Structural analysis of the complex of Keap1 with a prothymosin alpha peptide

Abstract

The Nrf2 transcription factor, which plays important roles in oxidative and xenobiotic stress, is negatively regulated by the cytoplasmic repressor Keap1. The beta-propeller/Kelch domain of Keap1, which is formed by the double-glycine repeat and C-terminal region domains (Keap1-DC), interacts directly with the Neh2 domain of Nrf2. The nuclear oncoprotein prothymosin alpha (ProTalpha) also interacts directly with Keap1 and may play a role in the dissociation of the Keap1-Nrf2 complex. The structure of Keap1-DC complexed with a ProTalpha peptide (amino acids 39-54) has been determined at 1.9 A resolution. The Keap1-bound ProTalpha peptide possesses a hairpin conformation and binds to the Keap1 protein at the bottom region of the beta-propeller domain. Complex formation occurs as a consequence of their complementary electrostatic interactions. A comparison of the present structure with recently reported Keap1-DC complex structures revealed that the DLG and ETGE motifs of the Neh2 domain of Nrf2 and the ProTalpha peptide bind to Keap1 in a similar manner but with different binding potencies.

Figure 1

Overall tertiary structure of the mouse Keap1-DC–ProTα complex. (a) Structure-based sequence alignment of the C-terminal region of Keap1, containing six DGR repeats and the CTR domain. Conserved amino-acid residues are shown in red. (b), (c) Ribbon models of the tertiary structure of the Keap1-DC β-propeller domain (blue to red) and the ProTα peptide (sticks) are shown in a side view and from a bottom view, respectively. All structure figures were generated with PyMOL (http://www.pymol.org).

Figure 2

The structure of the ProTα peptide in the Keap1-DC protein-bound form. (a) The refined structure of the ProTα peptide is shown, with its 2m|F _o| − D|F _c| (final) and m|F _o| − D|F _c| (initial) electron-density maps contoured at 1.0σ (in green) and 2.0σ (in red), respectively. (b) The peptide (stick model) binds to the highly basic bottom region of Keap1-DC. Surface acidic, basic and neutral residues are shown in red, blue and white, respectively.

Figure 3

Close-up views of the peptide-binding regions. The interacting residues are shown as sticks and hydrogen bonds are depicted by dashed lines. The Keap1-DC and the ProTα peptide are shown in salmon and dark slate, respectively. N and O atoms in the interacting residues are shown in blue and red, respectively.

Figure 4

Comparison of the Nrf2 complexes with the ETGE and DLG motif peptides. Close-up views of superpositions of the Keap1-DC–ProTα complex over the main-chain atoms of Keap1-DC (a) with the Keap1-DC–ETGE motif complex and (b) with the Keap1-DC–DLG motif complex. The colouring scheme for the ProTα complex is the same as in Fig. 3 ▶. For the ETGE and DLG complexes, the Keap1-DC interacting residues are shown in cyan and green, respectively, and the ETGE and DLG peptides in the complexes are shown in yellow and pink, respectively.