A NCAPG2-Derived Phosphopeptide Selectively Binds to the Polo-Box Domain of PLK1 and Inhibits Cancer Cell Proliferation

Abstract

Polo-like kinase 1 (PLK1) serves as a regulator of cell cycle progression and is overexpressed in various human cancer cells. PLK1 contains a conserved polo-box domain (PBD) as well as a kinase domain (KD), like other members of the PLK family. The PBDs of the PLK family interact with phosphopeptides that contain highly conserved Ser-pSer/pThr (S-pS/T) motifs and play significant roles in substrate recognition and subcellular localization. The PBD of PLK1 has been regarded as a promising therapeutic target for cancer therapy. In this study, we investigated whether the phosphopeptide derived from NCAPG2 has a high binding specificity for the PLK1 PDB over those other highly similar PBDs, including PLK2, PLK3, and PLK5. We showed that the PBDs of PLKs have different binding affinities against CDC25c-, PBIP-, and NCAPG2-derived phosphopeptides using the fluorescence polarization binding assay. Unlike CDC25c-phosphopeptide, NCAPG2-phosphopeptide specifically bound to the PLK1 PBD with nanomolar affinity (Kd ~ 48.68 nM), and its specificity was similar to that of the PBIP-derived phosphopeptide. Some of these phosphopeptide-PBD interactions can be explained by calculating the binding free energies using the Molecular Mechanics Poisson–Boltzmann surface area (MM/PBSA). Additionally, NCAPG2-phosphopeptide suppressed the proliferation of cancer cells. The NCAPG2-derived phosphopeptide can be applicable to the discovery of highly selective protein–protein interaction inhibitors targeting the PBD of PLK1.