Bora bridges Aurora-A activation and substrate recognition of PLK1

Overview

The study establishes the structural and mechanistic basis for PLK1 activation by Aurora-A in the context of mitotic entry, resolving previously uncharacterized interactions mediated by the intrinsically disordered protein Bora. The research demonstrates how Bora functions as a molecular bridge between the two kinases, facilitating proper substrate orientation and phosphorylation efficiency.

Methods and approach

Structural models of Aurora-A/Bora and Aurora-A/Bora/PLK1 complexes were generated and validated through multiple complementary approaches. Site-specific mutagenesis was employed to test predicted interactions, biochemical assays quantified phosphorylation kinetics and binding affinities, and nuclear magnetic resonance spectroscopy provided atomic-level characterization of complex formation and dynamics.

Results

Bora wraps around the N-lobe of Aurora-A, occupying binding pockets typically used by alternative Aurora-A activators. A CDK1-phosphorylated residue on Bora (Ser112) functionally mimics Aurora-A activation loop phosphorylation through a TPX2-like binding motif. In the ternary complex, Bora positions the PLK1 activation loop toward the Aurora-A catalytic site. A conserved Bora region (residues 56-66) establishes critical contacts with the PLK1 C-helix pocket, structurally analogous to the Aurora-A TPX2-binding Y-pocket. Aurora-A-mediated phosphorylation of Bora Ser59 generates an additional stabilizing interaction that enhances PLK1 phosphorylation efficiency.

Implications

These findings elucidate the structural mechanisms governing Aurora-A regulation by intrinsically disordered binding partners, providing a detailed framework for understanding how Bora-dependent signaling coordinates PLK1 activation during late G2 phase. The identification of specific phosphorylation sites and structural interfaces offers mechanistic insight into the hierarchical phosphorylation cascade that triggers mitotic entry. The functional characterization of the Bora/PLK1 interface may inform studies of cell cycle checkpoint control and the molecular basis of mitotic dysregulation in cancer.