Molecular basis for the disruption of Keap1–Nrf2 interaction via Hinge Latch mechanism

摘要

a Working hypothesis for the Hinge-Latch model of activation of Nrf2 by electrophilic and non-electrophilic inducers. This model was proposed based on the two-site binding model; DLGex and ETGE motifs of Nrf2 bind individually to the similar pocket structure in the Keap1-DC domain of the Keap1 homodimer. According to the Hinge-Latch model, DLGex site dissociation alone sufficiently disturbs the ubiquitin ligase activity of the Keap1 complex14. b Hypothetical statuses of the Keap1–Nrf2 complex. As ETGE motif has a 200-fold higher binding affinity to Keap1-DC domain than DLGex motif does, three statuses of the Keap1–Nrf2 protein complex can be postulated; two sites bound (left), only ETGE site bound (middle), and fully dissociated (right). c Rationale of the NMR titration technique. A schematic depiction of a 1D NMR peak is shown. The Neh2 domain of Nrf2 (98 amino acids) is intrinsically disordered and, upon binding to the large molecular weight protein Keap1 dimer (molecular weight ~140 kDa), the NMR signals corresponding to the Neh2 domain experience line-broadening. On the other hand, when released from Keap1 homodimer, the NMR signals corresponding to the binding sites of Neh2 domain recover by narrowing of linewidth. The NMR titration experiment enables us to detect the binding of each individual site and to validate the three conditions shown in b. d Scheme for induction and expression of recombinant full-length Keap1-6xHis protein. e Purification processes of recombinant full-length Keap1-6xHis protein. f Scheme for induction and expression of recombinant 6xHis-GST-13C15N Neh2 protein. g Purification processes of recombinant 6xHis-GST-13C15N Neh2 protein. The protein was purified by Ni2+ affinity column chromatography and gel filtration chromatography. For the preparation of Neh2 domain, after Ni2+ affinity column chromatography, 6xHis-GST tag was cleaved by turbo TEV protease and removed by second Ni2+ affinity chromatography (*). M molecular marker, L lysate, FT flow through, I input, E eluate.