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The structure of the Pan2–Pan3 core complex reveals cross-talk between deadenylase and pseudokinase

Nature Structural & Molecular Biology volume 21, pages 591–598 (2014)Cite this article

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Abstract

Pan2–Pan3 is a conserved complex involved in the shortening of mRNA poly(A) tails, the initial step in eukaryotic mRNA turnover. We show that recombinant Saccharomyces cerevisiae Pan2–Pan3 can deadenylate RNAs in vitro without needing the poly(A)-binding protein Pab1. The crystal structure of an active ~200-kDa core complex reveals that Pan2 and Pan3 interact with an unusual 1:2 stoichiometry imparted by the asymmetric nature of the Pan3 homodimer. An extended region of Pan2 wraps around Pan3 and provides a major anchoring point for complex assembly. A Pan2 module formed by the pseudoubiquitin-hydrolase and RNase domains latches onto the Pan3 pseudokinase with intertwined interactions that orient the deadenylase active site toward the A-binding site of the interacting Pan3. The molecular architecture of Pan2–Pan3 suggests how the nuclease and its pseudokinase regulator act in synergy to promote deadenylation.

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Figure 1: Deadenylation activity of a yeast Pan2–Pan3 core complex.
Figure 2: Structure of a yeast Pan2–Pan3 core complex.
Figure 3: Asymmetric interactions between a single Pan2 and two Pan3 subunits.
Figure 4: The Pan2 RNase: interaction with the pseudo-UCH domain and active site.
Figure 5: The Pan2 RNase and linker interact with the Pan3 pseudokinase domain.
Figure 6: The Pan3 A-binding site is juxtaposed with the Pan2 deadenylation site and affects deadenylation.

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Acknowledgements

We would like to thank A. Fischer for data in Figure 5b and C. Basquin for Thermofluor experiments (both Max Planck Institute (MPI) of Biochemistry); A. McCoy (Cambridge Institute for Medical Research) for advice on Phaser; the MPI Crystallization Facility for crystal screening and optimization; the MPI Core Facility for MS; and the beamline scientists at the Swiss Light Source (SLS) for excellent assistance with data collection. We also thank members of our laboratory for useful discussions and critical reading of the manuscript. This study was supported by the Max Planck Gesellschaft and grants of the European Research Council (ERC Advanced Investigator Grant 294371 and Marie Curie Initial Training Networks RNPnet) and the Deutsche Forschungsgemeinschaft (DFG SFB646, SFB1035, GRK1721, FOR1680 and CIPSM) to E.C.

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  1. Structural Cell Biology Department, Max Planck Institute of Biochemistry, Martinsried, Germany

    Ingmar B Schäfer, Michaela Rode, Fabien Bonneau, Steffen Schüssler & Elena Conti

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  1. Ingmar B Schäfer
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Contributions

I.B.S. performed molecular biology and biochemical analysis and determined structures. M.R. assisted with insect-cell culture. F.B. performed deadenylation assays. S.S. helped with protein purifications. I.B.S. and E.C. designed the study and wrote the paper.

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Correspondence to Elena Conti.

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Integrated supplementary information

Supplementary Figure 1 The WD40 domain of Pan2 directly contacts the Pan2–Pan3 core complex.

(a) RNase assays with Pan2fl–Pan3fl-H10 using different 15mer homo-oligonucleotides, carried out as in Fig. 1c. Abbreviation: conc. (concentration). (b) Protein co-precipitation of H10Pan3CR (His-tagged Pan3 C-terminal region, residues 275–679) and Pan2fl (Pan2 full-length). The Nickel pull-down assay was performed on the cell lysate in the presence of 300 mM salt and samples were separated on 4-12% SDS-PAGE. (c) Left panel: protein co-precipitation of Pan3ΔN-H10 (His tagged Pan3 residues 226–679) and Pan2N-3C-C (fl Pan2 with a 3C protease cleavage site engineered after the N-terminal WD40 domain, between residues 333 and 334). The Nickel pull-down assay (His-Select resin, Sigma) was carried as described in panel a. Central panel: analysis of the Pan3ΔN-H10– Pan2N-3C-C sample after 3C protease cleavage (lane 1). The cleaved sample was purified on an ion exchange column (AEC), which separated the excess of 3C protease (lanes 2-4) from three polypeptides which co-eluted in the same peak: Pan2N (residues 1–333), Pan2C (residues 334–1115) and Pan3ΔN-H10 (lane 5). The three peptides also co-eluted by size exclusion chromatography (SEC,lane 9). Right panel: chromatogram of the size-exclusion chromatography. Highlighted in red is the peak fraction corresponding to lane 9 of the gel on the left. Abbreviations: 3C-prot. (3C-protease), A280nm (Absorption at 280 nm).

Supplementary Figure 2 Structural and biochemical analysis of Pan2–Pan3.

