Molecular dynamics directed rational design and fluorescence binding assay of phosphopeptide ligands for PLK polo-box domain

Abstract

Polo-like kinase 1 (PLK1) plays a critical role during multiple stages of cell cycle progression and is involved in the development and metastasis of malignant tumours. The protein contains a regulatory polo-box domain (PBD) that can recognise and bind to a wide variety of phosphorylated substrates. Here, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was created based on the crystal structures of the domain in complex with its natural phosphopeptide ligands. With the profile we were able to explore the structural basis and energetic landscape of phosphopeptide binding to the domain. Moreover, in addition to domain peptide-binding pocket we also examined the intermolecular interaction between the N-terminal region of phosphopeptide and a newly discovered crystal packing site of the domain. All the harvested information was successfully integrated to guide the structural design and optimisation of phosphopeptide ligands with improved affinity and selectivity for the domain, which were then confirmed in vitro by fluorescence anisotropy assays. Structural analysis and energetic analysis revealed that the phosphopeptide ligand can be divided into three functionally independent sections: a N-terminal flexible tail, a C-terminal unbinding tail and a central binding region. The N-terminal tail possesses a high flexibility that can roll over on the surface of PBD crystal packing site, while the C-terminal tail points out of the peptide-binding pocket of the domain and no substantial interaction can be observed between them. In contrast, the central region binds tightly to the domain pocket and exhibits modest conformational change over different ligands.