Figure 3. Mode of interaction between Prx4 and Trx of P5.

(A) Activity of the C-terminally truncated Prx4 mutant (Prx4 ΔC) toward P5 a⁰. The experimental conditions were as described in Materials and Methods. Consumption of NADPH coupled to Prx4 catalysis was detected by monitoring absorbance at 340 nm (Abs₃₄₀). (B) Inhibition of Prx4-catalyzed oxidation of P5 a⁰ by the titration of the Prx4 C-terminal peptide. The experiments were carried out as in (a) in the presence of various concentrations (0, 10, 20, 30, 50, 100, 200, 300, 500 μM) of the Prx4 C-terminal peptide. (C) Crystal structure of P5 a⁰ (yellow) in a complex with the Prx4 C-terminal peptide (cyan) at 2.1 Å resolution. The Prx4 peptide is shown with sticks. Cys57 in the redox-active site of P5 a⁰ formed a mixed disulfide bond with the resolving cysteine (Cys248) of Prx4, as illustrated by orange sticks. The peptide-binding groove of P5 a⁰ is shown with a purple oval. (D) Close-up views of the interface of the P5 a⁰–Prx4 C-terminal peptide complex from two different angles. Electron density map at the 1.0 contour level of the Prx4 C-terminal peptide is indicated with blue mesh. (E) Modeled structure of the P5 a⁰–Prx4 decamer complex. The Prx4 C-terminal peptide bound to P5 a⁰ is superposed on the corresponding part of the oxidized Prx4 decamer such that the RMSD of the Cα atoms of Val247, Cys248, Pro249, and Ala250 is minimized. A dimeric unit of Prx4 that interacts with P5 a⁰ is highlighted in pink. (F) Close-up view of the interface of the P5 a⁰–Prx4 decamer complex shown in (E).