# Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress

Shiyun Cao, Sheena Faye Garcia, Huigang Shi, Ellie I James, Yuki Kito, Hui Shi, Haibin Mao, Sharon Kaisari, Gergely Rona, Sophia Deng, et al.

► To cite this version:

Shiyun Cao, Sheena Faye Garcia, Huigang Shi, Ellie I James, Yuki Kito, et al. Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress. Cell, 2024, 187 (26), pp.7568-7584. 10.1016/j.cell.2024.10.012. hal-05405068

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# Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Shiyun Cao, Sheena Faye Garcia, Huigang Shi, ..., Miklos Guttman, Michele Pagano, Ning Zheng

## Correspondence

michele.pagano@nyumc.org (M.P.), nzheng@uw.edu (N.Z.)

## In brief

The stability of BACH1 is controlled by two F-box proteins, FBXO22 and FBXL17. There are two forms of the BACH1 quaternary structure degrons that are encoded by its dimeric BTB domain. FBXO22 recognizes the intact dimeric BTB domain, whereas FBXL17 targets and remodels the BTB domain dimer when destabilized by S-nitrosylation.

## Highlights

* FBXO22 and FBXL17 play non-redundant roles in regulating the stability of BACH1

* FBXO22 binds a BACH1 quaternary structure degron presented by its dimeric BTB fold

* S-nitrosylation destabilizes the BACH1 BTB domain dimer and prevents FBXO22 binding

* Destabilized BACH1 BTB domain dimer is recognized and remodeled by a pair of FBXL17

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## Article

# Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress

Shiyun Cao,<sup>1,2,11</sup> Sheena Faye Garcia,<sup>3,4,11</sup> Huigang Shi,<sup>1,2,11</sup> Ellie I. James,<sup>5,6</sup> Yuki Kito,<sup>3,4</sup> Hui Shi,<sup>1,2,12</sup> Haibin Mao,<sup>1,2</sup>
Sharon Kaisari,<sup>3,4</sup> Gergely Rona,<sup>3,4,7,8</sup> Sophia Deng,<sup>3,4</sup> Hailey V. Goldberg,<sup>3,4</sup> Jackeline Ponce,<sup>3,4,9</sup> Beatrix Ueberheide,<sup>3,4,9</sup>
Luca Lignitto,<sup>3,4,10</sup> Miklos Guttman,<sup>5,6</sup> Michele Pagano,<sup>3,4,7,*</sup> and Ning Zheng<sup>1,2,13,*</sup>

## SUMMARY

Ubiquitin-dependent proteolysis regulates diverse cellular functions with high substrate specificity, which hinges on the ability of ubiquitin E3 ligases to decode the targets’ degradation signals, i.e., degrons. Here, we show that BACH1, a transcription repressor of antioxidant response genes, features two distinct unconventional degrons encrypted in the quaternary structure of its homodimeric BTB domain. These two degrons are both functionalized by oxidative stress and are deciphered by two complementary E3s. FBXO22 recognizes a degron constructed by the BACH1 BTB domain dimer interface, which is unmasked from transcriptional co-repressors after oxidative stress releases BACH1 from chromatin. When this degron is impaired by oxidation, a second BACH1 degron manifested by its destabilized BTB dimer is probed by a pair of FBXL17 proteins that remodels the substrate into E3-bound monomers for ubiquitination. Our findings highlight the multidimensionality of protein degradation signals and the functional complementarity of different ubiquitin ligases targeting the same substrate.

## INTRODUCTION

Ubiquitin-dependent protein degradation is a widespread mechanism regulating virtually every cellular function.<fnref n="1" /> The selectivity of this proteolytic process is exquisitely controlled by the specific degradation signal, also known as “degron,” which is encoded in the substrate protein and perceived by its cognate E3 ubiquitin ligase.<fnref n="2" />,<fnref n="3" /> Early studies have mapped the degrons of many classical substrate proteins to various short linear “motifs” (SLiMs) with specific consensus sequences.<fnref n="4" /> Many of these degrons are recognized by E3 ligases in their native form, while others require modulation by either post-translational modifications or hormonal signal to promote E3 binding. Although recent proteome-wide studies have further expanded the cellular repertoire of SLiM degrons,<fnref n="5" /> the

number of known degrons is still far below the predicted number of E3 ligases in most, if not all, eukaryotes.

The cullin-RING ubiquitin ligase (CRL) complexes constitute the largest superfamily of multi-subunit E3s in humans with more than 200 family members classified in five subfamilies: CRL1 (also known as SCF for SKP1, CUL1, F-box protein), CRL2, CRL3, CRL4A/B, and CRL5.<fnref n="6" /> Built with a modular architecture, each CRL subfamily employs a battery of substrate receptor (SR) subunits to recognize the unique degrons of their targets. For example, the CRL1/SCF SRs, β-TrCP and FBXW7, are two F-box proteins well known to recognize phosphorylated SLiM degrons.<fnref n="7" />,<fnref n="8" /> A panel of BC-box and DCAF proteins act as the CRL2 and CRL4 SRs to recognize various C-end degrons found at the extreme C terminus of substrate polypeptides.<fnref n="9" />,<fnref n="10" />

<footnote n="1"><sup>1</sup>Department of Pharmacology, University of Washington, Box 357280, Seattle, WA 98195, USA</footnote>

<footnote n="2"><sup>2</sup>Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA</footnote>

<footnote n="3"><sup>3</sup>Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY, 10016, USA</footnote>

<footnote n="4"><sup>4</sup>Laura and Isaac Perlmutter Cancer Center, New York University Grossman School of Medicine, New York, NY 10016, USA</footnote>

<footnote n="5"><sup>5</sup>Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA</footnote>

<footnote n="6"><sup>6</sup>Molecular Engineering & Science Institute, University of Washington, Seattle, WA 98195, USA</footnote>

<footnote n="7"><sup>7</sup>Howard Hughes Medical Institute, New York University Grossman School of Medicine, New York, NY 10016, USA</footnote>

<footnote n="8"><sup>8</sup>Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary</footnote>

<footnote n="9"><sup>9</sup>Proteomics Laboratory, Division of Advanced Research Technologies, New York University Grossman School of Medicine, New York, NY 10016, USA</footnote>

<footnote n="10"><sup>10</sup>Cancer Research Center of Marseille (CRCM), CNRS, Aix Marseille University, INSERM, Institut Paoli-Calmettes, Marseille, France</footnote>

<footnote n="11"><sup>11</sup>These authors contributed equally</footnote>

<footnote n="12"><sup>12</sup>Present address: Biortus Discovery Co., Ltd., 101 South Building, 99-3 Linhu Avenue, Xinwu District, Wuxi, Jiangsu, P.R. China</footnote>

<footnote n="13"><sup>13</sup>Lead contact</footnote>

<footnote>*Correspondence: michele.pagano@nyumc.org (M.P.), nzheng@uw.edu (N.Z.)</footnote>

<footnote>https://doi.org/10.1016/j.cell.2024.10.012</footnote>
CRLs also closely participate in redox homeostasis regulation. Oxidative stress results from an accumulation of oxygen and nitrogen-derived reactive species (ROS/RNS), which cause direct damage to cellular components and interfere with redox signaling.<sup>11,12</sup> To modulate the levels of prooxidant and anti-oxidant species, mammalian cells have evolved two antagonizing redox-sensitive transcription factors to dynamically control the expression of cytoprotective genes. BACH1, also known as BTB (broad-complex, tramtrack, and bric-à-brac) and CNC (cap "n" collar) homology 1, is a master transcriptional repressor in heme metabolism and redox homeostasis.<sup>13</sup> As a member of the basic leucine zipper (bZIP) protein family, BACH1 heterodimerizes with small musculoaponeurotic fibrosarcoma (sMAF) proteins and binds to the anti-oxidant response elements (AREs) of many anti-oxidant genes, including heme oxygenase 1 (HMOX1), to suppress their transcription under normal conditions.<sup>14–18</sup> Under oxidative stress, a critical transcription activator, NRF2 (nuclear factor erythroid-derived 2-like 2), accumulates and competes with BACH1 for binding sMAF and AREs. In doing so, NRF2 induces the expression of these genes, whose products go on to eliminate reactive species and restore redox homeostasis.<sup>19–22</sup>

