A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

Abstract

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

Kinetochore phosphatases suppress autonomous Polo-like kinase 1 activity to control the mitotic checkpoint

“…Therefore, we propose that the conserved hydrophobic amino acids within the MADBUB PBM act to stabilize PLK1 binding, and perhaps additionally to allow PLK1 dimerization and activation at kinetochores. In support of this idea, targeting the hydrophobic pocket on PLK1 directly, using mutations or small molecule inhibition, causes reduced PLK1 recruitment to kinetochores and a mitotic arrest associated with misaligned chromosomes ( Sharma et al, 2019 ). We hypothesize that impaired interaction of PLK1 with the BUB complex is at least partially responsible for these phenotypes.…”

“…A final striking feature of the core PBM is the conserved stretch of hydrophobic residues before and immediately after the Ser-Thr-Pro motif. PLK1 has recently been shown to use a conserved hydrophobic pocket that lies adjacent to the phospho-substrate binding groove to enhance substrate binding affinity ( Sharma et al, 2019 ). Although this pocket is often found in a closed conformation, it also exhibits a variety of crystal packing interactions with hydrophobic residues, demonstrating a high degree of flexibility ( Śledź et al, 2011 ).…”

Structure-Based Survey of the Human Proteome for Opportunities in Proximity Pharmacology

“…A number of non-catalytic pockets were also found that are targeted by weak ligands that may be valid starting points for the development of ProxPharm compounds (Table S1, confidence level 2). These include compounds and peptides found in the POLO-box domain of PLK1 (Figure 4) 29 and the PDZ domain of PTPN3 (Figure 6) 42 . Less reliable, but still promising are domains for which no ligand was reported in the context of the protein of interest, but that were shown to be chemically tractable in other proteins (confidence level 3).…”

Orchestrating serine/threonine phosphorylation and elucidating downstream effects by short linear motifs

“…However, the PBD confer specificity among the PLK family suggesting little redundancy in their functionality [ 66 ]. A recent report inferred the contribution of an additional tyrosine-rich pocket close to the phospho-peptide binding pocket to their substrate specificity [ 67 ].…”