Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division

Abstract

Polo-like kinases (Plks) are key cell cycle regulators. They contain a kinase domain followed by a polo-box domain that recognizes phosphorylated substrates and enhances their phosphorylation. The regulatory subunit of the Dbf4-dependent kinase complex interacts with the polo-box domain of Cdc5 (the sole Plk in Saccharomyces cerevisiae) in a phosphorylation-independent manner. We have solved the crystal structures of the polo-box domain of Cdc5 on its own and in the presence of peptides derived from Dbf4 and a canonical phosphorylated substrate. The structure bound to the Dbf4-peptide reveals an additional density on the surface opposite to the phospho-peptide binding site that allowed us to propose a model for the interaction. We found that the two peptides can bind simultaneously and non-competitively to the polo-box domain in solution. Furthermore, point mutations on the surface opposite to the phosphopeptide binding site of the polo-box domain disrupt the interaction with the Dbf4 peptide in solution and cause an early anaphase arrest phenotype distinct from the mitotic exit defect typically observed in cdc5 mutants. Collectively, our data illustrates the importance of non-canonical interactions mediated by the polo-box domain and provide key mechanistic insights into the combinatorial recognition of substrates by Polo-like kinases.

Figure 1

Cdc5 architecture and structure of its polo-box domain. (a) Sequence alignment of budding yeast Cdc5 (residues 451–705) and the drosophila (POLO, residues 333–576), zebrafish (zPLK1, residues 343–595) and human (hPLK1, residues 352–603) Plk1 homologs. Secondary structure elements are shown above the alignment and color red (polo cap), blue (first polo-box) and green (second polo box). Conserved hydrophobic (yellow), polar (green), positive charged (blue) and negative charged (red) residues are highlighted. Conserved residues involved in phosphopeptide binding are marked with (^). (b) Ribbon diagram of the structure of Cdc5 color coded as in (a). (c) Detail of the interactions between the ordered region of the α2-β7 loop (residues Thr602-Phe614) and strands β5 and β6 from the first polo-box. The Zn²⁺ metal ion is shown as a grey sphere with hydrogen bonds drawn as dashed lines.

Figure 2

Structures of the polo-box domain (PBD) of Cdc5 bound to Spc72^P. Opposite views of the PBD of Cdc5 bound to the Spc72^P peptide (magenta). Spc72^P binds at the groove defined by the two polo boxes forming a short antiparallel β-sheet with the β1 strand. The 2Fo-Fc electron density map around the Spc72^P peptide contoured at 1.2σ is shown as a grey mesh.

Figure 3

The Dbf4 peptide binds to the hydrophobic surface opposite to the pS/T-binding pocket. (a) Structure of the PBD of Cdc5 in the presence of the Dbf4 peptide. The Fo-Fc>0 electron density map around the hydrophobic pocket opposite to the phosphopeptide binding site is shown as a yellow mesh contoured at 2.5σ with a tetrapeptide modelled for reference (yellow sticks). (b) Isothermal calorimetry data and analysis for the titration of the Dbf4 into the polo-box domain of Cdc5^PBD (left) or Cdc5 ^PBD-A567W (right).

Figure 4

Cdc5 interacts simultaneously and non-competitively with Spc72^P and Dbf4. Isothermal calorimetry data and analysis for the titration of the Spc72^P peptide (a), its non-phosphorylated version (Spc72) (b), and the Dbf4 peptide (d) into the polo-box domain of Cdc5. (c) Titration of the Spc72^P peptide into the polo-box domain of Cdc5 pre-incubated with the Dbf4 at 1:4 molar excess. (e) Titration of the Dbf4 peptide into the polo-box domain of Cdc5 pre-incubated with the Spc72^P at 1:4 molar excess.

Figure 5

NMR spectra probing the Cdc5-substrate interactions. (a) One dimensional ¹H NMR spectra for Cdc5, Spc72^P, and Dbf4. The Cdc5-selective saturation frequency used in the STD experiments is indicated. (b) Aromatic expansion of the STD NMR spectrum of Scp72^P acquired in the presence of Cdc5. The 1D ¹H and STD reference (STR) spectra of Scp72^P in the absence and presence of Cdc5 are shown for comparison. (c) Overlay of the Cdc5 and Cdc5-Dbf4 STD spectra. Dashed lines highlight Cdc5-Dbf4 peaks with increased intensities that align with free Dbf4 (green) chemical shifts. (d) Overlay of the aromatic region of 1D ¹H NMR spectra for Cdc5 and Cdc5-Dbf4. (e) Aromatic expansion for the STD and STR spectra of the Cdc5-Scp72^P-Dbf4 complex. (f) Aliphatic region of the Cdc5-Spc72^P-Dbf4 STD spectra. 1D ¹H NMR spectra for each peptide and the STR spectrum of the Cdc5-Spc72^P- Dbf4 complex are shown for reference. (g) Overlay of the aromatic region of 1D proton spectra for the Cdc5-Spc72^P and Cdc5-Scp72^P-Dbf4 complexes. Arrows indicate chemical shift changes caused by Dbf4 binding.

Figure 6

Disruption of Cdc5 hydrophobic pocket generates conditional lethal mutants. Exponential culture of the indicated mutant and control cells were spotted on solid medium as fivefold dilution series. Cells were allowed to grow in temperature-controlled incubators for 36–72 hours until individual colonies were visible. (a) Effect of temperature on the proliferation of cdc5 mutants. Cells were incubated at 23 °C, 30 °C, and 37.5 °C on solid YPAD medium. (b) Sensitivity of cdc5 mutants to DNA replication inhibitor and DNA damaging agent. Cells were grown at 23 °C, and mutants defective in DNA repair (smc5-6) and Cdc5 kinase activity (cdc5-77) were included as controls. (n = 5).

Figure 7

Chromosome segregation defect in cdc5-S630Q mutants. Exponential cultures of cdc5-S630Q mutant and control cells were synchronized in G1 at 23 °C using α-factor. After synchronous release of cells in fresh YPAD medium at 37 °C, samples of culture were collected at regular intervals and processed to monitor the appearance of cell cycle landmarks. (a) Kinetics of bud formation and nucleus separation during the cell cycle. 100 cells were counted at each time point. (b) Micrographs showing representative nucleus morphology at the indicated times during the time-course experiment. White bar is 5 µm. Note that the 135 min micrograph for the cdc5-S630Q mutant shows a slightly larger surface area than other micrographs to allow visualization of 2 cells, hence the slightly shorter scale bar. (n = 4).

Figure 8

Comparison of yeast Cdc5 and zebrafish Plk1. (a) Ribbon diagram of the structure of zebrafish Plk1 in complex with Map205 (purple). The kinase domain (grey) and the polo-box domain (green) are labeled. (b) Ribbon diagram of the structure of the polo-box domain of Cdc5 in complex with the Spc72^P peptide shown in the same orientation as (a). The Spc72^P is shown as color-coded sticks and the Dbf4-binding interface indicated in orange. (c) Model depicting how the DDK complex may bind to Cdc5. Dbf4 interacts with the polo-box domain (PBD) of Cdc5, but additional interactions mediated by the Dbf4 or Cdc7 subunits of the DDK complex may contribute to the interaction and potentially alter the kinase activity of Cdc5.