Determination of Prdx6 binding sites for its interaction with SP‐A

Abstract

Our previous studies have shown that the PLA2 activity of Peroxiredoxin 6 (Prdx6) is inhibited by surfactant protein A (SP‐A) through direct protein‐protein interaction. In silico studies using the ZDOCK program (http://zlab.bu.edu/zdock/) suggested that the Prdx6 sequence (195EEEAKKLFPK204) is involved in the interaction with SP‐A. The ability of a peptide comprised of this sequence (Prdx6 peptide) and a scrambled peptide control (PAEKLKAFEKE) to bind to SP‐A were studied by Isothermal Titration Calorimetry at 25°C at pH 4 and pH 7. The resulting data revealed that Prdx6 peptide bound to SP‐A in an exothermic reaction, while the scrambled peptide did not bind. SP‐A reduced PLA2 activity of Prdx6 from 98±3 to 29±3 at pH 4 and from 50±3 to 30±2 nmol/min/mg at pH7. However, in the presence of the Prdx6 peptide, SP‐A inhibition of PLA2 activity was partially reversed (70±2 nmol/min/mg at pH 4 and 43±1 at pH 7). Results for phosphorylated Prdx6 (pPrdx6) were even more striking as SP‐A reduced the PLA2 activity of pPrdx6 from 1659±26 nmol/min/mg to 31±0.4 at pH 4 and from 1712±69 to 34±2 at pH 7 while the presence of the Prdx6 peptide largely reversed the inhibition by approximately 85%. The scrambled peptide had no effect at either pH. Thus, the peptide appears to block the SP‐A binding site on Prdx6 indicating that the identified Prdx6 sequence is involved in the interaction with SP‐A, which results in termination of PLA2 activity.