Active-site monovalent cations revealed in a 1.55-Å-resolution hammerhead ribozyme structure

Abstract

We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na(+) ion binding in the ribozyme's active site. At least two such Na(+) ions are observed. The first Na(+) ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na(+) ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na(+), but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na(+) directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank.

Keywords: PDB; Protein Data Bank; RNA; catalysis; metal ion; ribozyme.

Figure 1

An all-bond representation of the 1.55 Å refined crystal structure of the full-length hammerhead ribozyme with bound Na⁺ ions. The enzyme strand is depicted in red, the substrate strand in yellow, the cleavage-site nucleotide (C17) in green, and the various Na⁺ ions are represented as purple spheres. Coordinates are available in the PDB (3ZP8).

Figure 2

Atomic structure of the hammerhead ribozyme active site, with oxygen atoms depicted in red, nitrogen atoms in blue, phosphori in magenta, enzyme-strand carbon atoms in white, and substrate-strand carbon atoms in yellow. Na⁺ ions are shown as purple spheres. (a) A representative 1.55 Å resolution sigmaA-weighted 2F_o-F_c electron density map contoured at 2.0 r.m.s.d. reveals a hole in the six-membered aromatic ring of the general base, G12, in the active site. (b) The canonical A9 divalent metal ion binding site is occupied by Na⁺, (purple sphere), which forms inner-sphere interactions with the pro-R phosphate oxygen of A9, the N7 of G10.1, and four water molecules, three of which in turn form bridging interactions with other RNA atoms.(c) The Hoogsteen face of the general base, G12, within the active site, forms a second potentially catalytically relevant Na⁺ binding site (Na⁺ ion in purple). The O6 of G12 forms an inner-sphere interaction with the Na⁺, and one of the three unambiguously resolved water molecules it coordinates bridges to the N7 of G12. The other observed water molecules bridge to various phosphates near the active site. (d) A third potential ion or water molecule (orange sphere, see discussion in text) coordinates the pro-R oxygens of A9 and scissile phosphates, the 2′-O nucleophile of the cleavage site base, the 2′-O of G8 implicated in general acid catalysis, and a water molecule in a distorted octahedral complex.

Figure 3

Wall-eyed stereo diagrams illustrating the network of hydrogen bonding interactions and other close contacts within the active site, as well as Na⁺ ions, shown as purple spheres, and well-resolved water molecules, whose oxygen atoms are shown as red spheres. The octahedrally coordinated water molecule or ion is shown in orange and labeled with a question mark. The carbon atoms within the enzyme strand are white, and those of the substrate strand are yellow. Green dotted lines correspond to the potentially active hydrogen bonds of the general base (N1 of G12 to 2′O of C17) and general acid (2′OH of G8 to 5′O of C1.1). The orange dotted line is the presumed trajectory of bond formation between the 2′O of C17 and the adjacent phosphorus atom of C1.1. (The blue mesh of the top figure is a 1.55 Å resolution sigmaA-weighted 2F_o-F_c electron density contoured at 1.2 r.m.s.d. The bottom figure is the same view, with the electron density map removed for clarity.

Figure 4

Schematic diagram of a possible hammerhead ribozyme reaction mechanism, corresponding to the structural representation in Figure 2D. Na⁺ ions are indicated, as in the previous figures, in purple, and the potential Na⁺ or K⁺ ion binding site (M⁺) is indicated in orange. The Na⁺ ion associated with G12 may help to stabilize the negative charge associated with deprotonation of the N1 of G12, and the bridging potential Na⁺ or K⁺ ion may help to stabilize the close approach of two negatively-charged phosphates.