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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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Interactions of RNase E micro-domain and PNPase core. (a) Interaction of the RNase E recognition micro-domain (residues 1021–1061, red) with the solvent-exposed strand of an antiparallel β-sheet of PNPase (grey). The β-sheet is part of the C-terminal RNase PH-like subdomain of PNPase. The view is at the interface of two PNPase protomers, and the perspective is from the S1/KH side of the PNPase ring (i.e., from the bottom of the ring shown in Fig. 2b). (b) Overlay of the E. coli PNPase structure (grey) with the Rrp45-Rrp46 in the human exosome (yellow for Rrp46 and black for Rrp 45) showing an interaction for the two exosome subunits that is structurally homologous to that of PNPase core to RNase E.26 The perspective is from the same orientation as represented in (a).
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fig3: Interactions of RNase E micro-domain and PNPase core. (a) Interaction of the RNase E recognition micro-domain (residues 1021–1061, red) with the solvent-exposed strand of an antiparallel β-sheet of PNPase (grey). The β-sheet is part of the C-terminal RNase PH-like subdomain of PNPase. The view is at the interface of two PNPase protomers, and the perspective is from the S1/KH side of the PNPase ring (i.e., from the bottom of the ring shown in Fig. 2b). (b) Overlay of the E. coli PNPase structure (grey) with the Rrp45-Rrp46 in the human exosome (yellow for Rrp46 and black for Rrp 45) showing an interaction for the two exosome subunits that is structurally homologous to that of PNPase core to RNase E.26 The perspective is from the same orientation as represented in (a).

Mentions: PNPase core was co-crystallised with its RNase E recognition micro-domain (residues 1021–1061) under four different conditions, providing several independent views of the micro-domain–enzyme interaction (Table 1). The RNase E micro-domain was well resolved in the tetragonal crystal form, for which interpretable electron density was present for residues 1039–1061 in all three PNPase protomers that occupy the asymmetric unit. The backbone of the RNase E micro-domain forms hydrogen-bonding interactions with the solvent-exposed terminal ridge of an anti-parallel β-sheet within the amino-terminal RNase PH-like sub-domain of the PNPase core (residues 327–331). This interaction generates a pseudo-continuous extended β-sheet (Fig. 2a). The remaining portion of the RNase E micro-domain that is outside the sheet region has a distorted helical conformation (Fig. 3a). The RNase E micro-domain is also well resolved in the rhombohedral crystal form with Mn2+ but was poorly resolved in the second rhombohedral form grown in the absence of the metal (Table 1). The location of the micro-domain on the surface of PNPase is corroborated by an orthorhombic crystal form that, while diffracting to only limited resolution (roughly 3.6 Å), has four independent PNPase trimers in the asymmetric unit that reveal unbiased density for 12 copies of the RNase E micro-domain (results not shown).


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)

Interactions of RNase E micro-domain and PNPase core. (a) Interaction of the RNase E recognition micro-domain (residues 1021–1061, red) with the solvent-exposed strand of an antiparallel β-sheet of PNPase (grey). The β-sheet is part of the C-terminal RNase PH-like subdomain of PNPase. The view is at the interface of two PNPase protomers, and the perspective is from the S1/KH side of the PNPase ring (i.e., from the bottom of the ring shown in Fig. 2b). (b) Overlay of the E. coli PNPase structure (grey) with the Rrp45-Rrp46 in the human exosome (yellow for Rrp46 and black for Rrp 45) showing an interaction for the two exosome subunits that is structurally homologous to that of PNPase core to RNase E.26 The perspective is from the same orientation as represented in (a).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Interactions of RNase E micro-domain and PNPase core. (a) Interaction of the RNase E recognition micro-domain (residues 1021–1061, red) with the solvent-exposed strand of an antiparallel β-sheet of PNPase (grey). The β-sheet is part of the C-terminal RNase PH-like subdomain of PNPase. The view is at the interface of two PNPase protomers, and the perspective is from the S1/KH side of the PNPase ring (i.e., from the bottom of the ring shown in Fig. 2b). (b) Overlay of the E. coli PNPase structure (grey) with the Rrp45-Rrp46 in the human exosome (yellow for Rrp46 and black for Rrp 45) showing an interaction for the two exosome subunits that is structurally homologous to that of PNPase core to RNase E.26 The perspective is from the same orientation as represented in (a).
Mentions: PNPase core was co-crystallised with its RNase E recognition micro-domain (residues 1021–1061) under four different conditions, providing several independent views of the micro-domain–enzyme interaction (Table 1). The RNase E micro-domain was well resolved in the tetragonal crystal form, for which interpretable electron density was present for residues 1039–1061 in all three PNPase protomers that occupy the asymmetric unit. The backbone of the RNase E micro-domain forms hydrogen-bonding interactions with the solvent-exposed terminal ridge of an anti-parallel β-sheet within the amino-terminal RNase PH-like sub-domain of the PNPase core (residues 327–331). This interaction generates a pseudo-continuous extended β-sheet (Fig. 2a). The remaining portion of the RNase E micro-domain that is outside the sheet region has a distorted helical conformation (Fig. 3a). The RNase E micro-domain is also well resolved in the rhombohedral crystal form with Mn2+ but was poorly resolved in the second rhombohedral form grown in the absence of the metal (Table 1). The location of the micro-domain on the surface of PNPase is corroborated by an orthorhombic crystal form that, while diffracting to only limited resolution (roughly 3.6 Å), has four independent PNPase trimers in the asymmetric unit that reveal unbiased density for 12 copies of the RNase E micro-domain (results not shown).

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