A wealth of studies revealed a crucial role of the CRL3 SR KEAP1 (Kelch-like ECH-associated protein 1) in regulating NRF2 activity in a redox-sensitive manner.<sup>23</sup> Under steady-state conditions, KEAP1 recognizes two SLIM degrons of NRF2 and promotes its ubiquitination and degradation.<sup>24–26</sup> When cells are challenged by oxidative or electrophilic stress, covalent modification of highly reactive cysteine residues in KEAP1 impairs its E3 ligase activity, enabling newly synthesized NRF2 to rapidly accumulate and move to the nucleus to induce the expression of anti-oxidant genes.<sup>27,28</sup> Recent studies have identified two F-box proteins, FBXO22 and FBXL17, which mirror the function of KEAP1 to regulate the stability of BACH1 in response to elevated heme and ROS/RNS levels.<sup>29,30</sup> Both F-box proteins have been implicated in oxidative stress-induced degradation of BACH1. However, how FBXO22 and FBXL17 recognize BACH1 and together regulate its stability remains unknown.

In this study, we reveal that the stability of BACH1 is dictated by two mutually exclusive degrons, both of which are encoded in the quaternary structures of its dimeric BTB domain. The two BACH1 degrons are functionalized by different forms of oxidative stress and recognized by FBXO22 and FBXL17 via distinct and complementary mechanisms. By shedding light on the molecular basis of BACH1 regulation, our results define a previously undescribed class of protein degradation signals, which exploit the structural complexity of protein assembly to achieve substrate specificity.

## RESULTS

### Structure of the SCF<sup>FBXO22-BACH1-BTB</sup> complex

BACH1 contains an N-terminal BTB domain, which mediates its homodimerization and is critical for FBXO22 interaction (Figure 1A).<sup>30</sup> In a GST pull-down assay with purified recombinant proteins, we validated the direct binding between the BACH1 BTB domain and the full-length FBXO22 protein in complex with SKP1 (Figure 1B). We further confirmed the homodimeric

nature of the BACH1-BTB domain by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) (Figure 1C). Despite its expected symmetric architecture, the BACH1 BTB dimer appeared to be associated with only a single copy of FBXO22-SKP1.

To reveal the mechanism by which FBXO22 recognizes BACH1-BTB, we used cryoelectron microscopy (cryo-EM) to determine the structure of the SKP1-CUL1-RBX1-FBXO22 complex (also known as SCF<sup>FBXO22</sup>) bound to the BACH1 BTB dimer at 3.9 Å resolution (Figure S1; Table S1). The SCF<sup>FBXO22</sup>-BACH1 complex adopts a canonical SCF architecture<sup>31</sup> in which the FBXO22-SKP1 SR module, together with the BACH1 substrate, is docked to the N-terminal half of the cullin scaffold (Figure 1D). Unique among F-box proteins, FBXO22 features a C-terminal FIST (F-box and intracellular signal transduction proteins) domain responsible for substrate recruitment (Figure 1A). Consistent with our SEC-MALS analysis, the F-box protein forms an asymmetric complex with the BACH1 BTB dimer at a 1:1 molar ratio (Figure 1E).

The FIST domain is found in eukaryotic F-box proteins and prokaryotic proteins involved in signal transduction.<sup>32</sup> It has been proposed to function as a sensory domain, possibly binding small molecular ligands, although its fold was previously uncharacterized. Our structure reveals that the FBXO22 FIST domain is constructed by three structural repeats, each consisting of a central four-stranded β-sheet sandwiched by two α-helices on one side and a β-hairpin loop on the other (Figure 1F). The central β-sheet from each repeat packs against the other two repeats, giving rise to a compact globular fold with a pseudo 3-fold symmetry. A Dali search identified two classes of unrelated metabolic enzymes, chorismatases and cyanuric acid hydrolases, with a similar overall fold, which could have evolved from monomers of the trimeric YjgF superfamily (Figure S2A).<sup>33–35</sup> Different from the metabolic enzymes, the FIST domain of FBXO22 does not feature any obvious ligand-binding pocket. We name the three structural repeats of the FIST domain, FIST-1, FIST-2, and FIST-3. Noticeably, FIST-3 is distinguished from the other two repeats by having a longer β-hairpin loop, whose two β-strands, β15 and β16, perfectly align with the edge of the central β-sheet and extend it into a six-stranded sheet (Figures 1F and S2A).

### BACH1 quaternary structure degron

Similar to most BTB dimer structures, the BACH1 BTB domain dimer is characterized by a domain swapping architecture, in which the two protomers exchange their N-terminal β-strands.<sup>36,37</sup> In BACH1, the N-terminal β-strand (β1) of each protomer runs in antiparallel with the C-terminal α-helix (CTH) (α6) of the opposite protomer, extending the dimer interface beyond the core BTB domain (Figure 1G). Remarkably, one of these cross-protomer β1-α6 pairs within the BACH1-BTB dimer makes up the entire interface with the F-box protein by clinging to the edge of FBXO22 FIST-3 (Figure 2A).

The tri-molecular interface between FBXO22 and the BACH1-BTB dimer is stabilized by a hybrid of polar and hydrophobic interactions. Specifically, the β1 strand of one BACH1-BTB chain (BTB-B), together with the β5 strand of the second BACH1 BTB chain, is juxtaposed with the β15-β16 hairpin of FBXO22 FIST-3, creating an intermolecular anti-parallel β-sheet supported by a
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<figure><img src="image_11.png" /><figcaption>**Figure 1. Overall structure of the FBXO22-BACH1-BTB complex**</figcaption></figure>

(A) Domain composition of BACH1, FBXO22, and FBXL17.

(B) BACH1-BTB pull-down by GST-FBXO22.

(C) SEC-MALS analyses of BACH1-BTB, FBXO22-SKP1, and their complex with experimentally determined molecular weights. Monomeric BACH1-BTB and FBXO22-SKP1 have a theoretical molecular weight of 17 and 63 kDa, respectively.

(D) Structure model of SCF-FBXO22-BACH1-BTB fitted in the electron microscopy map. The RBX1 RING domain is not shown due to its flexibility.

(E) Electron microscopy map fit with the FBXO22-BACH1-BTB complex.

(F) Orthogonal views of the FBXO22 FIST domain with three repeats (FIST-1, 2, and 3).

(G) Structure of the BACH1 BTB domain dimer with select secondary structure elements indicated.
See also Figures S1 and S2 and Table S1.

network of backbone hydrogen bonds (Figure 2B). These secondary structure interactions are cemented by an adjacent hydrophobic core, which is nucleated by four amino acids from the BACH1-BTB β1-α6 pair (Phe9, Tyr11, Cys122, and Phe125) and three residues in the FBXO22 β15-β16 hairpin (Ile368, Phe373, and Leu375). Peripheral to the hydrophobic core, the FBXO22-BACH1-BTB dimer interface is further strengthened

by two salt bridges and intermolecular van der Waal packing made by additional residues from the F-box protein (Asp366, Arg308, Lys377, and Arg376) and the two protomers of the substrate (Glu12 and Phe128).

In support of a critical role played by the N-terminal β1 strand of BACH1-BTB at the interface, previous studies have shown that individual alanine mutations of its two aromatic residues,
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<figure><img src="image_23.png" /><figcaption>**Figure 2.** Recognition of the BACH1-BTB quaternary structure degron by FBXO22</figcaption></figure>

(A) Recognition of the BACH1 cross-protomer β1-α6 degron by FBXO22. BACH1-BTB(A) and BACH1-BTB(B) are shown as cartoons and colored in cyan and orange, respectively.

(B) Close-up view of the FBXO22-BACH1-BTB interface with key residues shown in sticks.

