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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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Structural changes associated with RNA binding to the PNPase core. (a) An overlay of the RNA-free (cyan) and RNA-bound (grey) forms of PNPase core viewed down the molecular 3-fold axis (left) and perpendicular to it (right). In the view on the right, the helical domain is on the top of the torus, and the S1 and KH domains (not shown) are on the bottom. (b) Expanded view of the central channel aperture in the RNA-free form. The aperture is occluded by the F77 and F78 side chain of the FFRR loop in this apo-structure, but the loop is less well ordered in the Mn2+apo structure and in the apo-structure reported by Shi et al.29 (c) The same view as in the left-hand panel but in the RNA-bound form; this shows that the aperture has dilated. It is not clear whether all three RNA-binding sites could be accommodated simultaneously. (d) RNA binds to the central aperture of both E. coli PNPase core (orange) and the S. solfataricus archaeal exosome (green), albeit at a different depth in the central channel. The view is with the molecular 3-fold oriented vertically.
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fig5: Structural changes associated with RNA binding to the PNPase core. (a) An overlay of the RNA-free (cyan) and RNA-bound (grey) forms of PNPase core viewed down the molecular 3-fold axis (left) and perpendicular to it (right). In the view on the right, the helical domain is on the top of the torus, and the S1 and KH domains (not shown) are on the bottom. (b) Expanded view of the central channel aperture in the RNA-free form. The aperture is occluded by the F77 and F78 side chain of the FFRR loop in this apo-structure, but the loop is less well ordered in the Mn2+apo structure and in the apo-structure reported by Shi et al.29 (c) The same view as in the left-hand panel but in the RNA-bound form; this shows that the aperture has dilated. It is not clear whether all three RNA-binding sites could be accommodated simultaneously. (d) RNA binds to the central aperture of both E. coli PNPase core (orange) and the S. solfataricus archaeal exosome (green), albeit at a different depth in the central channel. The view is with the molecular 3-fold oriented vertically.

Mentions: Previously reported crystal structures of the full-length S. antibioticus and E. coli PNPases identified two constricted points in the channel.19,29 One of these is closer to the channel entrance and the second is deeper within the channel and nearer the active site. The entrance-proximal aperture is formed by conserved residues, corresponding to E. coli residues F77-F78-R79-R80 (Fig. 5a and b). This loop had been predicted to form an RNA-binding site in the PNPases of many species.19,46 Confirming this hypothesis, our structure of PNPase in complex with RNA shows that F77 of each PNPase core monomer makes an aromatic stacking contact with an RNA base (Fig. 5c). The electron density in our structure is not sufficiently resolved to identify the bases, although the shape indicates that they are likely to be purines. F78 of the conserved FFRR loop supports the orientation of the base-contacting F77. The remainder of the RNA away from the stacking contact is disordered and the electron density here is poorly resolved, although it is clear that the pathway followed is along the central pore in the direction of the active site. We do not observe any interpretable electron density at the active site.


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)

Structural changes associated with RNA binding to the PNPase core. (a) An overlay of the RNA-free (cyan) and RNA-bound (grey) forms of PNPase core viewed down the molecular 3-fold axis (left) and perpendicular to it (right). In the view on the right, the helical domain is on the top of the torus, and the S1 and KH domains (not shown) are on the bottom. (b) Expanded view of the central channel aperture in the RNA-free form. The aperture is occluded by the F77 and F78 side chain of the FFRR loop in this apo-structure, but the loop is less well ordered in the Mn2+apo structure and in the apo-structure reported by Shi et al.29 (c) The same view as in the left-hand panel but in the RNA-bound form; this shows that the aperture has dilated. It is not clear whether all three RNA-binding sites could be accommodated simultaneously. (d) RNA binds to the central aperture of both E. coli PNPase core (orange) and the S. solfataricus archaeal exosome (green), albeit at a different depth in the central channel. The view is with the molecular 3-fold oriented vertically.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2723993&req=5

fig5: Structural changes associated with RNA binding to the PNPase core. (a) An overlay of the RNA-free (cyan) and RNA-bound (grey) forms of PNPase core viewed down the molecular 3-fold axis (left) and perpendicular to it (right). In the view on the right, the helical domain is on the top of the torus, and the S1 and KH domains (not shown) are on the bottom. (b) Expanded view of the central channel aperture in the RNA-free form. The aperture is occluded by the F77 and F78 side chain of the FFRR loop in this apo-structure, but the loop is less well ordered in the Mn2+apo structure and in the apo-structure reported by Shi et al.29 (c) The same view as in the left-hand panel but in the RNA-bound form; this shows that the aperture has dilated. It is not clear whether all three RNA-binding sites could be accommodated simultaneously. (d) RNA binds to the central aperture of both E. coli PNPase core (orange) and the S. solfataricus archaeal exosome (green), albeit at a different depth in the central channel. The view is with the molecular 3-fold oriented vertically.
Mentions: Previously reported crystal structures of the full-length S. antibioticus and E. coli PNPases identified two constricted points in the channel.19,29 One of these is closer to the channel entrance and the second is deeper within the channel and nearer the active site. The entrance-proximal aperture is formed by conserved residues, corresponding to E. coli residues F77-F78-R79-R80 (Fig. 5a and b). This loop had been predicted to form an RNA-binding site in the PNPases of many species.19,46 Confirming this hypothesis, our structure of PNPase in complex with RNA shows that F77 of each PNPase core monomer makes an aromatic stacking contact with an RNA base (Fig. 5c). The electron density in our structure is not sufficiently resolved to identify the bases, although the shape indicates that they are likely to be purines. F78 of the conserved FFRR loop supports the orientation of the base-contacting F77. The remainder of the RNA away from the stacking contact is disordered and the electron density here is poorly resolved, although it is clear that the pathway followed is along the central pore in the direction of the active site. We do not observe any interpretable electron density at the active site.

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