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Structural basis of mRNA-cap recognition by Dcp1–Dcp2

Nature Structural & Molecular Biology volume 23, pages 987–994 (2016)Cite this article

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Abstract

Removal of the 5′ cap on mRNA by the decapping enzyme Dcp2 is a critical step in 5′-to-3′ mRNA decay. Understanding the structural basis of Dcp2 activity has been a challenge because Dcp2 is dynamic and has weak affinity for the cap substrate. Here we present a 2.6-Å-resolution crystal structure of a heterotrimer of fission yeast Dcp2, its essential activator Dcp1, and the human NMD cofactor PNRC2, in complex with a tight-binding cap analog. Cap binding is accompanied by a conformational change in Dcp2, thereby forming a composite nucleotide-binding site comprising conserved residues in the catalytic and regulatory domains. Kinetic analysis of PNRC2 revealed that a conserved short linear motif enhances both substrate affinity and the catalytic step of decapping. These findings explain why Dcp2 requires a conformational change for efficient catalysis and reveals that coactivators promote RNA binding and the catalytic step of decapping, possibly through different conformational states.

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Figure 1: Cap binding requires a conformational change in Dcp2.
Figure 2: The 5′ cap is recognized by a composite nucleotide-binding site in Dcp2.
Figure 3: The cap-bound conformation of Dcp2 includes a positively charged tunnel that may bind RNA.
Figure 4: A conserved short linear motif enhances the catalytic step of decapping.
Figure 5: Proposed model for initial steps in the Dcp2 catalytic cycle.

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Acknowledgements

The authors thank X. Liu, J. Binning, and C. Waddling at UCSF for valuable help and advice on crystallography experiments, and J. Holton and G. Meigs at Lawrence Berkeley National Laboratory, Advanced Light Source beamline 8.3.1, for help with X-ray data collection. We also thank J. Kowalska for helpful discussions on the design and synthesis of the two-headed cap analog. This work was supported by the US National Institutes of Health (R01 GM078360 to J.D.G. and NRSA fellowship F32 GM105313 to J.S.M.) and the National Science Centre, Poland (grant no. UMO-2012/05/E/ST5/03893 to J.J. and fellowship no. UMO-2014/12/T/NZ1/00528 to M.Z.). The Advanced Light Source is supported by the US Department of Energy under contract no. DE-AC02-05CH11231.

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA

    Jeffrey S Mugridge, Marcin Ziemniak & John D Gross

  2. Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland

    Marcin Ziemniak

  3. Centre of New Technologies, University of Warsaw, Warsaw, Poland

    Jacek Jemielity

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  1. Jeffrey S Mugridge
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Contributions

J.S.M. designed and purified all protein constructs, carried out crystallization experiments, collected and refined crystallographic data, carried out decapping kinetics experiments, wrote the manuscript, and prepared the figures. M.Z. and J.J. designed and synthesized the two-headed cap analog. J.D.G. supervised the project and experimental design and guided manuscript preparation and editing. All authors read and commented on the manuscript.

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Correspondence to John D Gross.

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Integrated supplementary information

Supplementary Figure 1 Human PNRC2(91–121) activates fission yeast Dcp1–Dcp2.

