Identification of the Catalytic Mg²⁺ Ion in the Hepatitis Delta Virus Ribozyme

Abstract

The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pKa shifted towards neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the pre-cleavage ribozyme identified a Mg2+ ion that interacts through its partial hydration sphere with the G25•U20 reverse wobble. In addition, this Mg2+ ion is in position to directly coordinate the nucleophile, the 2’-hydroxyl of U(-1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2’-hydroxyl. To test the role of the active site Mg2+ ion, we replaced the G25•U20 reverse wobble with an isosteric A25•C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg2+ binding could be adversely affected by the mutation. Kinetic analysis and molecular dynamics of the A25•C20 double mutant suggest that this variant stably folds into an active structure. However, pH-rate profiles of the double mutant are inverted relative to the profiles for wild-type ribozyme, suggesting that the A25•C20 double mutant has lost the active site metal ion. Overall, these studies support a model wherein the partially hydrated Mg2+ positioned at the G25•U20 reverse wobble is catalytic and could serve as a Lewis acid, a Brønsted base, or both to facilitate deprotonation of the nucleophile.

Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM

“…Detailed biochemical and kinetic studies have revealed multiple functional divalent metal binding sites and demonstrated that molar concentrations of monovalent ions alone can support catalysis (Nakano et al 2001(Nakano et al , 2003. Sitebound metal ion interactions have been identified and characterized via crystallography (Ke et al 2004(Ke et al , 2007Chen et al 2010), spectroscopy ), chemical probing experiments (Chen et al , 2013Lévesque et al 2012;Thaplyal et al 2015), and molecular simulation (Lee et al 2011;Veeraraghavan et al 2011a,b;Chen et al 2013). Moreover, pH-rate profiles for the reaction in the absence of Mg 2+ and for mutants designed to disrupt binding of the proposed active site ion are inverted relative to the reaction of the native HDVr in Mg 2+ Chen et al 2013).…”

“…Sitebound metal ion interactions have been identified and characterized via crystallography (Ke et al 2004(Ke et al , 2007Chen et al 2010), spectroscopy ), chemical probing experiments (Chen et al , 2013Lévesque et al 2012;Thaplyal et al 2015), and molecular simulation (Lee et al 2011;Veeraraghavan et al 2011a,b;Chen et al 2013). Moreover, pH-rate profiles for the reaction in the absence of Mg 2+ and for mutants designed to disrupt binding of the proposed active site ion are inverted relative to the reaction of the native HDVr in Mg 2+ Chen et al 2013). Phosphorothioate interference studies have revealed sites of potential site-bound metal ion interactions via coordination to one or more nonbridging oxygens, including the pro-R P position of the scissile phosphate (Jeoung et al 1994;Fauzi et al 1997;Prabhu et al 1997;Raines and Gottlieb 1998;Oyelere et al 2002;Das and Piccirilli 2005;Thaplyal et al 2013).…”

Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

“…Thus, experimental studies and theoretical calculations have suggested that the nucleophile is activated in the HDV ribozyme through a divalent cation located in the active site, which can be partially hydrated, and would serve either as a Lewis acid, a Brønsted base or both [13,14]. In the case of the hammerhead, experimental and computational studies suggest that an adenine (A38) and a guanine (G8) have an important role in the catalytic mechanism, the later acting as an acid ("AH" in Scheme 1) whose hydroxyl O2′ is activated by the presence of a Mg 2+ cation [15,16].…”

Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme

“…1; Krasovska et al 2006;Chen et al 2010;Veeraraghavan et al 2011;Kapral et al 2014), which was not observed in the first two crystal structures (Ferre-D'Amare et al 1998;Ke et al 2004). This G25·U20 wobble is assumed to position the catalytic Mg 2+ ion in the active site, which comprises also C75, the catalytic cytosine (C57 in the CPEB3 ribozyme) (Chen et al 2013). N3-protonated C75 has been proposed to act as the general acid in the cleavage mechanism that protonates the 5 ′ -O leaving group with an estimated pK a of 6.4 (Gong et al 2007).…”

“…N3-protonated C75 has been proposed to act as the general acid in the cleavage mechanism that protonates the 5 ′ -O leaving group with an estimated pK a of 6.4 (Gong et al 2007). The catalytic Mg 2+ ion has recently been proposed to serve as Lewis acid, Brønsted base, or both to activate the 2 ′ -OH nucleophile, as well as to coordinate to the pro-R P oxygen of the scissile phosphodiester (Chen et al 2013;Thaplyal et al 2013).…”