(a) Snapshots of the simulated annealing Fo-Fc omit map countered at 2.5σ (mesh, in grey) for different segments of the Pan2 linker. The anomalous signal peak for the Selenomethionine residues is shown in red, contoured at 4.0σ. Abbreviation: UCH (ubiquitin C-terminal hydrolase). (b) Size exclusion chromatography profile of Pan2fl–Pan3ΔN with standard molecular weight markers. The calculated mass of the complex is shown on the right. Abbreviations: Pan3ΔN (residues 226–679), H10 (His-tag), fl (full length), MW (molecular weight), A280nm (Absorption at 280 nm).

Supplementary Figure 3 Conserved interactions centered on Pan3.

(a) Superposition of the atomic models of the individual protomers of a Pan3ΔN dimer from the yeast Pan2ΔN–Pan3ΔN structure (with the two chains in yellow and orange) with those of the N. crassa Pan3CR dimer28 (in gray, left panel) and of the D. melanogaster (in gray, right pane) in isolation. Note that helix αN (from the extension upstream of the pseudokinase domain) is not present in the D. melanogaster structure. Abbreviation: CR (C-terminal region, residues 275–679). (b) The surfaces of the knob domains are shown, colored according to sequence conservation from light (less conserved) to dark (conserved), with the Pan2 linker in ribbon representation. Indicated are the positions of the residues of yeast Pan3 corresponding to mutants previously described28. Abbreviation: N-term. (N-terminus).

Supplementary Figure 4 Comparison of Pan2ΔN–Pan3ΔN domains with related domains in other proteins.

(a) The UCH domain of Pan2 (left) is viewed in the same orientation as the UCH domain of HAUSP34, after optimal superposition. Both domains are colored with the Fingers in cyan, the Palm in blue and the Thumb in green. The active site of HAUSP and the equivalent position Pan2 CH are indicated with an asterisk. The active-site residues of HAUSP domain are shown in stick representation. The residues at the equivalent position of the inactive Pan2 UCH are also shown. Abbreviations: Pan2R (RNase domain of Pan2), Pan2U (ubiquitin C-terminal hydrolase of Pan2), UCH (ubiquitin C-terminal hydrolase). (b) The RNase domain of Pan2 (left panel) is viewed in the same orientation as the similar domains in the ISG20 RNase33 (central panel) and in yeast Caf1 (also known as Pop2)36 (right panel) after optimal superposition. Active site residues are highlighted in stick representation. The nucleotides in the active sites are also shown in stick representation, together with the ions as spheres. (c) The overall structure UCH–RNase module of Pan2 is essentially unchanged when in the Pan2ΔN–Pan3ΔN complex (UCH in blue, cyan and green, and RNase in pink) and in the structure of Pan2UR in isolation (in gray), with the exception of a loop that becomes ordered in the complex (corresponding to the loop shown in Supplementary Fig. 4d and in stick representation in Fig. 5a). (d) The N-terminal lobe of the Pan3bent pseudokinase (yellow) interacts with a loop of the Pan2 RNase domain (left and central panel) at a similar overall position used for interaction in either kinases, for example between Aurora-A (orange) and TPX2 (red)38 (right panel, shown in the same orientation after optimal superposition of the N-terminal lobes).

Supplementary Figure 5 Characterization of the Pan2–Pan3–nucleotide interactions.

(a) Thermofluor assay comparing the stability of Pan2ΔN–Pan3ΔN-ATP and Pan2ΔN–Pan3ΔN used in the assay in Fig. 6b. The curves show the thermal unfolding transition. The proteins were incubated with 3.5X of Sypro Orange dye (Invitrogen), which binds to hydrophobic patches exposed on the protein during unfolding). The proteins were subjected to thermal denaturation and the change in fluorescence intensity was measured with a real-time PCR system (Eppendorf). The apparent melting temperatures of Pan2ΔN–Pan3ΔN and Pan2ΔN–Pan3ΔN-ATP mutant measurements with the Thermofluor assay in the presence and absence of AMP are shown. Abbreviations: Pan2ΔN (residues 340–1115), Pan3ΔN (residues 226– 679), Pan3ATP (ATP binding mutant of Pan3). (b) Deadenylation assays using the protein samples shown in Fig. 6c in the absence (left panels) and presence of 1mM ATP. Abbreviations: M (size markers), conc. (concentration).

Supplementary Figure 6 Interaction sites in the Pan2–Pan3 complex.

Structure of the yeast Pan2ΔN–Pan3ΔN complex with the bound AMP molecule at the deadenylase active site. Also shown are the nucleotides (ATPγS) and the region corresponding to the Tryptophan-binding pocket from the structures of Pan3 orthologues28, after optimal superposition. Abbreviations: PK (pseudokinase), nt (nucleotide), UCH (ubiquitin C-terminal hydrolase)

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Schäfer, I., Rode, M., Bonneau, F. et al. The structure of the Pan2–Pan3 core complex reveals cross-talk between deadenylase and pseudokinase. Nat Struct Mol Biol 21, 591–598 (2014). https://doi.org/10.1038/nsmb.2834

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