(C) HA immunoprecipitation of BACH1 α6 mutants. HEK293T cells were transfected with either empty vector (EV) or HA-BACH1 wild type (WT) or mutants. Cells were then treated with MLN-4924 prior to HA immunoprecipitation. Proteins were immunoblotted as indicated.

*(legend continued on next page)*
Phe9 and Tyr11, are sufficient to abolish FBXO22-BACH1 interactions.³⁰ Similarly, replacing Phe125 in the BACH1 α6 helix with alanine or aspartate effectively abrogates E3-substrate complex formation (Figure 2C). Therefore, both secondary structure elements of the BACH1-BTB β1-α6 pair, which is shaped by the quaternary structure of the BTB dimer, are necessary to define the functional degron. In a second set of mutational analyses, we validated that the majority of the FBXO22 residues at the tri-molecular interface are indispensable for binding BACH1, suggesting that the edge of the third FBXO22 FIST repeat represents the primary docking site for BACH1 (Figure 2D). Noticeably, although the symmetric BACH1 BTB domain dimer contains two cross-protomer β1-α6 pairs, two FBXO22 molecules cannot simultaneously recognize the two identical BACH1 degrons without a severe steric collision (Figure S2B). A single SCF<sup>FBXO22</sup> E3 complex, therefore, appears responsible for ubiquitinating both protomers of a BACH1 dimer via their BTB domains (Figures S2C and S2D).

Mammalian BACH1 has a close paralog, BACH2, which is primarily expressed in the brain and spleen with well-documented roles in regulating immune cell differentiation.<sup>38-40</sup> The BTB domains of the two BACH proteins, including their terminal regions, are overall 62% identical in sequence (Figure 2E). Their homodimer structures can be superimposed with a root mean square deviation (RMSD) of 0.6 Å (Figure S2E).<sup>36,41</sup> Despite their sequence and structural similarity, only BACH1 but not BACH2 can be recognized by FBXO22 (Figure 2F). To identify key elements in BACH1 conferring this specificity, we made chimeric BACH proteins and tested their interactions with the F-box protein. Interestingly, replacing Phe9 in the N-terminal β1 strand of BACH1 with its counterpart in BACH2 (Tyr12) had little effect on FBXO22 binding. By contrast, swapping the C-terminal α6 helices of the two BACH proteins effectively switched their ability to engage the E3 (Figure 2F). A close examination of the BACH2 BTB domain structure reveals a non-helical nature of its C-terminal region (Figure S2E) distinct from the α6 helix of BACH1. This structural difference provides a possible explanation for BACH2's inability to bind FBXO22.

Together, these results reinforce the importance of the structural integrity of the β1-α6 degron of the BACH1 BTB dimer for FBXO22 recognition and underscore the capacity of the quaternary structure degron in conferring high E3-substrate selectivity, a previously undescribed characteristic of CRL substrates.

## Regulation of FBXO22-BACH1 interaction

Using biolayer interferometry (BLI), we determined the affinity of FBXO22 toward BACH1-BTB, which is ~90 nM and on par with other CRL-substrate interactions (Figure 2G).<sup>42</sup> Consistent with the lack of any obvious ligand-binding pocket in FBXO22 or BACH1-BTB, the binding between the two proteins is insensitive to heme (Figure 2H). Heme, therefore, does not appear to be physically required for the productive interaction between FBXO22 and BACH1 (Figures S2C and S2D) and most likely promotes BACH1 recognition by FBXO22 through an indirect mechanism. In previous studies, heme has been reported to block the DNA-binding activity of BACH1-sMAF heterodimers independent of the BACH1 BTB domain.<sup>19</sup> This raises the possibility that the quaternary structure degron of BACH1 might not be accessible to FBXO22 until the transcription factor is released from the DNA.

The BTB domain is widely found in CRL3 SRs and transcription factors.<sup>37</sup> Besides mediating protein oligomerization, the BTB domain of select transcription repressors, such as PLZF and BCL6, has been reported to interact with co-repressors.<sup>43,44</sup> In the case of BCL6, distinct SLiMs of SMRT/NCOR2 and BCOR co-repressors have been shown to form the same interface with the BCL6 BTB domain dimer, which is highlighted by an intermolecular β-sheet involving the N-terminal β1-strand of BCL6-BTB (Figure 2I).<sup>45,46</sup> Such a binding mode is remarkably analogous to the FBXO22-BACH1-BTB interface (Figure 2J). Similar to PLZF and BCL6, BACH1 has been previously shown to be functionally associated with nuclear co-repressor NCOR1, a close paralog of SMRT/NCOR2.<sup>47</sup> This prompted us to test whether NCOR1 can compete with FBXO22 for binding the BTB domain of BACH1. A central region of NCOR1 has been previously identified to interact with the BTB domain of multiple functionally unrelated transcriptional co-repressors.<sup>43</sup> Sequence alignment and structure prediction analyses indicate that this region contains several potential SLiMs conserved among NCOR1 orthologs, including the one used by NCOR2 for binding BCL6-BTB (Figure S2F). When co-expressed and co-purified from *E. coli*, this ~230 amino acid NCOR1 fragment was indeed sufficient to form a complex with the BACH1 BTB domain (Figure 2K). Importantly, the NCOR1 fragment was able to effectively block FBXO22-BACH1 binding in an AlphaScreen-based competition assay (Figure 2L), suggesting that the transcriptional co-repressor can occupy the quaternary structure degron of

<footnote>(D) FLAG immunoprecipitation of FBXO22 mutants. HEK293T cells were transfected with either EV or FLAG-FBXO22 WT or mutants. Cells were then treated with MLN-4924 prior to FLAG immunoprecipitation. Proteins were immunoblotted as indicated. Non-specific protein bands are labeled with asterisks.</footnote>

<footnote>(E) Sequence alignment of the N- and C-terminal regions of BACH1 and BACH2 BTB domains.</footnote>

<footnote>(F) FLAG immunoprecipitation of BACH1-BACH2 chimeras. HEK293T cells were transfected with either EV or FLAG-BACH1/BACH2 chimeras as indicated. Cells were then treated with MG-132 prior to FLAG immunoprecipitation. Proteins were immunoblotted as indicated. Non-specific protein bands are labeled with asterisks.</footnote>

<footnote>(G) Biolayer interferometry (BLI) measurements of FBXO22 and BACH1-BTB interaction. $K_d$-equilibrium, dissociation constant.</footnote>

<footnote>(H) BACH1-BTB pull-down by GST-FBXO22 in the presence and absence of 10 µM hemin. Non-specific protein bands are labeled with asterisks.</footnote>

<footnote>(I) Structure of BCL6-BTB dimer (green) in complex with the co-repressor SMRT (purple, PDB:1R2B) or BCOR (slate, PDB: 3BIM).</footnote>

<footnote>(J) Structure of BACH1-BTB dimer in complex with FBXO22 β15-β16 hairpin (purple).</footnote>

<footnote>(K) BACH1-BTB pull-down by GST-NCOR1.</footnote>

<footnote>(L) Inhibition of the FBXO22-BACH1-BTB interaction by NCOR1 fragments measured in AlphaLISA competition assays. Data are presented as mean ± SD. $IC_{50}$, half-maximum inhibitory concentration.</footnote>

<footnote>(M) Inhibition of SCF<sup>FBXO22</sup>-catalyzed BACH1 ubiquitination by an ~230 amino acid NCOR1 fragment. See also Figure S2.</footnote>
BACH1. Interestingly, when the N-terminal and C-terminal halves of the NCOR1 central fragment were individually tested, each sub-fragment was able to inhibit the interaction between FBXO22 and the BACH1 BTB dimer, albeit at a much lower potency (Figure 2L). Therefore, NCOR1 might employ more than one SLIM to simultaneously engage the two identical FBXO22-binding sites on BACH1-BTB to block the ubiquitination of the transcription repressor (Figure 2M). Together, these results imply that the NCOR1 co-repressor can shield the quaternary structure degron of chromatin-bound BACH1 from FBXO22 until BACH1 is released from DNA by heme.

