Rational redesign of a cation···π···π stacking at cardiovascular Fbw7–Skp1 complex interface and its application for deriving self-inhibitory peptides to disrupt the complex interaction

Abstract

The Fbw7–Skp1 complex is an essential component in the formation and development of the mammalian cardiovascular system; the complex interaction is mediated through binding of Skp1 C-terminal peptide (qGlu-peptide) to the F-box domain of Fbw7. By visually examining the crystal structure, we identified a typical cation ···π···π stacking system at the complex interface, which is formed by the Trp1159 residue of qGlu-peptide with the Lys2299 and His2359 residues of Fbw7 F-box domain. Both hybrid quantum mechanics/molecular mechanics (QM/MM) analysis of the real domain–peptide complex and electron-correlation ab initio calculation of the stacking system model suggested that the cation···π···π plays an important role in stabilizing the complex; substitution of peptide Trp1159 residue with aromatic Phe and Tyr would not cause a considerable effect on the configuration and energetics of cation···π···π stacking system, whereas His substitution seems to largely destabilize the system. Subsequently, the qGlu-peptide was stripped from the full-length Skp1 protein to define a so-called self-inhibitory peptide, which may rebind to the domain–peptide complex interface and thus disrupt the complex interaction. Fluorescence polarization (FP) assays revealed that the Trp1159Phe and Trp1159Tyr variants have a comparable or higher affinity (K _d = 41 and 62 μM) than the wild-type qGlu-peptide (K _d = 56 μM), while the Trp1159His mutation would largely impair the binding potency of qGlu-peptide to Fbw7 F-box domain (K _d = 280 μM), confirming that the cation···π···π confers both affinity and specificity to the domain–peptide recognition, which can be reshaped by rational molecular design of the nonbonded interaction system.