Abstract
The cell division cycle consists of a series of temporally ordered events. Cell cycle kinases and phosphatases provide key regulatory input, but how the correct substrate phosphorylation and dephosphorylation timing is achieved is incompletely understood. Here we identify a PxL substrate recognition motif that instructs dephosphorylation by the budding yeast Cdc14 phosphatase during mitotic exit. The PxL motif was prevalent in Cdc14-binding peptides enriched in a phage display screen of native disordered protein regions. PxL motif removal from the Cdc14 substrate Cbk1 delays its dephosphorylation, whereas addition of the motif advances dephosphorylation of otherwise late Cdc14 substrates. Crystal structures of Cdc14 bound to three PxL motif substrate peptides provide a molecular explanation for PxL motif recognition on the phosphatase surface. Our results illustrate the sophistication of phosphatase–substrate interactions and identify them as an important determinant of ordered cell cycle progression.
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Data availability
Atomic coordinates and structure factors have been deposited in the Protein Data Bank, with accession numbers 6G84 (Cdc14:Cbk1-C2), 6G85 (Cdc14:Cbk1-P212121) and 6G86 (Cdc14:Sic1). All additional data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank N. Davey and D. Morgan for bringing together the Kim and Uhlmann laboratories; the Diamond Light Source Synchrotron for access to beamlines IO3, IO4 and IO4-1 (mx13775); S. Federico, D. Joshi and N. O’Reilly, The Francis Crick Institute, London, UK for peptide synthesis; J. Diffley, The Francis Crick Institute, London, UK for the antibody to Orc2; V. Christodoulou, M. Hall, S. Kjaer, L. Masino, A. Purkiss and S. Touati for help and advice; and M. Godfrey, E. Weiss and members of our laboratory for discussions and critical reading of the manuscript. This work was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001198), the UK Medical Research Council (FC001198) and the Wellcome Trust (FC001198).
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M.K. and F.U. conceived the study; M.K. performed the experiments; S.M. and M.K. solved the crystal structures; M.-H.S., C.C.-V. and P.M.K. conducted the phage display screen; and M.K. and F.U. wrote the manuscript with input from all authors.
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Integrated supplementary information
Supplementary Figure 1 Biochemical characterization of the PxL motif.
(a), Recombinant Cdc14 preparations used for the phage display screen (His-Cdc14), peptide array (HA-Cdc14) and microscale thermophoresis experiments (GFP-Cdc14) were analyzed by SDS–PAGE and stained with Coomassie blue. A size marker is included. (b), Peptides recovered in the phage display screen are enriched in known Cdc14 substrates and Cdc14 interactors12,19 (hypergeometric test). (c), Mutational Cbk1 peptide array to probe the contribution of individual positions to Cdc14 binding. As in Fig. 1c, but a different membrane solvation was used (bottom), as indicated. A picture of the peptides stained with Ponceau S is also shown (top), as well as a control using the anti-HA antibody only (middle). (d), Microscale thermophoresis profiles of additional variant Cbk1-derived PxL motif peptides binding to GFP-Cdc14. Shown are the means and s.d. from two (F82G, V85G and Δ91–97) or three (L88G) independent experiments.
Supplementary Figure 2 Crystallization of Cdc14 with Cbk1 and Sic1 peptides.
(a), Schematic representation of the Cdc141–374 purification scheme, used for the crystallization studies. Purified protein was analyzed by SDS–PAGE followed by Coomassie blue staining. (b), Peptide docking to Cdc14 enhances its thermal stability. Thermal shift assays to determine the Cdc14 melting point (Tm) in the absence or presence of 200 μM Cbk1-derived PxL motif peptide or a scrambled derivative. Means and s.d. from three repeats of the experiment are shown and the melting points are listed together with the coefficient of determination (R2) that indicates the goodness of a fit to a Boltzmann sigmoidal function. (c), Photos of crystals obtained in the indicated space groups of Cdc14 bound by the Cbk1 and Sic1-derived PxL motif peptides.
Supplementary Figure 3 Comparison of budding yeast Cdc14 with human Cdc14B.
(a), Overlay of human Cdc14B (PDB ID: 1OHE; blue) and an S. cerevisiae Cdc14 protomer (gray), bound by the Cbk1 peptide (pink), demonstrates their similar overall fold (root-mean-square deviation of 0.89 Å over 242 Cα atoms). (b), Structure of the Cdc14-Cbk1 peptide complex. Each Cdc14 protomer (shades of gray) binds a Cbk1 peptide (pink). Close-up views of the MES buffer molecule bound to an active site and a zinc binding site are shown. (c), Anomalous difference electron density map indicating a density peak corresponding to zinc. The electron density is contoured at 7σ. For details regarding data collection, see Table 1. The zinc binding sites of both Cdc14 protomers are shown.
Supplementary Figure 4 Structures of the PxL motif peptides.
The difference electron density maps calculated by omitting the PxL peptides, contoured at 3σ, are displayed for Cbk1 and Sic1-derived PxL motif peptides bound to each Cdc14 protomer in the three crystal structures.
