2’-OH attacking the scissile phosphate (P), with O3’ acting as the leaving group. Distances between relevant atoms are labeled from d1 to d11. Mg2+ ions are labeled as M1 and M2, K+ as K1. b Helmholtz free energy (F) as a function of the reaction coordinate (RC). The reactant (R) and the product state (P) are separated by the phosphorane-like intermediate state (I). The reaction is characterized by two transition states (TS1 and TS2) and two proton transfer steps (PT1 and PT2). The free energy profile was obtained by integrating the mean constraint force along the RC. At each RC, the mean constraint force was obtained over 6000 frames. The corresponding errors were obtained from SD using error propagation. c Distances between atom pairs depicted in panel a as a function of the reaction coordinate. For clarity, only distances exhibiting a marked change as the reaction progressed are shown. Data are presented as mean values ± SD (n = 1000 frames). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Hydrogen bonding facilitates proton transfer from the nucleophile to the leaving group via the scissile phosphate.
The active site is shown in the reactant state (R), during the first proton transfer (PT1), in the intermediate state (I), during the second proton transfer (PT2), and in the product state (P). The intermediate state is stabilized by water-assisted hydrogen bonding.
Fig. 4
Fig. 4. Substitution of K+ ion by Li+ increases the activation barrier for the branching reaction.
a Active site in the pre-reaction state after equilibration with QM/MM MD. b Helmholtz free energy (F) as a function of the reaction coordinate (RC) in the presence of Li+ ion (in green) in comparison to the free energy profile obtained when K+ ion was bound in the active site instead (in violet). As in the case of the K+ ion, the reaction exhibits an intermediate state (I), two transition states (TS1 and TS2), and two proton transfer steps (PT1 and PT2). The arrows denote the activation barrier and the free energy difference between the reactant (R) and product state (P). The free energy profiles were obtained by integrating the mean constraint force over the RC. At each RC value, the mean constraint force was calculated from the last 6000 frames. The corresponding errors were obtained from SD using error propagation. c Distances between atom pairs depicted in panel a as a function of the reaction coordinate. Data are presented as mean values ± SD (n = 1000 frames). For clarity, only distances exhibiting a marked change during the reaction are shown. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Li+ ion negatively affects hydrogen bonding in the active site and hinders proton transfer.
a Snapshots of the active site during the branching reaction simulated in the presence of the Li+ ion. The active site is shown in the reactant state (R), during the first proton transfer (PT1), in the intermediate state (I), during the second proton transfer (PT2), and in the product state (P). b Frequency of hydrogen bonding between 2’-OH of BPA and G(+1)-O(Sp) before the branching reaction (hb1) and 3’-OH of G(−1) and G(+1)-O(Sp) after the reaction (hb2) as a function of the reaction coordinate. The violet and green bars report values obtained when K+ or Li+ ion, respectively, was bound in the active site. The distributions of distances and angles between relevant atoms, which served as the basis for calculating the frequency of hydrogen bonding, are shown in Supplementary Figs. 15 and 16. Source data are provided as a Source Data file.
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