Journal of Molecular Biology
Solution Structure of an Archaeal RNase P Binary Protein Complex: Formation of the 30-kDa Complex between Pyrococcus furiosus RPP21 and RPP29 Is Accompanied by Coupled Protein Folding and Highlights Critical Features for Protein–Protein and Protein–RNA Interactions
Introduction
Ribonuclease P (RNase P), a ribonucleoprotein (RNP) complex, catalyzes removal of the 5′ leader sequence during tRNA maturation.1, 2, 3 Across the three domains of life, it is composed of one RNA subunit and a varying number of protein subunits: one in bacteria, at least four in archaea, and nine in eukarya. The RNase P RNA (RPR) from each domain of life has been shown to be catalytic on its own in vitro under elevated monovalent and divalent ion concentrations.4, 5, 6 However, RNase P protein(s) (RPPs) enhance catalysis under near-physiological conditions by facilitating RPR folding, substrate recognition, and decrease in the Mg2+ requirement.7, 8, 9, 10 Interestingly, when the enzyme from the three domains of life is compared, an increase in protein content is associated with the loss of some RPR elements and a concomitant decrease in RPR activity.1, 4, 11, 12 Thus, RNase P is an appealing model to address how protein cofactors might have taken over the structural and functional attributes of RNAs during evolution from a putative RNA-centric world to the present protein-centric one. Towards the goal of understanding this progression, we have focused our efforts on the biochemically tractable and thermostable archaeal version of the RNase P enzyme from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu), whose RPPs are homologous to their eukaryal counterparts.13, 14
Bacterial RNase P is the best understood form of the enzyme.15 Based on the primary sequence and secondary structure of the RPRs, bacterial RNase P can be classified into two distinct types: A and B.16 In both types, the RPR is composed of two independently folding domains, termed the specificity and catalytic domains (S domain and C domain, respectively), held together by interdomain RNA–RNA contacts.17 To date, crystallographic structures have been reported for two S domains and two full-length RPRs, from bacterial RNase P of types A and B.18, 19, 20, 21 The structures of three homologous bacterial RPPs have been solved by crystallography or NMR spectroscopy as well.22, 23, 24 Although the structure of the bacterial RNase P holoenzyme complex has not yet been determined, models of A- and B-type RNPs have been built using a wealth of information from biochemical studies.25, 26, 27
Phylogenetic and biochemical studies have revealed that archaeal RNase P is a compositional intermediate between the bacterial and eukaryotic counterparts. Euryarchaeal RNase P can also be categorized into two groups based on their RPR sequences: A and M.16, 28 Euryarchaeal A-type RPR (e.g., Pfu) is similar to the bacterial A-type in terms of secondary structures and reported in vitro catalytic activity;5, 13 in contrast, the M-type RPR more closely resembles the eukaryotic RPR and has shown no catalytic activity on its own, although it can cleave a substrate tethered in cis.29 Though no high-resolution structure of the archaeal RPR is available, secondary-structure similarities and the presence of universally conserved nucleotides in the active site suggest that the archaeal RPR fold (especially the C domain) might resemble that of the bacterial RPR; however, the increased RPP content in archaeal RNase P suggests that RNA–protein interactions in the enzyme are likely to have replaced some of the intramolecular RNA–RNA interactions present in the bacterial RPR.12
Although the archaeal and eukaryotic RPRs share conserved structural features with their bacterial counterparts, none of the RPPs from archaeal or eukaryotic RNase P share sequence similarity with the single bacterial protein. At least four protein subunits are associated with Pfu RNase P and share sequence homology to the human RPPs: RPP21, RPP29, RPP30, and POP5.14 The structures of the four archaeal RPPs have been solved from different archaeal organisms by either NMR spectroscopy or X-ray crystallography, or both. These studies on the isolated proteins revealed the structures of the RPPs to fall within common nucleic acid binding protein families: an Sm-like fold (RPP29),30, 31, 32, 33 a zinc ribbon (RPP21),34, 35 an RRM-like fold (Pop5),36 and a TIM barrel (RPP30).37
Biochemical data have suggested that at least some of the archaeal and eukaryotic RPPs function in pairs. Yeast two-hybrid studies of proteins from both archaeal and eukaryotic RNase P confirmed the presence of two binary complexes: RPP21–RPP29 and RPP30–POP5.38, 39, 40 Reconstitution assays performed on Pfu RNase P have shown that either protein pair is sufficient to activate the RNA enzyme at lower ion concentrations, while no single protein can rescue the RNA enzyme under the same conditions.13 Although these studies established a role for RPP binary pairs in enhancing the catalytic activity of archaeal RPRs, the mechanistic basis for their actions is largely unclear without useful structural models of the archaeal RNase P holoenzyme. To that end, high-resolution structure determination of the binary complexes is a necessity.
