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.
Highlights
Processes that drive mitotic progression are under strict cellular regulation to ensure the faithful propagation of newly replicated genetic material
The side chains of Glu[553], His[569] and His[609], as well as that of His[524] from a symmetry related molecule, coordinate a zinc metal ion from the crystallization solution (Fig. 1c). This metal ion further stabilizes the conformation of the ordered portion of the loop and places the phenyl group of Phe[603] inside an exposed hydrophobic pocket delimited by Leu[546], Trp[555], Ile[557] and Ala[567] (Fig. 1b and Supplementary Fig. 1a)
We speculated that Ile[85] could occupy the hydrophobic pocket defined by Leu[546], Trp[555], Ile[557] and Ala[567] (Fig. 3a and Supplementary Fig. 1). If this was the case, Glu[86] would bind close to Asp[611] and Asp[631] from polo-box domain, and this could explain why a Dbf4-E86K peptide binds to the polo-box domain of Cdc[5] with higher affinity than the wild-type Dbf[4] peptide (Supplementary Fig. 1)[27]. To test whether this surface of the polo-box domain was important for the interaction with Dbf[4], we introduced a A567W mutation on the polo-box domain to occlude the hydrophobic pocket
Summary
Processes that drive mitotic progression are under strict cellular regulation to ensure the faithful propagation of newly replicated genetic material. Dbf[4] peptide, confirming that the extra density did correspond to the Dbf[4] peptide Mutations on this surface of the polo box domain result in early anaphase arrest phenotypes distinct from the mitotic exit defect typically observed in cdc[5] mutants. Based on these results, we propose a model for how binding to Dbf[4] may help release the auto-inhibition of the Cdc[5] kinase
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