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Crystal structure of Escherichia coli polynucleotide phosphorylase core bound to RNase E, RNA and manganese: implications for catalytic mechanism and RNA degradosome assembly.

Nurmohamed S, Vaidialingam B, Callaghan AJ, Luisi BF - J. Mol. Biol. (2009)

Bottom Line: At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites.Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state.We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, UK.

ABSTRACT
Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel beta-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

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A speculative model showing how metal can interact with the transition state in the E. coli PNPase core active site. The text describes the preparation of the model for the transition state using an overlay of ADP and poly(rA) bound structures of the Pyrococcus abyssi exosome (PDB codes 2PO0 and 2PO1, respectively).48 The β-phosphate of the ADP occupies the binding site for the inorganic phosphate and mimics its orientation in the transition state for either attack of the terminal phosphoester of RNA (for phosphorolysis) or the release of phosphate during polymerisation of NDP. The model was prepared by secondary structure superposition of E. coli Mn2+-bound PNPase structure (grey) with the active subunit of the P. abysssi exosome with ADP and poly(rA) bound. The Mn2+ (purple ball) coincides almost exactly with the alternative position of the β-phosphate atom in the ADP-bound form.
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fig7: A speculative model showing how metal can interact with the transition state in the E. coli PNPase core active site. The text describes the preparation of the model for the transition state using an overlay of ADP and poly(rA) bound structures of the Pyrococcus abyssi exosome (PDB codes 2PO0 and 2PO1, respectively).48 The β-phosphate of the ADP occupies the binding site for the inorganic phosphate and mimics its orientation in the transition state for either attack of the terminal phosphoester of RNA (for phosphorolysis) or the release of phosphate during polymerisation of NDP. The model was prepared by secondary structure superposition of E. coli Mn2+-bound PNPase structure (grey) with the active subunit of the P. abysssi exosome with ADP and poly(rA) bound. The Mn2+ (purple ball) coincides almost exactly with the alternative position of the β-phosphate atom in the ADP-bound form.

Mentions: To examine where the metal might lie with respect to an RNA substrate, we prepared an overlay of the structures of E. coli PNPase and Pyrococcus abyssi exosome Rrp41 subunit in separate complexes with RNA or ADP (PDB codes 2PO1 and 2PO0, respectively).46 Notably, an overlay of the ADP and RNA-bound forms of the P. abyssi exosome shows that the scissile phosphate of RNA is spatially coincident with one of two orientations for the α-phosphate of ADP, whereas the β-phosphate is positioned at a binding site for inorganic phosphate. Strikingly, an overlay of ADP and RNA forms resembles the pentavalent phosphate transition state proposed for many phosphotransfer reactions. Guided by this overlay, a model for the transition state of an RNA substrate under attack by PNPase was prepared. Firstly, the phosphate for the transition state was placed at the mean position of the phosphate atoms in the ADP and RNA-bound forms of the P. abyssi exosome. Secondly, the oxygen atoms that formed a trigonal planar arrangement were selected from either structure to generate a chimeric structure that resembles the transition state. Lastly, we docked the chimeric RNA structure from the P. abyssi exosome structures onto the corresponding position in our PNPase core-manganese structure. The overlay shows that the metal is in a good position to support the proposed transition state (Fig. 7). The metal is well orientated to interact with the carboxylates of D486 and D492, and the pro-chiral, non-esterified oxygens that are in the axial position of the bi-pyrimidal transition state. In the RNA-free structure, the metal is coordinated by water molecules at the positions that correspond to the phosphate oxygens in the transition state. Metal binding is anticipated to be linked favourably with substrate binding, and the metal can have a dual role to support general acid-base catalysis involving protons originating from the water molecules of its hydration shell and to offset charge build-up in the transition state. H403 is predicted to interact with an axial oxygen (Fig. 7), and substitution of this residue with alanine decreases catalytic activity of E. coli PNPase 10-fold or greater.7 The residues that contact the transition state are conserved in RNase PH and PNPase of all species and in the Rrp41 subunits of the archaeal exosome, suggesting that the mechanism of metal-assisted catalysis is conserved.