Here, we report the NMR-derived solution structure of the 30-kDa complex between two Pfu RPPs: the Sm-like RPP29 and the zinc-ribbon RPP21 proteins. This study complements recently reported crystal structures of the Pyrococcus horikoshii (Pho) POP5–RPP30 and RPP21–RPP29 complexes,41, 42 reveals dynamic features of the proteins and binding-coupled protein folding events, and identifies additional features important for protein–protein and protein–RNA interactions. Furthermore, footprinting studies allow us to map the RPP21–RPP29 complex onto the S domain of the RPR. Together with biochemical studies on the C domain and RPP30–POP5 complex from Pfu and Methanocaldococcus jannaschii (Mja),13, 29 this work represents an important step towards understanding the architecture and function of archaeal and eukaryal RNase P.
Section snippets
NMR spectroscopy of the RPP29–RPP21 complex
At lower temperatures (25 and 40 °C), NMR spectra of free Pfu RPP21 and RPP29 were of poor quality, with broad lines and highly variable peak intensities, suggesting that the proteins were poorly folded and/or aggregated at those temperatures. Spectra recorded at 55 °C yielded generally narrow lines and uniform peak intensities for both proteins, and thus, data were recorded at 55 °C for both the free proteins and the protein–protein complexes. The 15N-edited heteronuclear single quantum
Conclusion
The NMR data presented here indicate that formation of the Pfu RPP29–RPP21 complex involves binding-coupled folding of structural elements in both proteins. The RPP29–RPP21 complex is defined by a combination of conserved interfacial hydrophobic and polar residues, including apparent salt bridges. Outside of the structured cores, residues in the extreme N- and C-termini of both proteins indicate that these remain unstructured in the protein complex. Of particular interest are the long
Pfu RPP29
The Pfu RPP29/pET-33b plasmid13 was transformed into Escherichia coli BL21(DE3) Rosetta cells (Novagen). The cells were grown in 2-L flasks in a shaker–incubator at 37 °C in LB or minimal M9 media containing 1 g/L of 15NH4Cl and 2 g/L of 13C-glucose as the sole nitrogen and carbon sources, supplemented with 30 μg/L of kanamycin and 34 μg/L of chloramphenicol. Production of the recombinant proteins was induced by addition of 0.5 mM IPTG at an A600 of 0.6 and harvested after 4 h by
Acknowledgements
We thank C. Yuan and C. Cottrell (Campus Chemical Instrument Center) for assistance with the NMR data collection and R. C. Wilson, I. R. Kleckner, and the members of the V. Gopalan laboratory (especially Lien Lai) for reagents, encouragement, and helpful discussions. This work was supported by a grant from the National Institutes of Health to M.P.F and V.G. (GM067807).
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Cited by (0)
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C.D.A. and D.K.P. contributed equally to this work.
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Present addresses: C. D. Amero, Institut de Biologie Structurale, CNRS, Grenoble, France; D. K. Pulukkunat, Department of Chemistry, Columbia University, New York, NY, USA.