Assessing the Potential Effects of Active Site Mg2+ Ions in the glmS Ribozyme-Cofactor Complex

Abstract

Ribozymes employ diverse catalytic strategies in their self-cleavage mechanisms, including the use of divalent metal ions. This work explores the effects of Mg²⁺ ions in the active site of the glmS ribozyme-GlcN6P cofactor complex using computational methods. Deleterious and potentially beneficial effects of an active site Mg²⁺ ion on the self-cleavage reaction were identified. The presence of a Mg²⁺ ion near the scissile phosphate oxygen atoms at the cleavage site was determined to be deleterious, and thereby anticatalytic, due to electrostatic repulsion of the cofactor, disruption of key hydrogen-bonding interactions, and obstruction of nucleophilic attack. On the other hand, the presence of a Mg²⁺ ion at another position in the active site, the Hoogsteen face of the putative base, was found to avoid these deleterious effects and to be potentially catalytically favorable owing to the stabilization of negative charge and pK_a shifting of the guanine base.

Figure 1

(A) Structure of the glmS holoribozyme from Thermoanaerobacter tengcongensis (PDB ID 2Z75). Modified from ref (20). The crystal structure consists of two chains, an oligomer substrate (gray), and a motif from the glmS ribozyme RNA (blue). The active site consists of A-1 (red), G1 (black), G40 (green), and the cofactor (orange). The Mg²⁺ ions in the crystal structure are represented by pink spheres. (B) Illustration of the active site in the glmS ribozyme and the proposed self-cleavage mechanism indicated by the red arrows. The pro-R_P and pro-S_P oxygens of the scissile phosphate are labeled with subscripts R and S, respectively. Dashed black lines indicate important hydrogen bonds in the active site. The potential positions of the active site Mg²⁺ ion examined in this Letter are indicated by dotted circles. They are denoted “Site 1”, which is near the nonbridging oxygens of the scissile phosphate, and “Site 2”, which is near the Hoogsteen face of G40.

Figure 2

QM/MM optimized geometries for the canonical state (A) without a Mg²⁺ ion at the cleavage site, (B) with a Mg²⁺ ion near the pro-R_P oxygen, and (C) with a Mg²⁺ ion near the pro-S_P oxygen. For simplicity, the pro-R_P and pro-S_P oxygens are labeled as R and S respectively. Dashed black lines indicate the GlcN6P(O1):pro-R_P and A-1(O2′):G40(N1) hydrogen bonds when present.

Figure 3

QM/MM optimized geometries for the canonical state (A) without a Mg²⁺ ion at the Hoogsteen face of G40, (B) with a Mg²⁺ ion near G40(O6), and (C) with a Mg²⁺ ion near G40(N7). For simplicity, the pro-R_P and pro-S_P oxygens are labeled as R and S, respectively. Dashed black lines indicate GlcN6P(O1):pro-R_P and A-1(O2′):G40(N1) hydrogen bonds when present.