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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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High-resolution view of the inorganic phosphate binding site. (a) Unbiased Fo-Fc map at 3 sigma is shown in magenta and 2Fo-Fc map at 1 sigma in blue. The inorganic phosphate and contacting residues from the Rrp41 subunit are shown as sticks. (b) Schematic representation of the inorganic phosphate binding site with distances between the Pi ion and protein residues indicated. (c) Structures of the S. solfataricus and the M. thermautotrophicus exosomes superimposed reveal that the Pi-binding sites are very well conserved across different archaeal species.
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fig2: High-resolution view of the inorganic phosphate binding site. (a) Unbiased Fo-Fc map at 3 sigma is shown in magenta and 2Fo-Fc map at 1 sigma in blue. The inorganic phosphate and contacting residues from the Rrp41 subunit are shown as sticks. (b) Schematic representation of the inorganic phosphate binding site with distances between the Pi ion and protein residues indicated. (c) Structures of the S. solfataricus and the M. thermautotrophicus exosomes superimposed reveal that the Pi-binding sites are very well conserved across different archaeal species.

Mentions: Previously determined crystal structures of S. solfataricus exosomes crystallized in the absence of Pi displayed spherical density at the putative phosphate binding site compatible with a chloride ion [22]. The density observed for the Pi in the 1.8 Å structure presented here is clearly tetrahedral (proving that Pi and not Cl− is bound) and facilitates accurate refinement of the four oxygen atom positions of the phosphate as well as determination of binding geometry (Figure 2(a)). The small error of only 0.1 Å in coordinal positions for this structure (as estimated by the refinement program REFMAC) allows for accurate determination of the hydrogen bonding distances (Figure 2(b)). This analysis reveals that the Pi ion is bound by four residues from the Rrp41 subunit. Specifically, the phosphate is coordinated by the side chains of R99, R139, and S138 as well as the main chain amino groups of G137, S138, and R139 (numbering according to the S. solfataricus sequence). A total of eight contacts with binding distances of 2.6–3.2 Å are observed creating a strong phosphate anion binding site (Figure 2(b)). The only previously determined structure of an archaeal exosome in complex with phosphate is the 2.65 Å resolution structure of the M. thermautotrophicus RNase PH ring [25]. Comparison of this structure with the S. solfataricus exosome structure presented here reveals a high structural similarity with an RMSD of 1.2 Å after the two structures are superimposed. The two phosphate binding sites are almost identical and reveal very similar interactions between the protein and the phosphate (Figure 2(c)). The only significant difference is the presence of a threonine in M. thermautotrophicus Rrp41 (T136) in place of the S. solfataricus Rrp41 serine (S138). However, in both cases the side chain hydroxyl interacts with the Pi ion. The position of the Pi-binding site is highly conserved between archaeal exosomes and RNase PH [32] and PNPase enzymes [20] indicating a conserved mechanism of phosphate-dependent RNA degradation among phosphorolytic exosome-like complexes.


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

Lorentzen E, Conti E - Archaea (2012)

High-resolution view of the inorganic phosphate binding site. (a) Unbiased Fo-Fc map at 3 sigma is shown in magenta and 2Fo-Fc map at 1 sigma in blue. The inorganic phosphate and contacting residues from the Rrp41 subunit are shown as sticks. (b) Schematic representation of the inorganic phosphate binding site with distances between the Pi ion and protein residues indicated. (c) Structures of the S. solfataricus and the M. thermautotrophicus exosomes superimposed reveal that the Pi-binding sites are very well conserved across different archaeal species.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: High-resolution view of the inorganic phosphate binding site. (a) Unbiased Fo-Fc map at 3 sigma is shown in magenta and 2Fo-Fc map at 1 sigma in blue. The inorganic phosphate and contacting residues from the Rrp41 subunit are shown as sticks. (b) Schematic representation of the inorganic phosphate binding site with distances between the Pi ion and protein residues indicated. (c) Structures of the S. solfataricus and the M. thermautotrophicus exosomes superimposed reveal that the Pi-binding sites are very well conserved across different archaeal species.
Mentions: Previously determined crystal structures of S. solfataricus exosomes crystallized in the absence of Pi displayed spherical density at the putative phosphate binding site compatible with a chloride ion [22]. The density observed for the Pi in the 1.8 Å structure presented here is clearly tetrahedral (proving that Pi and not Cl− is bound) and facilitates accurate refinement of the four oxygen atom positions of the phosphate as well as determination of binding geometry (Figure 2(a)). The small error of only 0.1 Å in coordinal positions for this structure (as estimated by the refinement program REFMAC) allows for accurate determination of the hydrogen bonding distances (Figure 2(b)). This analysis reveals that the Pi ion is bound by four residues from the Rrp41 subunit. Specifically, the phosphate is coordinated by the side chains of R99, R139, and S138 as well as the main chain amino groups of G137, S138, and R139 (numbering according to the S. solfataricus sequence). A total of eight contacts with binding distances of 2.6–3.2 Å are observed creating a strong phosphate anion binding site (Figure 2(b)). The only previously determined structure of an archaeal exosome in complex with phosphate is the 2.65 Å resolution structure of the M. thermautotrophicus RNase PH ring [25]. Comparison of this structure with the S. solfataricus exosome structure presented here reveals a high structural similarity with an RMSD of 1.2 Å after the two structures are superimposed. The two phosphate binding sites are almost identical and reveal very similar interactions between the protein and the phosphate (Figure 2(c)). The only significant difference is the presence of a threonine in M. thermautotrophicus Rrp41 (T136) in place of the S. solfataricus Rrp41 serine (S138). However, in both cases the side chain hydroxyl interacts with the Pi ion. The position of the Pi-binding site is highly conserved between archaeal exosomes and RNase PH [32] and PNPase enzymes [20] indicating a conserved mechanism of phosphate-dependent RNA degradation among phosphorolytic exosome-like complexes.

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