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

摘要

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~(-4)Tyr 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~(-4)Ala), 82.1-fold (Trp~(-1)Ala) and 110.7-fold (Trp~(-1)Ala/Phe~(-4)Ala).