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 Zn(2+) bound to a P4 helix mimic is six-coordinate, with an average Zn-O/N bond distance of 2.08 A. 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(2+) 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 , Zn(2+), and 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.
Departments of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Kristin S
KS
Koutmou
Anette
A
Casiano-Negroni
Melissa M
MM
Getz
Samuel
S
Pazicni
Andrew J
AJ
Andrews
James E
JE
Penner-Hahn
Hashim M
HM
Al-Hashimi
Carol A
CA
Fierke
D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D043262:Ribonuclease P / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013052:Spectrometry, X-Ray Emission / Q000379:methods*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D008670:Metals; D043262:Ribonuclease P; D015032:Zinc; D005998:Glycine