Solvent Structure and Hammerhead Ribozyme Catalysis

Abstract

Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.

Role of Mg2+ in Hammerhead Ribozyme Catalysis from Molecular Simulation

“…Results are compared to the recent 2.2 Å crystal structure of the full-length hammerhead in the presence of Mg 2+ ions,20 and to the 2.0 Å crystal structure with resolved Mn 2+ binding sites and solvent structure. 24 The present simulations results are consistent with a hammerhead ribozyme model whereby the active site forms a region of local negative charge that requires electrostatic stabilization to preserve its structural integrity, and whereby this stabilization can be effected by divalent metal binding at the C-site or in a bridging position in the ground (pre-reactive) state. An Mg 2+ ion is observed to weakly bind at the C-site position at solvent separation with G10.1, facilitating the formation of near in-line attack conformations, particularly when in the bridging position where there is increased interaction between the nucleophile (C17:O 2′ ) and the implicated general base (G12).…”

“…15,20,24 The first initial Mg 2+ binding site, designated the "C-site'', is an implicated metal-ion binding site based on a very recent hammerhead RNA structure with resolved metal and solvent positions24 in which a Mn 2+ cation directly coordinates to both A9:O 2P and G10.1:N 7 . The second initial Mg 2+ binding site, designated the "Bridging'' site, is one in which the Mg 2+ ion bridges the A9 and scissile phosphates, directly coordinating the two nonbridging O P2 atoms that are 4.3 Å apart in the crystal structure.20 This type of coordination was inferred both from the O-O distance in the crystal structure and also the thio/rescue effect experiments that suggest a single divalent metal might bridge these positions in the transition state.…”

“…This stabilization can be effected by divalent metal ion binding24,29 or likely by high concentrations of monovalent cations (Li + , Na + or NH 4 + cations required concentrations 400-fold higher than those used in Mg 2+ to achieve comparable rates of cleavage).66-68 In the ground state, a divalent metal ion can occupy the C-site to effect such electrostatic stabilization. 24 In the case of Mn 2+ , the C-site formed by A9/G10.1 is a high-affinity binding site, as recently characterized by electron spin-echo envelope modulation spectroscopy.69 On the other hand, in the case of Mg 2+ , binding at the C-site does not appear to be strong, as suggested by the molecular simulations and the observed absence of resolved metal cations in that site in the full-length hammerhead crystal obtained in the presence of 1 mM Mg 2+ and > 1 M NH 4 +. 20 However, upon crystallization in the presence of 10 mM Mn 2+ , metal binding was observed in the C-site position,24 likely because the softer Mn 2+ has higher affinity than Mg 2+ for binding the N 7 of G10.1, as discussed in the section presenting results of DFT calculations.…”

“…Very recently, a crystal structure was obtained in the presence of the softer divalent cation Mn 2+ that allowed the identification of a divalent binding site involving A9 and G10.1 near the active site. 24 It remains an open question, however, as to what conformational events and changes in metal binding may occur in proceeding to the transition state from a precatalytic active conformation. Molecular simulation offers a powerful tool to probe the structure and dynamics along the chemical reaction coordinate and the role played by divalent metal ions in stabilizing the transition state.…”

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

“…These tertiary interactions result in a higher population of functionally folded ribozymes, leading to higher observed rates at significantly lower divalent metal concentrations in vitro, as well as activity in vivo (Khvorova et al 2003). New structures of constructs based on native HHRz's (2GOZ, 2OEU, 2QUS) largely satisfy predictions based on previous mutations and also provide new hypotheses concerning players in the HHRz mechanism (Martick and Scott 2006;Lee et al 2007;Chi et al 2008;Martick et al 2008). The conformation of the native HHRz in crystalforming conditions is significantly different from that of the truncated HHRz.…”