NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P
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 Zn2+ bound to a P4 helix mimic is six-coordinate, 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 Mn2+ 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 Graphic, Zn2+, and Graphicconfirm 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.
Proceedings of the National Academy of Sciences
National Academy of Sciences
Digital Object Identifier (DOI)
Koutmou, Kristin S.; Casiano-Negroni, Anette; Getz, Melissa M.; Pazicni, Samuel; Penner-Hahn, James E.; Al-Hashimi, Hashim M.; and Fierke, Carol A.* “NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P, ” Proceedings of the National Academy of Sciences, U.S.A. 2010, 107, 2479-2484
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