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Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly.

Hardwick SW, Gubbey T, Hug I, Jenal U, Luisi BF - Open Biol (2012)

Bottom Line: The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme.On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting.Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

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Interactions of Caulobacter crescentus PNPase with RNA. (a) (i) and (ii) correspond to the perspectives shown in figure 1b, with the three KH domains coloured green. The inset is an expanded view with the GSGG loops coloured red. (b) Schematic of the contacts with the RNA. Residues involved in phosphate backbone hydrogen bonding are shown on the left. Residues forming van der Waals contacts are in the far right margin, and the residues hydrogen bonding to bases or sugar 2′-OH groups are in the middle-right group. The diamonds are colour coded for the three PNPase chains. All contacts shown are from the KH domain or from the FFKR loop at the entry aperture of the central channel. Base 7 is disordered and has not been modelled in the final structure. (c) Electron density map for endogenous RNA chain. An omit map of the RNA chain contoured at 1σ is shown as grey mesh. RNA is shown as orange and green cartoon, and the three KH domains are shown as semi-transparent cartoons, coloured separately as in figure 2b.
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RSOB120028F2: Interactions of Caulobacter crescentus PNPase with RNA. (a) (i) and (ii) correspond to the perspectives shown in figure 1b, with the three KH domains coloured green. The inset is an expanded view with the GSGG loops coloured red. (b) Schematic of the contacts with the RNA. Residues involved in phosphate backbone hydrogen bonding are shown on the left. Residues forming van der Waals contacts are in the far right margin, and the residues hydrogen bonding to bases or sugar 2′-OH groups are in the middle-right group. The diamonds are colour coded for the three PNPase chains. All contacts shown are from the KH domain or from the FFKR loop at the entry aperture of the central channel. Base 7 is disordered and has not been modelled in the final structure. (c) Electron density map for endogenous RNA chain. An omit map of the RNA chain contoured at 1σ is shown as grey mesh. RNA is shown as orange and green cartoon, and the three KH domains are shown as semi-transparent cartoons, coloured separately as in figure 2b.

Mentions: In the course of preparing recombinant C. crescentus PNPase, it was found that oligonucleotide originating from the E. coli expression host remained avidly associated with the protein during chromatographic purification. Extensive incubation with phosphate to promote phosphorolysis, followed by gel filtration, liberated nucleoside diphosphate; however, the limit digest product still remained associated with the PNPase. On the basis of absorbance at 260 nm, an estimated 20–30 nucleotides were bound to each PNPase trimer. It was possible to liberate a fraction of the protein from RNA by chromatography, and this material was used to prepare the apo-form crystals described in §4.1. Crystals of the RNA-bound material were obtained in space group P22121 that contain a PNPase trimer in the asymmetric unit (table 2). The structure was solved by molecular replacement using as search models the RNase PH-like core and a KH domain from the apo structure. The other two KH domains were found in the unbiased maps. It was also clear from the early electron density maps that RNA was present and interacting with the KH domains (figure 2a). The KH domain is structurally similar to the human poly(C) binding protein, and the GxxG loop makes similar contacts with nucleic acids (electronic supplementary material, figure S2).Figure 2.


Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly.

Hardwick SW, Gubbey T, Hug I, Jenal U, Luisi BF - Open Biol (2012)

Interactions of Caulobacter crescentus PNPase with RNA. (a) (i) and (ii) correspond to the perspectives shown in figure 1b, with the three KH domains coloured green. The inset is an expanded view with the GSGG loops coloured red. (b) Schematic of the contacts with the RNA. Residues involved in phosphate backbone hydrogen bonding are shown on the left. Residues forming van der Waals contacts are in the far right margin, and the residues hydrogen bonding to bases or sugar 2′-OH groups are in the middle-right group. The diamonds are colour coded for the three PNPase chains. All contacts shown are from the KH domain or from the FFKR loop at the entry aperture of the central channel. Base 7 is disordered and has not been modelled in the final structure. (c) Electron density map for endogenous RNA chain. An omit map of the RNA chain contoured at 1σ is shown as grey mesh. RNA is shown as orange and green cartoon, and the three KH domains are shown as semi-transparent cartoons, coloured separately as in figure 2b.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376730&req=5

RSOB120028F2: Interactions of Caulobacter crescentus PNPase with RNA. (a) (i) and (ii) correspond to the perspectives shown in figure 1b, with the three KH domains coloured green. The inset is an expanded view with the GSGG loops coloured red. (b) Schematic of the contacts with the RNA. Residues involved in phosphate backbone hydrogen bonding are shown on the left. Residues forming van der Waals contacts are in the far right margin, and the residues hydrogen bonding to bases or sugar 2′-OH groups are in the middle-right group. The diamonds are colour coded for the three PNPase chains. All contacts shown are from the KH domain or from the FFKR loop at the entry aperture of the central channel. Base 7 is disordered and has not been modelled in the final structure. (c) Electron density map for endogenous RNA chain. An omit map of the RNA chain contoured at 1σ is shown as grey mesh. RNA is shown as orange and green cartoon, and the three KH domains are shown as semi-transparent cartoons, coloured separately as in figure 2b.
Mentions: In the course of preparing recombinant C. crescentus PNPase, it was found that oligonucleotide originating from the E. coli expression host remained avidly associated with the protein during chromatographic purification. Extensive incubation with phosphate to promote phosphorolysis, followed by gel filtration, liberated nucleoside diphosphate; however, the limit digest product still remained associated with the PNPase. On the basis of absorbance at 260 nm, an estimated 20–30 nucleotides were bound to each PNPase trimer. It was possible to liberate a fraction of the protein from RNA by chromatography, and this material was used to prepare the apo-form crystals described in §4.1. Crystals of the RNA-bound material were obtained in space group P22121 that contain a PNPase trimer in the asymmetric unit (table 2). The structure was solved by molecular replacement using as search models the RNase PH-like core and a KH domain from the apo structure. The other two KH domains were found in the unbiased maps. It was also clear from the early electron density maps that RNA was present and interacting with the KH domains (figure 2a). The KH domain is structurally similar to the human poly(C) binding protein, and the GxxG loop makes similar contacts with nucleic acids (electronic supplementary material, figure S2).Figure 2.

Bottom Line: The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme.On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting.Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

Show MeSH