## Degradation of S-nitrosylated BACH1 by FBXO22 and FBXL17

FBXL17 is a leucine-rich repeats (LRRs)-containing F-box protein (Figure 1A) that, similarly to FBXO22, has been implicated in the degradation of BACH1 in response to heme-mediated oxidative stress.²⁹ Furthermore, it has been proposed that FBXL17 can recognize non-functional heterodimers of BTB proteins and sequester their monomeric BTB domains to ubiquitiate and degrade the mismatched substrate proteins.⁴⁸,⁴⁹ How FBXL17 captures BACH1 upon oxidative stress, presumably by recognizing its homodimeric BTB domain, remains unclear. We first investigated the functional relationship between FBXL17 and FBXO22 in regulating BACH1 stability in the context of BACH1 S-nitrosylation. Nitric oxide (NO) is a free radical in the cell that is either produced endogenously or exogenously.⁵⁰ By reacting with cysteine residues of selective targets, NO induces protein S-nitrosylation and further disulfide formation, which contribute to redox signaling that protects the cell against oxidative stress.⁵¹,⁵² NOR3, an NO donor, has recently been shown to induce BACH1 degradation, an effect that can be augmented by a garlic-derived sulfur amino acid, S-1-propenylcysteine (S1PC).⁵³ We first confirmed by biotin switch assay that BACH1 can be modified by two additional NO donors, S-nitrosoglutathione (GSNO) and spermine NONOate (sNONO) (Figure 3A). Using FBXL17 and FBXO22 individual and double knockout (KO) HCT-116 cells (Figures S3A and S3B), we verified that both FBXO22 and FBXL17 contribute to BACH1 turnover at steady state, although FBXO22 appears to play a predominant role (Figures S3C-S3E). By contrast, in response to NO treatment, BACH1 degradation is similarly slowed down when either FBXO22 or FBXL17 is missing (Figures 3B-3D, S3F, and S3G). The two F-box proteins, therefore, appear to play nonredundant roles in regulating BACH1 stability in response to stress.

The BTB domain of BACH1 contains five cysteine residues, two of which, Cys107 and Cys122, are located at the β1-α6 degron recognized by FBXO22 (Figure 3E). Consistent with a recent proteome-wide S-nitrosylation site mapping study,⁵⁶ mass spectrometry (MS) analysis of the recombinant BACH1 BTB domain treated with the NO donor NOR3-S1PC identified Cys107 but not Cys55 as an S-nitrosylation site (Figure S3H). Due to incomplete sequence coverage, whether Cys34, Cys109, and Cys122 could be modified by NO remained unclear. As a residue on the α5 helix of BACH1-BTB making direct contact with the α6 helix (Figure 3E), covalent modification of Cys107 is expected to perturb the local structure of the β1-α6 degron, thereby impairing FBXO22 binding. Indeed, even though

the NOR3-S1PC-treated BACH1 BTB domain still retains its homodimeric form in solution (Figure S3I), it can no longer interact with FBXO22 in a $Ni^{2+}$ NTA pull-down assay (Figure 3F). By contrast, FBXL17 showed robust activity in binding the NOR3-S1PC-treated BACH1 BTB domain (Figure 3G). Likewise, NO treatment reduced the amount of FBXO22 that can be co-immunoprecipitated by BACH1, while the opposite is true for FBXL17 (Figures 3H and 3I). These results suggest that FBXL17 might have been evolved to recognize and ubiquitiate BACH1 when BACH1 is in a form incompatible with FBXO22 binding (Figures S3J and S3K).

Interestingly, treatment with either an NO donor or hemin induced the release of BACH1 from chromatin (Figure 3J). Accordingly, sNONO reduced the binding of BACH1 to MafF and NCOR1 in the cell (Figure 3K). Similar to hemin,¹⁹ an NO donor can effectively abrogate the ability of the BACH1 bZIP domain to super-shift sMAF-bound DNA in an electrophoretic mobility shift assay (Figure S3L). These results implicate that, in addition to its BTB domain, the bZIP DNA-binding domain of BACH1, which is rich in cysteine residues, might also be susceptible to S-nitrosylation. We speculate that a subpopulation of the BACH1 proteins, depending on their cellular context, might be modified by S-nitrosylation within their bZIP domain but not the BTB domain, which could explain why BACH1-FBXO22 complex could still be detected in NO-treated cells (Figure 3H) and, in comparison to the parental cells, BACH1 was partially stabilized in FBXO22 KO cells despite NO treatment (Figures 3B-3D).

## FBXL17 preferentially remodels NO-modified BACH1-BTB dimer

From the single time point (30 min) affinity pull-down assay, we noticed that FBXL17 preferentially interacts with the NO-modified BACH1 BTB domain over the native polypeptide (Figure 3G). To further dissect the activity of FBXL17 in BACH1-BTB recognition, we used BLI to monitor the kinetics of their interaction with or without treating BACH1-BTB with NOR3-S1PC. In agreement with the affinity pull-down results, compound-treated BACH1-BTB interacted strongly with FBXL17 with an “apparent $K_d$” of ~110 nM, whereas the native BACH1-BTB protein only showed a detectable affinity toward the F-box protein with an apparent $K_d$ greater than 2.4 µM (Figures 4A and 4B). Noticeably, with compound-treated BACH1-BTB immobilized on the probe, the maximum binding signal appeared far from plateauing within the time window of the measurement, even though the amplitude of the association curves started to show saturation when the FBXL17 concentration reached the µM range (Figure 4A). The same phenomenon held true when we lengthened the time window to 10 min (Figure S4A). This feature hinted at a minimally biphasic nature of the interaction, which might involve complex rearrangement in addition to simple binding equilibrium.

To further characterize the interaction between FBXL17 and compound-treated BACH1-BTB, we used SEC-MALS to analyze samples of the two proteins mixed for different periods of time. In congruence with the reported activity of FBXL17 in forming stable complexes with monomeric BTB domains,⁴⁸ the F-box protein was found to bind a single copy of BACH1-BTB after the two proteins were mixed for 24 h (Figure 4C). Interestingly, it took a relatively long incubation time for such a stable 1:1 complex to form,
<figure><img src="image_24.png" /><figcaption>Figure 3. Degradation of BACH1 by FBXO22 and FBXL17</figcaption></figure>

(A) Biotin switch assay to assess BACH1 S-nitrosylation. HCT-116 cells were treated with either DMSO (NT) or NO donors (GSNO and sNONO) before lysates were subjected to S-nitrosylation biotin switch. KEAP1 and SKP1 were included as positive and negative controls, respectively.⁵⁴,⁵⁵ Biotinylated proteins were purified and immunoblotted as indicated.

(B and C) Assessing the stability of endogenous BACH1 in parental, KO cells upon NO treatment. Proteins were immunoblotted as indicated. The graph in (C) shows the quantification of BACH1 protein levels relative to that at 0 h.

(D) Quantification of BACH1 protein levels measured in CHX chase assays in HCT-116 parental KO cells with/without NO treatment. Data are presented as mean ± SD. The graph is created based on data shown in Figures S3F and S3G.

(E) Five cysteine residues in BACH1-BTB with their side chains shown in dot spheres.

(F and G) Effects of BACH1-BTB S-nitrosylation on FBXO22 and FBXL17 binding investigated by Ni²⁺ pull-down assay. Non-specific protein bands are labeled with asterisks.

(H) Co-immunoprecipitation of FBXO22 by FLAG-BACH1 with/without NO treatment.

(I) Co-immunoprecipitation of FBXL17 by HA-BACH1 with/without NO treatment.

(J) Western blots monitoring the amount of endogenous BACH1 in the soluble and chromatin fractions of HCT-116 cells upon hemin or NO treatment.

(K) HEK293T cells were transfected with empty vector (EV) or HA-BACH1. Co-immunoprecipitation of NCOR1 and MafF by HA-BACH1 with/without NO treatment. Proteins were immunoblotted as indicated.

