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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 107; no. 6; pp. 2479 - 2484
• Main Authors: Koutmou, Kristin S, Casiano-Negroni, Anette, Getz, Melissa M, Pazicni, Samuel, Andrews, Andrew J, Penner-Hahn, James E, Al-Hashimi, Hashim M, Fierke, Carol A
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 09.02.2010; National Acad Sciences
• Subjects: Bacillus subtilis - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Biochemistry; Biological Sciences; Catalysis; Catalytic Domain; Chemical equilibrium; Coordination polymers; Glycine - chemistry; Glycine - genetics; Glycine - metabolism; Ligands; Magnetic Resonance Spectroscopy - methods; Metal ions; Metals; Metals - chemistry; Metals - metabolism; Mutation; NMR; Nuclear magnetic resonance; Nucleic Acid Conformation; Nucleotides; Protein Binding; Ribonuclease P - chemistry; Ribonuclease P - genetics; Ribonuclease P - metabolism; RNA; Spectrometry, X-Ray Emission - methods; Spectrum analysis; Transfer RNA; Zinc; Zinc - chemistry; Zinc - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0906319107

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 Formula , Zn²⁺, and Formula 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.