(a) Complexes of Hs PNRC2(91-121)–Sp Dcp1–Dcp2(1-243) (purple) decap a 355-mer RNA substrate faster than Sp Dcp1–Dcp2 alone (green). kobs is plotted against protein concentration to determine kinetic constants. Errors are s.d. on individual fits to determine kobs. (b) Bar graph showing kmax (red) and KM (blue) values determined from fits of kinetic data shown in (a). For Hs PNRC2(91-121)–Sp Dcp1–Dcp2(1-243) kmax = 1.22 ± 0.09 min-1 and KM = 27 ± 4 µM, for Sp Dcp1–Dcp2(1-243) kmax = 0.15 ± 0.02 min-1 and KM = 26 ± 5 µM. This corresponds to an 8-fold activation in catalysis by PNRC2(91-121). Errors are s.d. of fits to determine kmax and KM. (c) Single chain constructs where PNRC2(91-121) is tethered to the N-terminus of Dcp2 by a flexible GGGGS linker (Dcp1–scPNRC2Dcp2) are more active than co-purified complexes of PNRC2(91-121)–Dcp1–Dcp2. Plots of fraction m7GDP product versus time are shown for decapping reactions for both complexes under saturating protein concentrations (25 µM). (d) Bar graph showing kobs values determined from the decapping time course shown in (c). For the co-purified complex PNRC2(91-121)–Dcp1–Dcp2, kobs = 0.72 ± 0.08 min-1; for the single chain Dcp1–scPNRC2Dcp2 complex, kobs = 1.33 ± 0.07 min-1. Errors are s.d. of the individual exponential fits to obtain kobs shown in (c).

Supplementary Figure 2 Crystal structure of the apo Dcp1–scPNRC2Dcp2 complex.

(a) Asymmetric unit of Dcp1–scPNRC2Dcp2. The Dcp1–scPNRC2Dcp2 complex crystallizes as a nearly symmetric domain-swapped dimer, in which the PNRC2 peptide tethered to the N-terminus of Dcp2 binds in trans to Dcp1 in the adjacent protomer. PNRC2 peptides are colored the same as the Dcp2 regulatory domain to which they are tethered; a dotted line shows where the flexible GGGGS linker connects the C-terminus of PNRC2(91-121) to the N-terminus of Dcp2. (b) Alignment of apo Dcp1–scPNRC2Dcp2 structure (colored, PNRC2 not shown) from this study, with closed, ATP-bound Sp Dcp1–Dcp2 structure from PDB 2QKM20 (Dcp1–Dcp2 in light gray, ATP in dark gray, Dcp2 residues 1-243 are shown). Dcp1–Dcp2(1-90) was used to align the structures; backbone RMSD is 0.7 Å over 213 residues. (c) Alignment of apo Dcp1–scPNRC2Dcp2 structure (colored, PNRC2 not shown) from this study, with Sp Dcp1–Dcp2 structure from PDB 5J3Y39 (light gray). Dcp1–Dcp2(1-90) was used to align the structures; backbone RMSD is 1.2 Å over 214 residues.

Supplementary Figure 3 Two-headed cap analog binds both molecules of the Dcp1–scPRNC2Dcp2 asymmetric unit in a different conformation.

(a) Asymmetric unit of Dcp1–scPNRC2Dcp2 with bound cap analogs. One Dcp1–Dcp2 protomer undergoes a conformational change to bind cap analog at a composite active site (colored in blues; this binding mode and conformational change are described in the main text). The second protomer of Dcp1–Dcp2 has the same conformation as in the apo structure, but with cap analog bound to exclusively the Nudix domain of Dcp2 (colored in reds). (b) Cap analog binds exclusively to the Nudix domain of Dcp2 by bridging Y220 and W117. Fo-Fc omit map shown at 1.5σ. (c) Although the electron density for the cap analog bound to only the Nudix domain is weak and discontinuous, likely due to weak binding and flexibility in the phosphate chain, this binding mode agrees perfectly with previous NMR chemical shift mapping experiments in which cap analog was titrated into the isolated Nudix domain.40 The cap analog-bound Nudix domain is shown here with previously determined NMR chemical shift perturbations (green) and resonance broadening (cyan) mapped to the surface.

Supplementary Figure 4 Cap-binding residues are occluded or separated by large distances in previous structures of Dcp1–Dcp2.

