Structural Basis for Variations in Polo-like Kinase 1 Conformation and Intracellular Stability Induced by ATP-Competitive and Novel Noncompetitive Abbapolin Inhibitors

Polo-like kinase 1 (PLK1) is an essential protein kinasewith multipleroles in mitotic progression. PLK1 consists of a kinase domain (KD)and a phosphopeptide-binding polobox domain (PBD), which is responsiblefor substrate recognition and subcellular localization. The regulationof PLK1 involves an autoinhibitory conformation in which KD and PBDinteract. Our previous work identified PBD-binding molecules termedabbapolins that inhibit the cellular phosphorylation of a PLK1 substrateand induce the loss of intracellular PLK1. Here, we describe a comparisonof the abbapolin activity with that of KD inhibitors to gain insightinto conformational features of PLK1. As measured by a cellular thermalshift assay, abbapolins produce ligand-induced thermal stabilizationof PLK1. In contrast, KD inhibitors decreased the soluble PLK1, suggestingthat catalytic-site binding causes a less thermally stable PLK1 conformation.Binding measurements with full-length PLK1 and a KD inhibitor alsodemonstrated a conformational change. Interestingly, the cellularconsequences of KD versus PBD engagement contrast as KD binding causesthe accumulation of intracellular PLK1, whereas PBD binding producesa striking loss of nuclear PLK1. These data are consistent with therelief of autoinhibited PLK1 by KD binders; an explanation for theseobservations is presented using structures for the catalytic domainand full-length PLK1 predicted by AlphaFold. Collectively, the resultshighlight an underappreciated aspect of targeting PLK1, namely, conformationalperturbations induced by KD versus PBD binding. In addition to theirsignificance for PBD-binding ligands, these observations have implicationsfor the development of ATP-competitive PLK1 inhibitors because catalyticinhibitors may conversely promote PLK1 noncatalytic functions, whichmay explain their lack of clinical efficacy to date.