Crystal structure of Escherichia coli polynucleotide phosphorylase core bound to RNase E, RNA and manganese: implications for catalytic mechanism and RNA degradosome assembly.

Nurmohamed S, Vaidialingam B, Callaghan AJ, Luisi BF - J. Mol. Biol. (2009)

A speculative model showing how metal can interact with the transition state in the E. coli PNPase core active site. The text describes the preparation of the model for the transition state using an overlay of ADP and poly(rA) bound structures of the Pyrococcus abyssi exosome (PDB codes 2PO0 and 2PO1, respectively).48 The β-phosphate of the ADP occupies the binding site for the inorganic phosphate and mimics its orientation in the transition state for either attack of the terminal phosphoester of RNA (for phosphorolysis) or the release of phosphate during polymerisation of NDP. The model was prepared by secondary structure superposition of E. coli Mn2+-bound PNPase structure (grey) with the active subunit of the P. abysssi exosome with ADP and poly(rA) bound. The Mn2+ (purple ball) coincides almost exactly with the alternative position of the β-phosphate atom in the ADP-bound form.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2723993&req=5

fig7: A speculative model showing how metal can interact with the transition state in the E. coli PNPase core active site. The text describes the preparation of the model for the transition state using an overlay of ADP and poly(rA) bound structures of the Pyrococcus abyssi exosome (PDB codes 2PO0 and 2PO1, respectively).48 The β-phosphate of the ADP occupies the binding site for the inorganic phosphate and mimics its orientation in the transition state for either attack of the terminal phosphoester of RNA (for phosphorolysis) or the release of phosphate during polymerisation of NDP. The model was prepared by secondary structure superposition of E. coli Mn2+-bound PNPase structure (grey) with the active subunit of the P. abysssi exosome with ADP and poly(rA) bound. The Mn2+ (purple ball) coincides almost exactly with the alternative position of the β-phosphate atom in the ADP-bound form.
Mentions: To examine where the metal might lie with respect to an RNA substrate, we prepared an overlay of the structures of E. coli PNPase and Pyrococcus abyssi exosome Rrp41 subunit in separate complexes with RNA or ADP (PDB codes 2PO1 and 2PO0, respectively).46 Notably, an overlay of the ADP and RNA-bound forms of the P. abyssi exosome shows that the scissile phosphate of RNA is spatially coincident with one of two orientations for the α-phosphate of ADP, whereas the β-phosphate is positioned at a binding site for inorganic phosphate. Strikingly, an overlay of ADP and RNA forms resembles the pentavalent phosphate transition state proposed for many phosphotransfer reactions. Guided by this overlay, a model for the transition state of an RNA substrate under attack by PNPase was prepared. Firstly, the phosphate for the transition state was placed at the mean position of the phosphate atoms in the ADP and RNA-bound forms of the P. abyssi exosome. Secondly, the oxygen atoms that formed a trigonal planar arrangement were selected from either structure to generate a chimeric structure that resembles the transition state. Lastly, we docked the chimeric RNA structure from the P. abyssi exosome structures onto the corresponding position in our PNPase core-manganese structure. The overlay shows that the metal is in a good position to support the proposed transition state (Fig. 7). The metal is well orientated to interact with the carboxylates of D486 and D492, and the pro-chiral, non-esterified oxygens that are in the axial position of the bi-pyrimidal transition state. In the RNA-free structure, the metal is coordinated by water molecules at the positions that correspond to the phosphate oxygens in the transition state. Metal binding is anticipated to be linked favourably with substrate binding, and the metal can have a dual role to support general acid-base catalysis involving protons originating from the water molecules of its hydration shell and to offset charge build-up in the transition state. H403 is predicted to interact with an axial oxygen (Fig. 7), and substitution of this residue with alanine decreases catalytic activity of E. coli PNPase 10-fold or greater.7 The residues that contact the transition state are conserved in RNase PH and PNPase of all species and in the Rrp41 subunits of the archaeal exosome, suggesting that the mechanism of metal-assisted catalysis is conserved.

Bottom Line: At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites.Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state.We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, UK.

ABSTRACT
Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel beta-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

Show MeSH
Related in: MedlinePlus