See also Figure S3.
<figure><img src="image_25.png" /><figcaption>Figure 4. FBXL17 preferentially remodels S-nitrosylated BACH1-BTB</figcaption></figure>

(A and B) BLI measurements of FBXL17 and BACH1-BTB interaction with and without NOR3-S1PC treatment. The apparent K<sub>d-equilibrium</sub> values are indicated.

(C) SEC-MALS analyses of NO-treated BACH1-BTB, FBXL17-SKP1, and their mixture at various incubation time with the experimentally determined molecular weights. The theoretical molecular weights of MBP-FBXL17-SKP1 and monomeric BACH1-BTB are 108 and 17 kDa, respectively. A larger amount of protein sample was applied to BACH1-BTB alone run (gray).

(D) The time-courses of the interactions between FBXL17 and BACH1-BTB with or without NOR3-S1PC treatment monitored by affinity pull-down. t<sub>1/2</sub>: time needed for 50% complex formation.

(E and F) BLI measurements of the interaction between FBXL17 and BACH1-BTB (WT and mutants) with/without NOR3-S1PC treatment. The effect of compound treatment was diminished by C107A and C122A but was enhanced by C109A, which by itself is insufficient to stimulate FBXL17 binding.

See also Figures S3 and S4.

even though BLI could instantaneously detect the interaction between FBXL17 and BACH1-BTB. Given that compound-treated BACH1-BTB remains a stable dimer even after being diluted to ~3 µM (Figure S3I), we speculate that the biphasic binding curve detected by BLI reflects both the initial contact between FBXL17 and dimeric BACH1-BTB and the final complex formation between FBXL17 and monomeric BACH1-BTB after the BACH-BTB dimer is remodeled by the F-box protein.

To fully assess the binding kinetics of FBXL17 and BACH1-BTB, we returned to the affinity pull-down assay and monitored the interaction with a time window up to 1–2 days. As expected from our BLI and SEC-MALS results, it took about 1 h for half of the maximal amount of FBXL17 to become stably associated with compound-treated BACH1-BTB under the tested experimental condition (Figure 4D). By contrast, the amount of FBXL17 captured by untreated BACH1-BTB did not saturate
A

<figure><img src="image_26.png" /></figure>

B

<figure><img src="image_27.png" /></figure>

C

<figure><img src="image_28.png" /><figcaption>**Figure 5.** S-nitrosylation-induced structural changes of BACH1-BTB dimer</figcaption></figure>

(A) Thermostability of native and NO-treated BACH1-BTB determined by differential scanning fluorimetry. T~m~ is melting temperature and is presented as mean ± SD.

(B) Summary of HDX data overlayed onto BACH1-BTB dimer. Red and orange regions indicate faster exchange observed in the NO-treated sample. Red regions display correlated EX1 kinetics, likely reflective of dimer reconfiguration over longer time points (see Figures S4D and S4E). Blue regions show a subpopulation

(legend continued on next page)
even after 2 days. Such a strong preference of FBXL17 to capture BACH1-BTB monomers from the compound-treated dimer over the intact non-treated dimer raised the possibility that NO treatment destabilizes BACH1 to promote FBXL17 binding and subsequent ubiquitination.

Importantly, this effect of NO is attributable to the modification of two specific cysteine residues on the BACH1 BTB domain. Without impacting the resistance of native BACH1-BTB dimer to FBXL17 binding (Figure 4E), individual alanine mutations of Cys107 and Cys122 but not the other three cysteine residues significantly reduced the ability of FBXL17 to release monomeric BACH-BTB from its NO donor-treated dimeric form (Figures 4F, S4B, and S4C). This result allows us to map Cys122, an amino acid also residing within the β1-α6 degron, as another key residue sensitive to S-nitrosylation.

## Global destabilization of BACH1-BTB dimer by S-nitrosylation

To confirm that S-nitrosylation indeed destabilizes BACH1-BTB dimerization, we first used differential scanning fluorometry (DSF) to show that NO treatment reduced the thermostability of BACH1-BTB by almost 10 degrees Celsius (Figure 5A). Subsequent hydrogen-deuterium-exchange MS (HDX-MS) experiments allowed us to map the secondary structure elements within the BACH1-BTB dimer that are structurally destabilized. Strikingly, while the α6 and β1 terminal regions are intrinsically more susceptible to deuteration in both native and NO donor-treated samples, multiple secondary structure elements within the BTB core domain, including those at the central dimer interface, are impacted the most by NO treatment (Figures 5B, S4D, and S4E). Because BACH1-BTB remains as a dimer upon NO treatment (Figure S3I), these HDX results hint at an altered topology of the BTB dimer induced by S-nitrosylation. In support of this notion, several peptides detected in our HDX-MS analysis displayed a bimodal exchange profile in the NO-treated samples (Figures 5C and S4F), indicating that the BACH1-BTB dimer might exist in two distinct populations where one population underwent faster deuteration exchange than the other. For some peptides, the two species were also detectable in the native sample. NO treatment appeared to shift the equilibrium between the two populations, increasing the fast exchange species, which is structurally less stable. These results are in full agreement with the global destabilizing effect of NO treatment detected by the DSF thermal shift assay (Figure 5A).

By directly detecting two S-nitrosylated peptides from our LC-MS analysis of the NOR3-S1PC-treated sample, we further confirmed that C107 and C122 are the only two NO-modified amino acids within the BACH1 BTB domain (Figures S5A-S5C). Akin to NO treatment, a C122E mutation in BACH1 mimicking the effect of S-nitrosylation reduced the thermostability of the BTB domain (Figure S5D). Both *in vitro* and in cells, the BACH1 C122E mutant was no longer recognized by

FBXO22 but increased its ability to bind FBXL17 (Figures S5E-S5G). This could explain the similar half-lives of the BACH1 mutant and the wild-type (WT) protein (Figure S5H), as the impairment of its FBXO22-mediated turnover is compensated by FBXL17. Based on these observations, we conclude that S-nitrosylation of the two cysteine residues in BACH1 reduces the structural stability of the BACH1-BTB dimer by reconfiguring the dimer arrangement, thereby facilitating FBXL17 binding.

## Transient remodeling of BACH1-BTB dimer by a pair of SCF<sup>FBXL17</sup> proteins

To reveal the mechanism by which FBXL17 recognizes and remodels NO-modified homodimeric BACH1-BTB, we mixed NO donor-treated BACH1-BTB with the SCF<sup>FBXL17</sup> complex for different periods of time and subjected the samples to cryo-EM analysis. 2D classification of the particles imaged at three incubation times (30 s, 10 min, and 24 h) indicated that the predominant observable species were either in the form of free SCF<sup>FBXL17</sup> or the E3 complex bound to the monomeric BACH1 BTB domain (hereafter referred to as mSCF<sup>BTB</sup>) (Figures S6A-S6C). Strikingly, a small fraction of the particles appeared to contain a pair of parallel SCF<sup>FBXL17</sup> complexes with the FBXL17 LRR domains facing each other, likely sandwiching a BACH1-BTB dimer in the middle (Figure 6A). Although these minor classes, which possibly represent a transient intermediate species of the BACH1-BTB dimer remodeled by the E3 complex, yielded two 7.8 Å 3D reconstructions (Figure S6C), the numbers of particles were too limited to determine the structures at a higher resolution.

