Distinct Perception Mechanisms of BACH1 Quaternary Structure Degrons by Two F-box Proteins under Oxidative Stress

SUMMARY The transcription factor BACH1 regulates heme homeostasis and oxidative stress responses and promotes cancer metastasis upon aberrant accumulation. Its stability is controlled by two F-box protein ubiquitin ligases, FBXO22 and FBXL17. Here we show that the homodimeric BTB domain of BACH1 functions as a previously undescribed quaternary structure degron, which is deciphered by the two F-box proteins via distinct mechanisms. After BACH1 is released from chromatin by heme, FBXO22 asymmetrically recognizes a cross-protomer interface of the intact BACH1 BTB dimer, which is otherwise masked by the co-repressor NCOR1. If the BACH1 BTB dimer escapes the surveillance by FBXO22 due to oxidative modifications, its quaternary structure integrity is probed by a pair of FBXL17, which simultaneously engage and remodel the two BTB protomers into E3-bound monomers for ubiquitination. By unveiling the multifaceted regulatory mechanisms of BACH1 stability, our studies highlight the abilities of ubiquitin ligases to decode high-order protein assemblies and reveal therapeutic opportunities to block cancer invasion via compound-induced BACH1 destabilization.


Supplementary
C. The flowchart of single particle analysis of the SCF FBXO22- BACH1 complex.D. The angular distribution of particles used in the final reconstruction.E, Fourier shell correlation (FSC) curves for SCF  .At the Gold-standard threshold of 0.143, the resolution is 3.9 Å. F. Representative density in local refined EM map.

Figure S3
. Structural and biochemical analyses of FBXO22-BACH1 interaction and sequence analysis of NCOR1/2. A. Steric hindrance prevents the formation of a BACH1-BTB-FBXO22 complex with a 2:2 ratio.The asymmetric complex formed between a BACH1 dimer (protomer A: light blue; subunit B: orange) and FBXO22 (purple) is shown as cartoon diagram.A second copy of FBXO22 shown in slate surface representation is modeled onto the BACH1-BTB dimer and is in clash with the other FBXO22 macromolecule.B. In vitro ubiquitination of BACH1 by SCF FBXO22 .C. Superposition of the crystal structures of BACH1-BTB (PDB:2IHC) and BACH2-BTB (PDB:3OHU).BACH2 C-terminal region is disordered and highlighted in red.D. Sequence alignment of five NCOR1 vertebrate orthologs and human NCOR2.Highly conserved short linear motifs (SLiMs) are underlined with gray bars.The BCL6 BTB-interacting motif found in PDB:1R2B is underlined with a purple bar.

Figure S4
. BLI analysis of FBXL17-BACH1-BTB interactions and the schematic workflow of single particle reconstruction of the SCF FBXL17-BACH1 complex.A. BLI measurements of the binding between FBXL17 and NOR3-S1PC-treated BACH1-BTB with a 10-minutes association step.Kd, dissociation constant.B. & C. BLI measurements of the interaction between FBXL17 and BACH1-BTB (wild type and C34A mutant) untreated or treated with NOR3-S1PC.In the absence of compound treatment, C34A enhanced FBXL17 binding.This effect is exaggerated upon compound treatment.The C34 residue, therefore, is not required for S-nitrosylation.D. A representative cryo-EM micrograph for the sample containing a mixture of SCF FBXL17 and BACH1-BTB treated with NOR3-S1PC.E. Typical 2D averages.F. The flowchart of single particle analysis of the sample containing a mixture of SCF FBXL17 and BACH1-BTB treated with NOR3-S1PC.

Figure S5
. Cryo-EM single particle analysis workflow of wild type SCF FBXL17-BACH1 .A. A representative cryo-EM micrograph.B. Typical 2D averages revealing the most populated monomeric complex.C. The flowchart of single particle analysis of the sample containing a mixture of SCF FBXL17 and BACH1-BTB.D. The particle angular distribution and FSC curves of dSCF FBXL17-BACH1-I with local refinement.E. The particle angular distribution and FSC curves of dSCF FBXL17-BACH1-II .F. The angular distribution and FSC curves of the monomeric SCF FBXL17- BACH1 complex.

Figure S1 .
Figure S1.The schematic workflow of single particle reconstruction of the SCF FBXO22- BACH1 complex.A. A representative cryo-EM micrograph.B. Typical 2D averages of the cryo-EM dataset.Scale bar 10 nm. C. The flowchart of single particle analysis of the SCF FBXO22- BACH1 complex.D. The angular distribution of particles used in the final reconstruction.E, Fourier shell correlation (FSC) curves for SCF FBXO22-BACH1 .At the Gold-standard threshold of 0.143, the resolution is 3.9 Å. F. Representative density in local refined EM map.

Figure S2 .
Figure S2.Sequence and structural analysis of FBXO22. A. Sequence alignment of five vertebrate FBXO22 orthologues with second structure annotations.The sequences of the three FIST domains are underlined in different colors (slate, salmon, and purple).Unique secondary structure elements in each repeat are highlighted in red.B. A comparison of the three FIST domains of FBXO22 and homologous structure of YabJ from the YjgF superfamily (PDB:1QD9).
Figure S1 Figure S3 Figure S4 I C