Human DPP III – Keap1 Interactions: A Combined Experimental And Computational Study

Abstract

Kelch-like ECH associated protein 1 (Keap1) is a cellular sensor for oxidative stress and a negative regulator of the transcription factor Nrf2. Keap1 and Nrf2 control expression of nearly 500 genes with diverse cytoprotective functions and the Nrf2-Keap1 signaling pathway is a major regulator of cytoprotective responses to oxidative and electrophilic stress. It was found that the metallopeptidase dipeptidyl peptidase III (DPP III) contributes to Nrf2 activation by binding to Keap1, probably by binding to the Kelch domain, and thereby influences Nrf2 activity in cancer. We here first determined that the KD of the DPP III-Kelch domain complex is in the submicromolar range. In order to elucidate the molecular details of the DPP III -Kelch interaction we then built models of the complex between human DPP III and the Keap1 Kelch domain and performed coarse-grained and atomistic simulations of the complexes. In the most stable complexes, the ETGE motif in the DPP III flexible loop binds near the central pore of the six-blade β-propeller Kelch domain. According to the preliminary HD exchange experiments DPP III binds to the more unstructured end of Kelch domain. According to the results of MD simulations DPP III binding to the Kelch domain does not influence the overall DPP III structure or the long-range domain fluctuations. We can conclude that DPP III forms the stable complexes with the Keap1 Kelch domain by inserting the flexible loop into the entrance to the central pore of the six blade β-propeller Kelch domain at its more unstructured, N-terminus.

Oxidase or peptidase? A computational insight into a putative aflatoxin oxidase from Armillariella tabescens

“…Calculated RMSF values show the Phe454–Thr487 loop to be highly flexible. This structural motif is also present in the human orthologue and it is suspected to be the site of the DPP III–Keap1 interaction . The proposed “hinge” region that connects the two domains also proved to be highly flexible, while the inner core of the protein is mostly rigid (Supporting Information Figure S8).…”

“…This structural motif is also present in the human orthologue and it is suspected to be the site of the DPP III-Keap1 interaction. 69 The proposed "hinge" region that connects the two domains 30 also proved to be highly flexible, while the inner core of the protein is mostly rigid (Supporting Information Figure S8). This data agrees well with the B-factors of the crystal structure (Supporting Information Figure S9).…”

Aprotinin interacts with substrate-binding site of human dipeptidyl peptidase III

“…In our study, for the first time, the protein-protein interaction of human DPP III involving the substrate binding region of this enzyme has been demonstrated. Up to date, only hDPP III interaction with the Keap1 protein (Gundi c et al, 2016;Hast et al, 2013) has been described. DPP III binds to Kealch domain of Keap1 via the E 480 TGE 483 motif situated in the flexible loop on the surface of the "upper" domain and remote from the substrate binding site.…”

Interdisciplinary Study of the Effects of Dipeptidyl-Peptidase III Cancer Mutations on the KEAP1-NRF2 Signaling Pathway

“…Previously, we have shown that the binding of DPP III to Kelch is a two-step process, which involves separation of the ETGE motif, which is located at the tip of the flexible loop in the upper domain of DPP III, from the protein body and insertion of the loop with ETGE motif into the binding site of the Kelch domain. We showed that the DPP III-R623W variant, in which the ETGE loop is less tightly bound to the protein body, has a substantially higher affinity towards the Kelch domain than the WT [16,17]. In this work, we extended our investigations to some other mutations that either shift the equilibrium process of loop detachment or alter the binding affinity of the ETGE loop of DPP III to the Kelch domain.…”