To overcome the challenge of resolving these presumably unstable intermediates, we turned to the native BACH1-BTB sample, which is still susceptible to reconfiguration by FBXL17, except at a much slower rate (Figure 4D). We reasoned that the higher resistance of the native BACH1-BTB sample to FBXL17-mediated monomerization might offer an experimental window to capture the intermediate steps of the reaction. Remarkably, cryo-EM analysis of a sample of SCF<sup>FBXL17</sup> incubated with native BACH1-BTB unveiled four species, free SCF<sup>FBXL17</sup>, mSCF<sup>BTB</sup>, and two distinct dimeric SCF<sup>FBXL17-BACH1-BTB</sup> super-assemblies (hereafter referred to as dSCF<sup>BTB-I</sup> and dSCF<sup>BTB-II</sup>) (Figure S7). The structure of the mSCF<sup>BTB</sup> complex resolved at 3.2 Å resolution is highly analogous to the previously reported crystal structure of FBXL17 in complex with the monomeric mutant KEAP1 BTB domain,⁴⁸ except that no clear density is present for the β1 strand of the BACH1 BTB domain (Figure 6B). In both structures, the canonical dimer interface of the BTB domains is blocked by the FBXL17 CTH. Importantly, both Cys107 and Cys122 of BACH1-BTB are solvent-exposed and far away from the BACH1-FBXL17 interface, indicating that they are not directly recognized by the F-box protein. Modification of these two residues in the NO donor-treated BACH1 sample, therefore, likely accelerated FBXL17 binding by destabilizing the BACH1-BTB dimer.

<footnote>that was slightly more protected in the NO-treated sample. White indicates no observable differences. Individual uptake plots show the exchange for unmodified (blue) and NO modified (red). Data are presented as mean ± SD.
(C) Peptides 6–21 and 79–89 exhibit bimodal exchange behavior but with different extents in the unmodified and NO-modified states. Individual populations within the spectra are shown in purple and green. By contrast, peptides 113–121 do not exhibit bimodal profiles.
See also Figures S4 and S5.</footnote>
<figure><img src="image_29.png" /><figcaption>Native BACH1-BTB + SCF<sup>FBXL17</sup>
Parallel dimer</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>Anti-parallel dimer</figcaption></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

HEK293T
WCE
IP: α-FLAG

EV
+ - + +
+ - + -
+ - + -

FLAG-FBXL17
HA-FBXL17
HA-FBXL17 ΔCTH
HA-FBXL17 (α-HA)
FLAG-FBXL17 (α-FLAG)
SKP1
GAPDH

<figure><img src="image_38.png" /></figure>

1.5
+ -
- +
+ +

8
+ -
+ +
+ +

24
+ -
+ +
+ +

hours
Biotin-BACH2-BTB
Biotin-BACH1-BTB
MBP-FBXL17-SKP1

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>Figure 6. Structural analysis of BACH1-BTB remodeling by SCF<sup>FBXL17</sup></figcaption></figure>

(A) Representative 2D classes of dimeric SCF-FBXL17-BACH1-BTB complexes found in the native and NOR3-S1PC-treated samples.

(B) Structure of FBXL17-LRR in complex with monomeric BACH1-BTB. Structurally disordered β1 strand of BACH1-BTB is shown in a dashed line.

(C) Two dimeric SCF-FBXL17-BACH1-BTB complexes fitted in their electron microscopy maps.

(D) Close-up views of BACH1-BTB dimer interface in the dimeric FBXL17-BACH1 complex. Local refinement-improved electron microscopy map is fitted with a BACH1-BTB dimer and two copies of FBXL17-LRRs.

(E) Conformational changes of the BACH1-BTB dimer during the transition from the free to the dimeric SCF-FBXL17-bound form. Two arrowed dashed lines indicate the two-step rotations of the protomer A (cyan) of BACH1-BTB.

(F) Co-immunoprecipitation of HA-FBXL17 by FLAG-FBXL17. FLAG-FBXL17 was co-transfected with full-length or C-terminally truncated HA-FBXL17 into HEK293T cells. Proteins were immunoblotted as indicated. CTH, C-terminal helix.

(G) FBXL17 pull-down by biotinylated BACH1-BTB but not BACH2-BTB.

(legend continued on next page)
Although the 3D reconstructions of dSCF<sup>BTB-I</sup> and dSCF<sup>BTB-II</sup> suffered from a lower resolution (4.4 Å) (Figure 6C), local refinement improved the density map and enabled us to resolve the secondary structures of the two BACH1 BTB domains in the middle of the dimeric SCF (Figures 6D, S7C, and S7D). In dSCF<sup>BTB-I</sup>, FBXL17 is engaged with BACH1-BTB through the same interface as found in mSCF<sup>BTB</sup>. However, in addition to the β1 strand, the α6 helix in each BACH1-BTB protomer is also structurally disordered. Remarkably, the β5 strands of the two protomers join each other and form an intermolecular anti-parallel β sheet, structurally mimicking the β1-β5 cross-protomer two-stranded β sheet found in the isolated BACH1-BTB dimer (Figure 6E). This new dimer interface is further substantiated by the cross-promoter packing of a coiled region transformed from the mostly melt α5 helices. A similar dimer interface, albeit being slightly twisted, is found in dSCF<sup>BTB-II</sup>. As a result, the C-terminal lobes of the two CUL1-RBX1 catalytic scaffolds become separate from each other (Figure 6C). The dimeric arrangement of these dSCF<sup>BTB</sup> complexes, therefore, is predominantly, if not solely, stabilized by the BACH1 BTB domains. Importantly, the two structure models, namely dSCF<sup>BTB-I</sup> and dSCF<sup>BTB-II</sup>, derived from the native BACH1-BTB sample, fit snuggly into the 7.8 Å 3D reconstruction maps obtained with the NO-treated sample, indicating that they belong to common species found in the two specimens (Figures S6D and S6E). Taken together, we postulate that these dimeric SCF<sup>FBXL17-BACH1-BTB</sup> super-assemblies represent intermediate states of BACH1-BTB dimer remodeling, which is simultaneously catalyzed by two SCF<sup>FBXL17</sup> E3 complexes. This notion is further supported by co-immunoprecipitation analysis, which revealed physical association between two differentially tagged FBXL17 when co-expressed in the cell (Figures 6F and S5I). Their complexation was largely abrogated when the CTH, which is responsible for BTB binding, was removed from one of the FBXL17 constructs (Figure 6F), suggesting that the association of the two FBXL17 proteins is dependent on BTB binding.

To assess whether efficient substrate remodeling indeed requires the synergy of two copies of the E3 complexes, we isolated a BACH1-BACH2 BTB domain heterodimer with tandem affinity purification upon co-expression. Lacking the key residues for FBXL17 binding,⁴⁸ homodimeric BACH2-BTB is completely resistant to remodeling by the F-box protein (Figure 6G). Interestingly, even with an incubation time of about 1 day, FBXL17 can barely capture BACH1-BTB from the BACH1-BACH2 heterodimer, which is in stark contrast to the homodimeric BACH1-BTB (Figure 6H). Of note, because the BACH1-BACH2 heterodimer retains a functional β1-α6 quaternary structure degron (Figure 2F), it can still be recognized by FBXO22 (Figure 6I). Similarly, FBXL17 can sequester WT BACH1-BTB from a WT BACH1-BTB homodimer far more efficiently than a BACH1-BTB heterodimer consisting of a WT

BTB domain and a mutant BTB defective for binding FBXL17 (Figures 6J and 6K). These results strongly suggest that the homodimeric BACH1 BTB dimer is indeed inspected by a pair of SCF<sup>FBXL17</sup> proteins, which is distinct from SCF<sup>FBXO22</sup>. If the structural integrity of the substrate dimer is compromised by oxidative modifications, such as S-nitrosylation, SCF<sup>FBXL17</sup> can efficiently seize monomeric BACH1.

