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Crystal structure of a 9-subunit archaeal exosome in pre-catalytic states of the phosphorolytic reaction.

Lorentzen E, Conti E - Archaea (2012)

Bottom Line: The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms.These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries.The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

ABSTRACT
The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms. The archaeal exosome is functionally similar to bacterial polynucleotide phosphorylase (PNPase) and RNase PH enzymes as it uses inorganic phosphate (Pi) to processively cleave RNA substrates releasing nucleoside diphosphates. To shed light on the mechanism of catalysis, we have determined the crystal structures of mutant archaeal exosome in complex with either Pi or with both RNA and Pi at resolutions of 1.8 Å and 2.5 Å, respectively. These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries. In the structure with both Pi and RNA bound, the Pi closely approaches the phosphate of the 3'-end nucleotide of the RNA and is in a perfect position to perform a nucleophilic attack. The presence of negative charge resulting from the close contacts between the phosphates appears to be neutralized by conserved positively charged residues in the active site of the archaeal exosome. The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

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Model of the archaeal exosome bound to RNA, Pi, and Mn++. (a) The model shows the active site of the archaea exosome (S. solfataricus coordinates from this study) with a divalent cation (magenta ball) from E. coli PNPase (pdb code 3GME) after superimposing the coordinating aspartate residues. (b) Schematics of the model shown in (a) with interaction distances indicated as derived from the structure with RNA∗Pi bound presented here. A magnesium ion instead of a manganese ion is shown as the S. solfataricus exosome is known to be significantly more active with magnesium [26]. The divalent cation is positioned between the Pi and the 3′-end phosphate of the RNA but accurate coordination distances are not known.
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fig4: Model of the archaeal exosome bound to RNA, Pi, and Mn++. (a) The model shows the active site of the archaea exosome (S. solfataricus coordinates from this study) with a divalent cation (magenta ball) from E. coli PNPase (pdb code 3GME) after superimposing the coordinating aspartate residues. (b) Schematics of the model shown in (a) with interaction distances indicated as derived from the structure with RNA∗Pi bound presented here. A magnesium ion instead of a manganese ion is shown as the S. solfataricus exosome is known to be significantly more active with magnesium [26]. The divalent cation is positioned between the Pi and the 3′-end phosphate of the RNA but accurate coordination distances are not known.

Mentions: The S. solfataricus exosome was recombinantly expressed in E. coli, purified and crystallized as previously described [4, 27]. The 1.8 Å resolution structure with one Pi ion bound was obtained after soaking crystals with 1 mM 7 mer poly(A) RNA and 10 mM Pi for 20 min followed by flash cooling in liquid nitrogen. Electron density maps calculated at 1.8 Å resolution revealed very clear density for the Pi ion but only spurious density for RNA. The 2.5 Å resolution structure with RNA and Pi bound was obtained by first soaking crystals for 48 h with 1 mM 7 mer poly(A) RNA followed by a 20 min soak with 10 mM Pi and immediate flash cooling in liquid nitrogen. X-ray diffraction data were collected at the Swiss Light Source beamline X06SA and processed with the program XDS [28]. The structure was determined using the available Rrp4/41/42 structure as a starting model followed by iterative cycles of refinement in REFMAC (Pi bound structure) [29] or PHENIX (RNA∗Pi bound structure) [30] and model building in COOT [31]. All figures were made in the program PyMOL (http://www.pymol.org/). Figure 4(a) was prepared by superposing the native Rrp41/42 structure (pdb code 2BR2) onto Rrp4/41D182A/42 mutant exosome to obtain the conformation of the D182 side chain. The position of the divalent cation was obtained by superimposing the coordinating aspartates (D486 and D492) of E. coli PNPase structure (pdb code 3GME) onto the equivalent residues (D182 and D188) of the S. solfataricus exosome.


Crystal structure of a 9-subunit archaeal exosome in pre-catalytic states of the phosphorolytic reaction.

Lorentzen E, Conti E - Archaea (2012)

Model of the archaeal exosome bound to RNA, Pi, and Mn++. (a) The model shows the active site of the archaea exosome (S. solfataricus coordinates from this study) with a divalent cation (magenta ball) from E. coli PNPase (pdb code 3GME) after superimposing the coordinating aspartate residues. (b) Schematics of the model shown in (a) with interaction distances indicated as derived from the structure with RNA∗Pi bound presented here. A magnesium ion instead of a manganese ion is shown as the S. solfataricus exosome is known to be significantly more active with magnesium [26]. The divalent cation is positioned between the Pi and the 3′-end phosphate of the RNA but accurate coordination distances are not known.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539426&req=5

fig4: Model of the archaeal exosome bound to RNA, Pi, and Mn++. (a) The model shows the active site of the archaea exosome (S. solfataricus coordinates from this study) with a divalent cation (magenta ball) from E. coli PNPase (pdb code 3GME) after superimposing the coordinating aspartate residues. (b) Schematics of the model shown in (a) with interaction distances indicated as derived from the structure with RNA∗Pi bound presented here. A magnesium ion instead of a manganese ion is shown as the S. solfataricus exosome is known to be significantly more active with magnesium [26]. The divalent cation is positioned between the Pi and the 3′-end phosphate of the RNA but accurate coordination distances are not known.
Mentions: The S. solfataricus exosome was recombinantly expressed in E. coli, purified and crystallized as previously described [4, 27]. The 1.8 Å resolution structure with one Pi ion bound was obtained after soaking crystals with 1 mM 7 mer poly(A) RNA and 10 mM Pi for 20 min followed by flash cooling in liquid nitrogen. Electron density maps calculated at 1.8 Å resolution revealed very clear density for the Pi ion but only spurious density for RNA. The 2.5 Å resolution structure with RNA and Pi bound was obtained by first soaking crystals for 48 h with 1 mM 7 mer poly(A) RNA followed by a 20 min soak with 10 mM Pi and immediate flash cooling in liquid nitrogen. X-ray diffraction data were collected at the Swiss Light Source beamline X06SA and processed with the program XDS [28]. The structure was determined using the available Rrp4/41/42 structure as a starting model followed by iterative cycles of refinement in REFMAC (Pi bound structure) [29] or PHENIX (RNA∗Pi bound structure) [30] and model building in COOT [31]. All figures were made in the program PyMOL (http://www.pymol.org/). Figure 4(a) was prepared by superposing the native Rrp41/42 structure (pdb code 2BR2) onto Rrp4/41D182A/42 mutant exosome to obtain the conformation of the D182 side chain. The position of the divalent cation was obtained by superimposing the coordinating aspartates (D486 and D492) of E. coli PNPase structure (pdb code 3GME) onto the equivalent residues (D182 and D188) of the S. solfataricus exosome.

Bottom Line: The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms.These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries.The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

ABSTRACT
The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms. The archaeal exosome is functionally similar to bacterial polynucleotide phosphorylase (PNPase) and RNase PH enzymes as it uses inorganic phosphate (Pi) to processively cleave RNA substrates releasing nucleoside diphosphates. To shed light on the mechanism of catalysis, we have determined the crystal structures of mutant archaeal exosome in complex with either Pi or with both RNA and Pi at resolutions of 1.8 Å and 2.5 Å, respectively. These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries. In the structure with both Pi and RNA bound, the Pi closely approaches the phosphate of the 3'-end nucleotide of the RNA and is in a perfect position to perform a nucleophilic attack. The presence of negative charge resulting from the close contacts between the phosphates appears to be neutralized by conserved positively charged residues in the active site of the archaeal exosome. The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

Show MeSH
Related in: MedlinePlus