Rational Improvement of Peptide Affinity to Human Pregnancy-Related Serine Protease HtrA3 PDZ Domain by Introducing a Halogen Bond to the Domain–Peptide Complex Interface

Abstract

The pregnancy-related serine protease HtrA3 plays an important role in human placental development and has recently been recognized as a potential therapeutic target in the treatment of cancer. Previously, a C-terminal pentapeptide FGRWV–COOH was identified to bind at the PDZ domain of HtrA3 with a moderate affinity. Here, based on the high-resolution complex crystal structure of HtrA3 PDZ domain with the pentapeptide ligand we successfully introduced a rationally designed halogen bond to the complex interface by substituting R4-hydrogen atom of the indole moiety of peptide Trp-1 residue with a halogen atom. High-level theoretical calculations suggested that bromine is the best choice that can render strong interaction energy for the halogen bond and can confer high affinity to the PDZ–peptide complex. Fluorescence spectroscopy characterizations revealed that the resulting R4-brominated peptide (Kd = 0.15 ± 0.03 μM) exhibited 12-fold affinity improvement relative to its nonhalogenated counterpart (Kd = 1.8 ± 0.4 μM). In contrast, the PDZ-binding affinity of R6-brominated peptide (Kd = 1.2 ± 0.1 μM), a negative control that was unable to form the halogen bond according to theoretical investigations, did not change substantially as compared to the nonhalogenated peptide.