Limits...
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.

Show MeSH

Related in: MedlinePlus

Calorimetric analysis of the interaction of E. coli PNPase and RNase E recognition micro-domain (residues 1021–1061). Top panel: The isothermal calorimetry profile showing the heat released upon titrating RNase E micro-domain with PNPase core. Bottom panel: The integrated heats after correction for heat of dilution. The data are best fit with a single binding site model, yielding parameters N =  1.02  ±  0.02, Ka 9.56 ( ±  0.71) × 105 M-1, ΔH =  –21.48  ±  0.52 kcal mol–1, ΔS = -44.7 cal mol–1 K–1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2723993&req=5

fig4: Calorimetric analysis of the interaction of E. coli PNPase and RNase E recognition micro-domain (residues 1021–1061). Top panel: The isothermal calorimetry profile showing the heat released upon titrating RNase E micro-domain with PNPase core. Bottom panel: The integrated heats after correction for heat of dilution. The data are best fit with a single binding site model, yielding parameters N =  1.02  ±  0.02, Ka 9.56 ( ±  0.71) × 105 M-1, ΔH =  –21.48  ±  0.52 kcal mol–1, ΔS = -44.7 cal mol–1 K–1.

Mentions: Because two RNase PH-like domains of the PNPase protomer are related by an internal pseudo-dyad, there are potentially two sites within each protomer that could form an extended sheet-like interaction with the RNase E micro-domain; however, only the amino-terminal RNase PH sub-domain forms an interaction with RNase E. The observed stoichiometry of one PNPase monomer binding to one RNase E micro-domain in the crystal structures is consistent with data from mass spectrometry,39 and isothermal titration calorimetry, which show that one RNase E micro-domain binds to each PNPase protomer (Fig. 4). The observed binding affinity by calorimetry is roughly 0.9 μM and is therefore very weak for a macromolecular interaction. The interaction may be stronger in the context of the full-length RNase E, since that molecule is a tetramer and therefore the PNPase binding sites will be spatially co-localised.


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)

Calorimetric analysis of the interaction of E. coli PNPase and RNase E recognition micro-domain (residues 1021–1061). Top panel: The isothermal calorimetry profile showing the heat released upon titrating RNase E micro-domain with PNPase core. Bottom panel: The integrated heats after correction for heat of dilution. The data are best fit with a single binding site model, yielding parameters N =  1.02  ±  0.02, Ka 9.56 ( ±  0.71) × 105 M-1, ΔH =  –21.48  ±  0.52 kcal mol–1, ΔS = -44.7 cal mol–1 K–1.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Calorimetric analysis of the interaction of E. coli PNPase and RNase E recognition micro-domain (residues 1021–1061). Top panel: The isothermal calorimetry profile showing the heat released upon titrating RNase E micro-domain with PNPase core. Bottom panel: The integrated heats after correction for heat of dilution. The data are best fit with a single binding site model, yielding parameters N =  1.02  ±  0.02, Ka 9.56 ( ±  0.71) × 105 M-1, ΔH =  –21.48  ±  0.52 kcal mol–1, ΔS = -44.7 cal mol–1 K–1.
Mentions: Because two RNase PH-like domains of the PNPase protomer are related by an internal pseudo-dyad, there are potentially two sites within each protomer that could form an extended sheet-like interaction with the RNase E micro-domain; however, only the amino-terminal RNase PH sub-domain forms an interaction with RNase E. The observed stoichiometry of one PNPase monomer binding to one RNase E micro-domain in the crystal structures is consistent with data from mass spectrometry,39 and isothermal titration calorimetry, which show that one RNase E micro-domain binds to each PNPase protomer (Fig. 4). The observed binding affinity by calorimetry is roughly 0.9 μM and is therefore very weak for a macromolecular interaction. The interaction may be stronger in the context of the full-length RNase E, since that molecule is a tetramer and therefore the PNPase binding sites will be spatially co-localised.

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