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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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Detailed view of the S. solfataricus exosome active site in complex with RNA and inorganic phosphate. (a) RNA, Pi and contacting residues are shown as sticks (blue for Rrp41 residues and green for Rrp42 residues). An unbiased Fo-Fc electron density map at 3 sigma is displayed in magenta. Two orientations related by a 180 degree rotation around the vertical axis are shown. (b) Position of RNA substrates in the active sites of exosomes from S. solfataricus (RNA displayed in cyan, Rrp41 in blue and Rrp42 in green) and P. abyssi (RNA in magenta, Rrp41 in yellow and Rrp42 in orange) after superimposing the backbone C-alpha atoms. The position of the inorganic phosphate as observed in the S. solfataricus exosome is shown in red. The position of the RNA is very similar in the two different archaeal exosomes.
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fig3: Detailed view of the S. solfataricus exosome active site in complex with RNA and inorganic phosphate. (a) RNA, Pi and contacting residues are shown as sticks (blue for Rrp41 residues and green for Rrp42 residues). An unbiased Fo-Fc electron density map at 3 sigma is displayed in magenta. Two orientations related by a 180 degree rotation around the vertical axis are shown. (b) Position of RNA substrates in the active sites of exosomes from S. solfataricus (RNA displayed in cyan, Rrp41 in blue and Rrp42 in green) and P. abyssi (RNA in magenta, Rrp41 in yellow and Rrp42 in orange) after superimposing the backbone C-alpha atoms. The position of the inorganic phosphate as observed in the S. solfataricus exosome is shown in red. The position of the RNA is very similar in the two different archaeal exosomes.

Mentions: The 2.5 Å difference map obtained from S. solfataricus exosome crystals soaked with RNA for 48 h followed by a quick Pi soak displayed clear density for the 4 most 3′-end nt of RNA as well as one Pi ion revealing a pre-catalytic state with both substrates bound (Figure 3(a)). The RNA binding mode is similar to previously structures of the S. solfataricus Rrp41/42 RNase PH ring (not shown) and P. abyssi Rrp41/42 in the absence of Pi (Figure 3(b)) confirming that mainly sequence-independent phosphate-backbone interactions hold the RNA substrate in place. Phosphorolytic degradation of RNA requires the nucleophilic attack of a negatively charged Pi on the negatively charged phosphate of the most 3′-end nt of the RNA substrate. In the structure presented here, the Pi ion is positioned only 3.4 Å from the 3′-end phosphate of the RNA substrate. The phosphorolytic mechanism of the archaeal exosome results in the build up of negative charge that needs to be neutralized. The close proximity of the phosphates is facilitated by two conserved arginine residues (R139 and R99 of S. solfataricus Rrp41) that neutralize the charge of the phosphates (Figure 4). Only smaller conformational changes in active residues of maximal 0.8 Å occur upon RNA binding in the RNA∗Pi-bound structure as compared to the Pi-bound structure. Interestingly, the 3′-OH of the 3′-end ribose of the RNA makes a close 2.5 Å contact with the Pi anion (Figure 3(a)) suggesting that the nucleophilic attack of the Pi may be RNA-substrate-assisted. From the electron density and the distance of 3.4 Å between the Pi ion and the phosphate of the 3′ end of the RNA substrate, it is clear that a nucleophilic attack has not yet taken place. As the structure presented here is of the Rrp41D182A mutant, we conclude that D182 is required for initiation of the nucleophilic attack in the phosphorolytic mechanism of the archaeal exosome (further discussed in the following section).


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

Lorentzen E, Conti E - Archaea (2012)

Detailed view of the S. solfataricus exosome active site in complex with RNA and inorganic phosphate. (a) RNA, Pi and contacting residues are shown as sticks (blue for Rrp41 residues and green for Rrp42 residues). An unbiased Fo-Fc electron density map at 3 sigma is displayed in magenta. Two orientations related by a 180 degree rotation around the vertical axis are shown. (b) Position of RNA substrates in the active sites of exosomes from S. solfataricus (RNA displayed in cyan, Rrp41 in blue and Rrp42 in green) and P. abyssi (RNA in magenta, Rrp41 in yellow and Rrp42 in orange) after superimposing the backbone C-alpha atoms. The position of the inorganic phosphate as observed in the S. solfataricus exosome is shown in red. The position of the RNA is very similar in the two different archaeal exosomes.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Detailed view of the S. solfataricus exosome active site in complex with RNA and inorganic phosphate. (a) RNA, Pi and contacting residues are shown as sticks (blue for Rrp41 residues and green for Rrp42 residues). An unbiased Fo-Fc electron density map at 3 sigma is displayed in magenta. Two orientations related by a 180 degree rotation around the vertical axis are shown. (b) Position of RNA substrates in the active sites of exosomes from S. solfataricus (RNA displayed in cyan, Rrp41 in blue and Rrp42 in green) and P. abyssi (RNA in magenta, Rrp41 in yellow and Rrp42 in orange) after superimposing the backbone C-alpha atoms. The position of the inorganic phosphate as observed in the S. solfataricus exosome is shown in red. The position of the RNA is very similar in the two different archaeal exosomes.
Mentions: The 2.5 Å difference map obtained from S. solfataricus exosome crystals soaked with RNA for 48 h followed by a quick Pi soak displayed clear density for the 4 most 3′-end nt of RNA as well as one Pi ion revealing a pre-catalytic state with both substrates bound (Figure 3(a)). The RNA binding mode is similar to previously structures of the S. solfataricus Rrp41/42 RNase PH ring (not shown) and P. abyssi Rrp41/42 in the absence of Pi (Figure 3(b)) confirming that mainly sequence-independent phosphate-backbone interactions hold the RNA substrate in place. Phosphorolytic degradation of RNA requires the nucleophilic attack of a negatively charged Pi on the negatively charged phosphate of the most 3′-end nt of the RNA substrate. In the structure presented here, the Pi ion is positioned only 3.4 Å from the 3′-end phosphate of the RNA substrate. The phosphorolytic mechanism of the archaeal exosome results in the build up of negative charge that needs to be neutralized. The close proximity of the phosphates is facilitated by two conserved arginine residues (R139 and R99 of S. solfataricus Rrp41) that neutralize the charge of the phosphates (Figure 4). Only smaller conformational changes in active residues of maximal 0.8 Å occur upon RNA binding in the RNA∗Pi-bound structure as compared to the Pi-bound structure. Interestingly, the 3′-OH of the 3′-end ribose of the RNA makes a close 2.5 Å contact with the Pi anion (Figure 3(a)) suggesting that the nucleophilic attack of the Pi may be RNA-substrate-assisted. From the electron density and the distance of 3.4 Å between the Pi ion and the phosphate of the 3′ end of the RNA substrate, it is clear that a nucleophilic attack has not yet taken place. As the structure presented here is of the Rrp41D182A mutant, we conclude that D182 is required for initiation of the nucleophilic attack in the phosphorolytic mechanism of the archaeal exosome (further discussed in the following section).

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