Supplementary Figure 5 Overlay of Cbk1 and Sic1-derived PxL motif peptides.
(a), Overlay of the Cbk1-derived PxL motif peptides bound to the hydrophobic pockets of the two Cdc14 protomers in the orthorhombic crystal. (b), Overlay of the Cbk1-derived PxL motif peptides bound to the hydrophobic pockets of the two Cdc14 protomers in the monoclinic crystal. (c), Overlay of the Sic1-derived PxL motif peptides bound to the hydrophobic pockets of the two Cdc14 protomers. (d), Overlay of the Cbk1 and the Sic1-derived PxL motif peptides bound to one of the hydrophobic Cdc14 pockets in the two orthorhombic crystals.
Supplementary Figure 6 Characterization of the Cdc14 Q106L and Cdc14 W108R mutant proteins.
(a), Cdc14 Q106L and Cdc14 W108R show uncompromised enzymatic activity. GFP-Cdc14, GFP-Cdc14 Q106L and GFP-Cdc14 W108R were purified and analyzed by SDS–PAGE followed by Coomassie blue staining. Velocity of p-NPP hydrolysis by GFP-Cdc14, GFP-Cdc14 Q106L and GFP-Cdc14 W108R was recorded as a function of the indicated substrate concentrations in three independent experiments. The means are shown; error bars indicate s.d. (b), GFP-Cdc14 Q106L and GFP-Cdc14 W108R fail to bind the Cbk1-derived PxL motif peptide. The means and s.d. from three independent microscale thermophoresis experiments to measure Cbk1-derived PxL motif peptide binding to the two Cdc14 variants are shown.
Supplementary Figure 7 Conservation of structurally identified Cdc14 features in its orthologs.
Multiple-sequence alignment of Cdc14 paralogs in different species. Secondary structure elements within the S. cerevisiae protein are indicated. Cdc14 residues, forming the PxL motif binding pocket and the dimer interface, are highlighted with blue and green stars, respectively. The proline residues found at the focal point of the dimer interface are highlighted with a green background. The residues coordinating zinc in S. cerevisiae Cdc14 are indicated by red stars; the catalytic cysteine residue in the active site is indicated by a gold star.
Supplementary Figure 8 The Cdc14 GETS dimer interface mutant.
(a), Additional illustration of the dimer interface within Cdc14, spanning both domains A and B and comprising extensive interactions between the two protomers. Residues at the dimer interface are displayed as sticks; a network of hydrogen bonds between the protomers is indicated. (b), Equal amounts of purified recombinant His6-Cdc14 and of the dimer interface mutant His6-Cdc14-GETS were analyzed by SDS–PAGE followed by Coomassie blue staining. (c), Analytical gel filtration profiles of Cdc14 and Cdc14-GETS using a Superdex 200 10/300 Increase column. The increased elution volume of Cdc14-GETS is suggestive of dimer disruption. (d), Velocity of p-NPP hydrolysis by 100 nM Cdc14 and Cdc14-GETS at the indicated substrate concentrations suggests that dimerization is required for full enzymatic activity of the phosphatase. The means and s.d. of three independent experiments are shown.
Supplementary Figure 9 Mechanism of PxL-motif-promoted dephosphorylation.
(a), Depiction of the distances from the C-terminal end of the resolved PxL motif peptide to the MES molecule found in the phosphatase active site in the same (cis) or adjacent (trans) protomer. (b), No evidence for allosteric phosphatase activation by the PxL motif. Velocity of p-NPP hydrolysis by Cdc14 was recorded in the presence of the indicated concentrations of wild-type or scrambled Cbk1-derived PxL motif peptide. The means and s.d. from three independent experiments are shown. (c), Kinetics analysis of peptide dephosphorylation by Cdc14 using an optimal phospho-SPxKK substrate containing an upstream functional or mutant PxL motif. (d), A PxL motif facilitates threonine dephosphorylation by Cdc14. Dephosphorylation velocities were determined using 1 μM Cdc14 and the indicated phosphopeptide concentrations, containing a pTP site preceded by a functional or mutant PxL motif.
Supplementary Figure 10 Distribution of PxL motifs relative to Cdk phosphorylation sites.
Relative positions of PxL motifs and known Cdk phosphorylation sites (based on PhosphoGRID, https://phosphogrid.org/, and the Saccharomyces Genome Database, https://www.yeastgenome.org/) on a representation of the ten top PxL-motif-containing phage display hits, together with a representation of the Orc6 PxL fusion protein.
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Supplementary Text and Figures
Supplementary Figures 1–10 and Supplementary Tables 2–4
Supplementary Table 1
Peptides recovered in the phage display screen
Supplementary Dataset 1
Uncropped blot images
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Kataria, M., Mouilleron, S., Seo, MH. et al. A PxL motif promotes timely cell cycle substrate dephosphorylation by the Cdc14 phosphatase. Nat Struct Mol Biol 25, 1093–1102 (2018). https://doi.org/10.1038/s41594-018-0152-3
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DOI: https://doi.org/10.1038/s41594-018-0152-3