Large rotations of the Nudix domain are required for all previously identified closed conformations of Dcp1–Dcp2 to access the cap-binding conformation described in this study. Dcp1 is yellow, Dcp2 regulatory domain is purple, Dcp2 Nudix domain is green, Dcp2 dorsal RNA binding helix is blue, cap-binding residues are red. (a) cap analog-bound conformation of Sp Dcp1–Dcp2 from this study (top; PNRC2 not shown), and close-up view of W43 / Y220 residues (bottom). Y92 points toward the interior of the protein to bind cap, W43 and Y220 from the regulatory and Nudix domains of Dcp2 are positioned close to one another and are surface exposed in order to sandwich cap. (b) Closed, ATP-bound conformation of Sp Dcp1–Dcp2 from PDB 2QKM (top),20 and close-up view of W43 / Y220 residues (bottom). Y92 points toward solvent, W43 is occluded by residue R167 and unavailable for cap binding, and Y220 binds ATP but is not aligned with W43 for cap binding. A 30° rotation of the Nudix domain is required to bind cap with the composite active site depicted in (a). (c) Structure of Sp Edc1–Dcp1–Dcp2 from PDB 5J3T (top)39 and close-up of W43 / Y220 residues (bottom); Sp Edc1 peptide is colored orange. Y92 points toward solvent, W43 is buried in the interface with Sp Edc1 and the Nudix domain of Dcp2 and is unavailable to bind cap, and W43 and Y220 are separated by nearly 20 Å. A 90° rotation of the Nudix domain is required to form the composite active site as in (a).

Supplementary Figure 5 Interaction diagram showing contacts between two-headed cap analog and Dcp2 residues.

Residues on the Dcp2 regulatory domain (1-95) are shown in purple, residues on the Dcp2 Nudix domain (96-243) are shown in green. All distances are in Angstroms, and are heteroatom-heteroatom distances. Curved bold lines denote aromatic stacking interactions. Charges on protein sidechains and the ligand phosphate backbone are omitted in this schematic diagram.

Supplementary Figure 6 Alternative RNA binding path on Dcp1–Dcp2.

An alternative pathway for RNA binding to Dcp1–Dcp2 in the cap analog-bound conformation is shown on the electrostatic surface (a), or on a cartoon with RNA binding residues highlighted in blue (b). In this putative RNA binding path, RNA binds the dorsal helix of the Nudix domain (Box B motif), follows a flexible, lysine-rich loop (K206-K216) along the exterior of the protein, Y92 binds the first transcribed nucleotide, and m7G of cap is positioned near the center of the enzyme and bound by W43/Y220/D47.

Supplementary Figure 7 Comparison of decapping activation kinetics by Hs PNRC2 and Sp Edc1, and purification of PNRC2–Dcp1–Dcp2 complexes for kinetics experiments.

(a) Sp Edc1 strongly activates mRNA decapping by Dcp1–Dcp2. Comparison of decapping activation kinetics for complexes of Hs PNRC2(91-121) or Sp Edc1 with Sp Dcp1–Dcp2. Sp Edc1 is a stronger activator than Hs PNRC2 at 25 µM enzyme complex concentration; decapping kinetics with Sp Edc1 are too fast to extract kobs under these conditions by manual pipetting. (b) Size exclusion chromatography of the WT Hs PNRC2(1-121) – Sp Dcp1–Dcp2 complex; Hs PNRC2 binds to Sp Dcp1–Dcp2 and the proteins co-elute (top), as visualized by SDS-PAGE (bottom). (c) SDS-PAGE of purified Hs PNRC2(1-121) – Sp Dcp1–Dcp2 complexes with PNRC2 point mutants in the YAGxxF Dcp2-activating motif used in kinetic experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1815 kb)

Supplementary Data Set 1

Kinetic data associated with Figure 4 and Supplementary Figure 1a,b. (XLSX 10 kb)

Conformational change in Dcp1–Dcp2 accompanied by cap-analog binding.

This video shows a morph between the apo Dcp1–Dcp2 conformation (PDB 5KQ1) and the two-headed cap analog-bound Dcp1–Dcp2 conformation (PDB 5KQ4). (MPG 4732 kb)

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Mugridge, J., Ziemniak, M., Jemielity, J. et al. Structural basis of mRNA-cap recognition by Dcp1–Dcp2. Nat Struct Mol Biol 23, 987–994 (2016). https://doi.org/10.1038/nsmb.3301

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