Rational Molecular Design of Potent PLK 1 PBD Domain‐binding Phosphopeptides Using Preferential Amino Acid Building Blocks

Polo‐like kinase 1 ( PLK 1) is an important regulator in diverse aspects of the cell cycle and proliferation. The protein has a highly conserved polo‐box domain ( PBD ) present in C‐terminal noncatalytic region, which exhibits a relatively broad sequence specificity in recognizing and binding phosphorylated substrates to control substrate phosphorylation by the kinase. In order to elucidate the structural basis, thermodynamic property, and biological implication underlying PBD ‐substrate recognition and association, a systematic amino acid preference profile of phosphopeptide interaction with PLK 1 PBD domain was established via virtual mutagenesis analysis and mutation energy calculation, from which the contribution of different amino acids at each residue position of two reference phosphopeptides to domain–peptide binding was characterized comprehensively and quantitatively. With the profile, we are able to determine the favorable, neutral, and unfavorable amino acid types for each position of PBD ‐binding phosphopeptides, and we also explored the molecular origin of the broad sequence specificity in PBD ‐substrate recognition. To practice computational findings, the profile was further employed to guide rational design of potent PBD binders; three 6‐ mer phosphopeptides ( i.e ., IQS p SPC , LQS p TPF , and LNS p TPT ) were successfully developed, which can efficiently target PBD domain with high affinity ( K d = 5.7 ± 1.1, 0.75 ± 0.18, and 7.2 ± 2.6 μ m , resp.) as measured by a fluorescence anisotropy assay. The complex structure of PLK 1 PBD domain with a newly designed, potent phosphopeptide LQS p TPF as well as diverse noncovalent chemical forces, such as H‐bonds and hydrophobic interactions at the complex interface, were examined in detail to reveal the molecular mechanism of high affinity and stability of the complex system.