## DISCUSSION

### Degrons beyond SLiMs

Ubiquitin ligases have been well known to recruit substrates by recognizing their SLiM degrons.² Although portability has long been implicated as an inherent property of these degradation signals, it is not an essential feature originally used to define the term degron.³ Recent studies have shown that the targeting signals of a number of CRL-substrate proteins are constructed by their tertiary folds.⁵⁷,⁵⁸ This study expands the degron space into the quaternary structures of protein oligomers, which allow E3s to differentiate targets based on signature motifs encrypted only in high-order assemblies. In the case of the β1-α6 degron of the dimeric BACH1-BTB recognized by FBXO22, substrate specificity is dictated by the proper association of the two identical subunits of the substrate. By contrast, the degradation signal of the BACH1-BTB dimer decoded by FBXL17 is determined by the strength of protein-protein interaction within the homodimeric assembly, a physical property that can be affected by post-translation modifications associated with oxidative stress. These examples highlight the diverse nature of degradation signals recognized by ubiquitin ligases and underscore the complex mechanisms governing E3-substrate specificity in protein degradation. We speculate that the number of degrons with high-order structural complexity beyond simple primary amino acid sequence will continue growing, especially as more potential CRL E3 substrates are deconvoluted.⁵⁹⁻⁶¹

### Substrate repertoire of FBXO22 and FBXL17

FBXO22 has been suggested to ubiquitinate several substrate proteins with no structural similarity to BACH1.⁶² How the F-box protein recognizes these targets, presumably also through its FIST domain, remains to be determined. With a role in protein dimerization quality control, FBXL17 has been implicated in ubiquitinating a subset of BTB-domain-containing proteins when they form mismatched heterodimers through their BTB domains.⁴⁸,⁴⁹ However, only the structure of FBXL17 in complex with a monomeric BTB domain has been captured so far, leading to a paradox in the mechanism of action of this E3. We propose that it is not the mismatched nature of the BTB domain heterodimers but the strength of protein-protein interaction at the dimeric interface that is perceived by FBXL17. This distinction explains why the

(H) A comparison of the remodeling efficiency of BACH1-BTB homodimer and BACH1-BTB-BACH2-BTB heterodimer by FBXL17 as monitored by the time course of BACH1-BTB-MBP-FBXL17 association.

(I) Binding of the BACH1-BTB-BACH2-BTB heterodimer to FBXO22 by MBP pull-down assay. MSB is a protein solubilizing tag unrelated to MBP.

(J) FBXL17 pull-down by biotinylated wild-type (WT) BACH1-BTB but not a mutant (MT) defective in FBXL17 binding.

(K) A comparison of the remodeling efficiency of WT BACH1-BTB homodimer and a WT and mutant BACH1-BTB heterodimer by FBXL17 as monitored by the time course of BACH1-BTB-MBP-FBXL17 association.

See also Figures S6 and S7 and Table S1.
<figure><img src="image_43.png" /></figure>

same F-box protein can also sequester protomers derived from the homodimeric BTB domain of BACH1 when given long enough incubation time or when the stability of the homodimer is compromised by oxidative stress. Moreover, the structures of the FBXL17-BTB dimer intermediates indicate that efficient clearance of these substrate proteins requires the synergistic actions of a pair of SCF<sup>FBXL17</sup>, which can remodel its homodimeric BACH1 substrate beyond a simple monophasic binding event. Interpretation of the degradation signal by FBXL17, therefore, involves at least two, if not more, steps of action taken by the F-box protein

<caption>**Figure 7.** A schematic model of oxidative-stress-induced BACH1 ubiquitination by FBXO22 and FBXL17</caption>

Under homeostatic conditions, BACH1 homodimerizes via its BTB domain and interacts with small MAF proteins through its bZIP domain to repress gene expression by binding to the antioxidant response elements (AREs) of oxidative stress response genes. The interaction between BACH1 and components of the transcriptional co-repressor complex (i.e., NCOR1 and other histone modifiers) blocks the recognition of the β1-α6 quaternary structure degron of BACH1 by FBXO22. Under oxidative stress, reactive oxygen species (ROS) covalently modify cysteine residues on the bZIP domain of BACH1 and release it from chromatin. If the BTB domain of BACH1 remains intact, its β1-α6 degron is recognized by FBXO22, which promotes its ubiquitination and degradation. If the pro-oxidant also modifies the BACH1 BTB domain and compromises the structural integrity of the cross-protomer β1-α6 degron, thereby weakening the BTB dimer, a pair of FBXL17 will transiently associate with the BACH1 BTB dimer and remodel it into stably bound monomer for ubiquitination and degradation.

beyond simple recognition. Consistent with this notion, a minor population of dimeric SCF<sup>FBXL17-BACH1-BTB</sup> particles configured in an antiparallel manner can be found in the 2D class averages of the native BACH1-BTB sample (Figure 6A). Unlike conventional E3s, FBXL17 appears to be able to enzymatically remodel and promote the degradation of its substrates.

## BACH1 as a sensor for oxidative stress

As a key transcription factor regulating heme metabolism and oxidative stress, BACH1 is tightly regulated at its protein level. FBXO22 and FBXL17 appear to have been co-evolved to play complementary roles in promoting BACH1 ubiquitination. Our studies not only reveal the distinct structural basis of BACH1 recognition by the two F-box proteins but also shed light on the regulatory mechanisms

by which different forms of oxidative stress make the transcriptional repressor discernible to the E3s (Figure 7). With an unusually large number of cysteine residues, BACH1 appears to function as a redox sensor, which can modulate its own stability in response to pro-oxidants. While heme or pro-oxidants releases BACH1 from the chromatin and exposes its β1-α6 degron to FBXO22, NO modifies two cysteine residues in the BACH1 BTB domain and makes the BTB dimer susceptible to FBXL17-mediated remodeling by weakening the dimer interface. Outside the BTB domain, a series of reactive cysteine residues of
BACH1, including several in the bZIP domain, have been identified by proteome-wide analyses to be sensitive to modifications by H₂O₂ and NO donors.⁵⁶,⁶³ It is plausible that these cysteine residues of BACH1 could also serve as sensors for a variety of pro-oxidants and possibly affect the function and stability of the transcriptional repressor via unknown mechanisms.⁶⁴,⁶⁵

Beyond its role in the oxidative stress response pathway, BACH1 also targets genes involved in cell cycle control, apoptosis, and metabolism.¹⁶,⁴⁷,⁶⁶ By positively regulating the expression of multiple pro-metastatic genes, BACH1 has been shown to promote cancer cell invasiveness.⁶⁷⁻⁶⁹ Our studies suggest that compounds that could modify the BTB or DNA-binding domains of BACH1 have the potential to promote the ubiquitination and degradation of BACH1 by FBXL17 or FBXO22, thereby ameliorating BACH1-mediated cancer metastasis. Similar strategies to reshape or re-surface domains and complexes of disease-promoting target proteins with small molecules hold the promise to create “neo” tertiary or quaternary structure degrons for targeted protein degradation as an emerging therapeutic modality.⁷⁰,⁷¹

## Limitations of the study

The affinity of the central NCOR1 fragment toward the BACH1-BTB domain is much lower than that of FBXO22, raising the question as to how the transcriptional co-repressor could compete with the E3 ubiquitin ligase before BACH1 is released from chromatin. We hypothesize that binding of NCOR1 to BACH1 might be reinforced by multivalent interactions between other subunits of the co-repressor complex and chromatin. The nature of these interactions remains to be elucidated. Future studies are also needed to dissect the sub-populations of BACH1, which might be differentially modified by S-nitrosylation at different sites and at different times after induction of oxidative stress. Validation of this hypothesis could help explain why, in addition to FBXL17, FBXO22 also plays a role in promoting the degradation of BACH1 upon NO treatment, despite its inability to bind the S-nitrosylated BACH1 BTB domain. Finally, future structural studies might reveal additional intermediate states of BACH1-BTB dimer recognized by FBXL17.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Ning Zheng (nzheng@uw.edu).

### Materials availability

All unique/stable reagents generated in this study are available upon request.

### Data and code availability

* Original western blot images have been deposited at Mendeley at https://dx.doi.org/10.17632/vf73rr8p5n.1 and are publicly available as of the date of publication.

* The atomic coordinates and EM maps of protein complexes have been deposited at the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) with accession codes listed in the *key resources table*. They are publicly available as of the date of publication.

* HDX-MS data have been deposited at ProteomeXchange as PXD053923. Proteomics data have been deposited at ProteomeXchange as PXD056513. They are publicly available as of the date of publication.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the *lead contact* upon request.

