Structural analysis and rational design of orthogonal stacking system in an E. coli DegP PDZ1–peptide complex

Abstract

The DegP is essential for clearance of denatured or aggregated components from the inner-membrane and periplasmic space in Escherichia coli (E. coli). The enzyme contains two regulatory PDZ domains that have been shown to act as substrate specificity determinant by binding to the C-terminal hydrophobic stretch of substrate proteins. Here, the complex structure of E. coli DegP PDZ1 domain with a phage-displayed C3H1 pentapeptide is modeled and examined using peptide grafting, virtual mutagenesis, and QM/MM calculation. An orthogonal stacking system is identified at the domain–peptide complex interface, which consists of a T-shaped cation-π stacking (t-stacking) and a paralleled cation-π stacking (p-stacking) formed from domain cationic residue R325 to peptide aromatic residues Trp⁻¹ and Phe⁻⁴, respectively. A synergistic effect between t-stacking and p-stacking is observed; π-electron conjugation is primarily responsible for the synergistic effect. Subsequently, the two peptide aromatic residues are systematically replaced by other aromatic amino acids as well as a non-aromatic alanine to optimize the synergistic effect, from which the binding affinities of wild-type C3H1 peptide and seven variants to E. coli DegP PDZ1 domain are determined at micromolar level using fluorescence-based assay. A good linear correlation between experimental binding affinities and calculated binding energies is derived, with a Pearson’s correlation coefficient r_p = 0.928. The aromatic Phe⁻⁴Tyr substitution can considerably improve peptide binding potency by 8.7-fold, whereas the non-aromatic substitutions at each of t-stacking and p-stacking or both can largely impair the peptide affinity by 20.7-fold (Phe⁻⁴Ala), 82.1-fold (Trp⁻¹Ala) and 110.7-fold (Trp⁻¹Ala/Phe⁻⁴Ala).