NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P.
Proc Natl Acad Sci U S A
; 107(6): 2479-84, 2010 Feb 09.
Article
en En
| MEDLINE
| ID: mdl-20133747
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(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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Espectrometría por Rayos X
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Proteínas Bacterianas
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Espectroscopía de Resonancia Magnética
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Ribonucleasa P
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Año:
2010
Tipo del documento:
Article
País de afiliación:
Estados Unidos