FC0075
Prothymosin α (ProT α)  -  Kelch-like ECH-associated protein 1 (Keap1), Kelch domain

Biological function
Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription.

Structural evidence
Nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. In the bound state, amino acids in the region 35–53 had SSP scores ranging only from −0.34 to 0.17, indicating a lack of stable secondary structure in this region. No dramatic increases in protection factor values were observed upon addition of Kelch. Even though moderate increases in protection factor values for N38, E45, N50, and G60 were observed, these values were significantly smaller than those observed in folded proteins. The disordered nature of the ProTα–Kelch complex is also supported by the observations that the resonance signals of ProTα in the ¹H–¹⁵N HSQC experiments were clustered in a narrow range from 7.9 ppm to 8.6 ppm, both in the free and bound states. Significant chemical shift changes were observed only for amino acids 35–50. In the Kelch complex the NOE values of ProTα remained predominantly negative upon binding to Kelch, indicative of a flexible protein lacking secondary structure.

Biochemical evidence
To evaluate the contributions of ProTα residues distant from the Kelch-binding motif to the affinity of binding, the affinity of Kelch to the full-length ProTα and an 11-mer peptide spanning residues 38– 48 were compared. The dissociation constant of the full-length ProTα– Kelch complex was determined to be 2.6 ± 0.4 μM by ITC. The 11-mer ProTα peptide bound slightly tighter (K_d = 1.79 ± 0.09 μM) to Kelch compared to full-length ProTα. Despite the similar K_d values, the ΔH and ΔS of these two binding processes were very different. The results indicate a larger entropy loss when the ProTα peptide bound to Kelch. The more negative ΔS value was compensated for by the more favorable ΔH, resulting in similar K_d values for both full-length ProTα and its peptide.

Mechanism category
competitive binding

Significance
Fuzziness of the ProTα - Keap1 complex enables ProTα to interact with other targets simultaneously. In addition, the fuzzy complex formed allows ProTα to quickly dissociate from Keap1 after the latter is translocated into the nucleus.