NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

Abstract

Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5′ end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn 2þ bound to a P4 helix mimic is sixcoordinate, with an average Zn-O/N bond distance of 2.08 Å. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn 2þ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with CoðNH 3 Þ 3þ 6 , Zn 2þ , and CoðNH 3 Þ 3þ 6 ∕Zn 2þ confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.manganese | ribozyme | RNase P | X-ray absorption spectroscopy | zinc R NA molecules are large polyanions that associate with numerous divalent metal ions that stabilize their structure and promote catalysis. Identification of the RNA moieties involved in metal-binding and discerning the nature of these interactions is an important outstanding question in the field (1, 2). Whereas RNA-bound metals can be characterized at the atomic level in the solid-state by X-crystallography there are not yet techniques for characterizing the binding sites and coordination schemes for RNA-bound metals in solution. Techniques based on NMR, EPR, and Raman spectroscopy as well as nucleotide analog interference mapping (NAIM) and phosphorothioate substitution exist for identifying RNA residues involved in metal-binding; however the geometry of the bound metal and the nature of the coordination scheme cannot be resolved based on these experiments alone.RNase P is a metal-dependent ribozyme that catalyzes precursor tRNA (pre-tRNA) maturation by cleaving a specific phosphodiester bond (3). In RNase P, metal ions stabilize the folded RNase P RNA (PRNA) structure, enhance ligand affinity, and stabilize the transition state for cleavage (4); in vivo the metal requirement is fulfilled by Mg 2þ ions (5, 6). A majority of the ∼150 divalent metal ions associated with PRNA bind nonspecifically via electrostatic interactions whereas only a handful of ions form specific contacts with RNase P (5, 7). Discerning the position and structure of the few divalent ions that site-specifically interact with RNA is a majo...

Of proteins and RNA: The RNase P/MRP family

“…Divalent ions (naturally magnesium) play crucial roles in RNase P RNA folding (Baird et al 2007(Baird et al , 2010Qu et al 2008;Kazantsev et al 2009, and references therein) and catalysis (Kirsebom and Trobro 2009, and references therein). Due to the insufficient resolution of available crystal structures, the precise location of the magnesium ions could not be reliably determined, although valuable information was obtained using anomalous scattering by other ions (Krasilnikov et al 2004;Kazantsev et al 2009), as well as by a combination of NMR and extended X-ray absorption fine structure (EXAFS) spectroscopy (Koutmou et al 2010a) and other approaches (Christian et al 2006;Getz et al 2007, and references therein).…”

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

“…Nonetheless, higher-affinity binding sites, like the ones in the G-quadruplex channel [171] or in a short RNase P helix P4 model [172] can be characterized in remarkable detail, not to forget complexes of kinetically more inert metal ions [173,174].…”

“…The large magnetic moment of an unpaired electron of a paramagnetic metal ion species efficiently relaxes nuclei in their immediate environment ( Figure 5b) [172,217,218]. The effect is strictly distance-dependent (relative to r -6 ) and leaves less room for ambiguity than chemical shift perturbations.…”

“…Paramagnetic line broadening information can even be included as weak distance restraints in molecular dynamics calculations [197]. [223] and has been used in a wide range of studies [172,200,201,[224][225][226].…”

Zinc Bells Rang in Jerusalem!A report on the Meeting of the International Society for Zinc Biology, Jerusalem, Israel, 1 to 5 December 2009.

“…Using two-photon and fluorescence microscopy as well as x-ray imaging to dynamically monitor the subcellular localization of zinc, Christoph Fahrni (Georgia Institute of Technology, USA) demonstrated that changes in zinc concentrations accompany various phases of the cell cycle (74). Finally, Carol Fierke (University of Maryland School of Medicine, USA) described a new x-ray and fluorescence microprobe for imaging zinc that revealed the subcellular distribution of zinc in yeast cells (75). …”