## ACKNOWLEDGMENTS

The authors would like to thank R. Yan, X. Zhao, J. Jung, and Z. Yu at the Cryo-EM Facility on the Janelia Research Campus of the Howard Hughes Medical Institute and J.D. Quispe and S. Dickinson at the Arnold and Mabel Beckman Cryo-EM Center at the University of Washington for their assistance in electron microscopy data acquisition. The authors thank Mengxi Liu for her contribution to this work. We also thank the NYU Proteomics Lab (supported in part by the NYU School of Medicine and the Laura and Isaac Perlmutter Cancer Center Support grant P30CA016087 from the National Cancer Institute). In addition, the mass spectrometric experiments were supported with a shared instrumentation grant from the NIH (1S10OD010582-01A1 for the purchase of an Orbitrap Eclipse) for S-nitrosylation site-mapping experiments. The authors also thank T.R. Hinds for help on BLI data analysis and members of the Zheng Lab and Pagano Lab for discussion. N.Z. and M.P. are Howard Hughes Medical Institute Investigators. This work is also supported by NIH grant GM136250 to M.P. and NIH NIGMS grant S10OD030237 and the National Science Foundation award 2304707 to M.G.

## AUTHOR CONTRIBUTIONS

S.C., S.F.G., M.P., and N.Z. conceived the project. S.C. constructed and purified all protein samples in this study with inputs from S.F.G. and H.M. S.C. performed BLI, AlphaScreen, EMSA, DSF, and *in vitro* pull-down and ubiquitination experiments and prepared the sample for cryo-EM analysis. S.F.G. generated CRISPR KO cell lines, designed plasmid constructs, and performed all of the cell-based co-immunoprecipitations and protein stability assays, with notable contributions from Y.K., H.V.G., S.D., S.K., G.R., and L.L. J.P. and B.U. performed the mass spectrometric sample preparation, data acquisition, and analysis. Huigang Shi performed the majority of cryo-EM grid preparation, specimen screening, data collection, and processing tasks with early contributions from Hui Shi and S.C. E.I.J. and M.G. designed and performed the HDX-MS experiments. The manuscript was written with inputs from all authors. The order of the second and third co-first authors was chosen randomly.

## DECLARATION OF INTERESTS

N.Z. and M.P. are scientific cofounders of SEED Therapeutics with financial interests. They also received research funding from and are shareholders in Kymera Therapeutics. N.Z. serves on the scientific advisory boards of Synthex, Molecular Glue Labs, and Differentiated Therapeutics with financial interests. M.P. is a consultant and/or serves on the scientific advisory boards of CullGen, Sibylla Biotech, and Triana Biomedicines with financial interests. The findings presented in this manuscript were not shared with these companies.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Cell lines

* Source organism

* METHOD DETAILS

* Protein preparation

* Affinity pull-down assay

* BioLayer interferometry

* AlphaLISA

* SEC-MALS

* Differential scanning fluorimetry (DSF)

* In vitro ubiquitination assay

* Electrophoretic mobility shift assay (EMSA)

* Cryo-EM sample preparation and data collection
* Image processing and 3D reconstruction

* Model building and refinement

* Plasmids and antibodies

* Drug Treatment Procedures

* Co-immunoprecipitation assay

* Cell-based protein stability assay

* CRISPR-Cas9 genome editing

* Chromatin Fractionation Assay

* Biotin Switch Assay

* S-nitrosylation site mapping

* HDX-MS analyses

## QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cell.2024.10.012.

Received: October 12, 2023

Revised: August 25, 2024

Accepted: October 10, 2024

Published: November 5, 2024

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74. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605-1612. https://doi.org/10.1002/jcc.20084.

75. Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486-501. https://doi.org/10.1107/S0907444910007493.

76. Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948-1954. https://doi.org/10.1107/S0907444902016657.

77. Afonine, P.V., Poon, B.K., Read, R.J., Sobolev, O.V., Terwilliger, T.C., Urzhumtsev, A., and Adams, P.D. (2018). Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531-544. https://doi.org/10.1107/S2059798318006551.

78. Guttman, M., Weis, D.D., Engen, J.R., and Lee, K.K. (2013). Analysis of overlapped and noisy hydrogen/deuterium exchange mass spectra. J. Am. Soc. Mass Spectrom. 24, 1906-1912. https://doi.org/10.1007/s13361-013-0727-5.

79. Suloway, C., Pulokas, J., Fellmann, D., Cheng, A., Guerra, F., Quispe, J., Stagg, S., Potter, C.S., and Carragher, B. (2005). Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41-60. https://doi.org/10.1016/j.jsb.2005.03.010.

80. Mastronarde, D.N. (2005). Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36-51. https://doi.org/10.1016/j.jsb.2005.07.007.

81. Wu, C., Huang, X., Cheng, J., Zhu, D., and Zhang, X. (2019). High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift. J. Struct. Biol. 208, 107396. https://doi.org/10.1016/j.jsb.2019.09.013.

82. Guo, H., Franken, E., Deng, Y., Benlekbir, S., Singla Lezcano, G., Janssen, B., Yu, L., Ripstein, Z.A., Tan, Y.Z., and Rubinstein, J.L. (2020). Electron-event representation data enable efficient cryoEM file storage with full preservation of spatial and temporal resolution. IUCrJ 7, 860-869. https://doi.org/10.1107/S205225252000929X.

83. Punjani, A., Rubinstein, J.L., Fleet, D.J., and Brubaker, M.A. (2017). cryo-SPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290-296. https://doi.org/10.1038/nmeth.4169.

84. Zhang, K. (2016). Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1-12. https://doi.org/10.1016/j.jsb.2015.11.003.

85. Bepler, T., Morin, A., Rapp, M., Brasch, J., Shapiro, L., Noble, A.J., and Berger, B. (2019). Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153-1160. https://doi.org/10.1038/s41592-019-0575-8.

86. Punjani, A., Zhang, H., and Fleet, D.J. (2020). Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214-1221. https://doi.org/10.1038/s41592-020-00990-8.

87. Punjani, A., and Fleet, D.J. (2021). 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J. Struct. Biol. 213, 107702. https://doi.org/10.1016/j.jsb.2021.107702.

88. Rohou, A., and Grigorieff, N. (2015). CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216-221. https://doi.org/10.1016/j.jsb.2015.08.008.

89. Murphree, T.A., Vorauer, C., Brzoska, M., and Guttman, M. (2020). Imidazolium Compounds as Internal Exchange Reporters for Hydrogen/Deuterium Exchange by Mass Spectrometry. Anal. Chem. 92, 9830-9837. https://doi.org/10.1021/acs.analchem.0c01328.

90. Watson, M.J., Harkewicz, R., Hodge, E.A., Vorauer, C., Palmer, J., Lee, K.K., and Guttman, M. (2021). Simple Platform for Automating Decoupled LC-MS Analysis of Hydrogen/Deuterium Exchange Samples. J. Am. Soc. Mass Spectrom. 32, 597-600. https://doi.org/10.1021/jasms.0c00341.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>BACH1 (1:1000)</td><td>Bethyl</td><td>Cat# A303-058A; RRID: AB_10894145</td></tr><tr><td>FBXO22 (1:1000)</td><td>Proteintech</td><td>Cat# 13606-1-AP; RRID: AB_2104403</td></tr><tr><td>NRF2 (1:1000)</td><td>Cell Signaling</td><td>Cat# 12721S; RRID: AB_2715528</td></tr><tr><td>KEAP1 (1:10 000)</td><td>Proteintech</td><td>Cat# 10503-2-AP; RRID: AB_2132625</td></tr><tr><td>HMOX1 (1:1000)</td><td>Bethyl</td><td>Cat# A303-662A; RRID: AB_11205464</td></tr><tr><td>SKP1 (1:5000)</td><td>Michele Pagano's lab</td><td>N/A</td></tr><tr><td>CUL1 (1:1000)</td><td>Thermo Fisher Scientific</td><td>Cat# 718700; RRID: AB_2534002</td></tr><tr><td>HA (1:1000)</td><td>Sigma-Aldrich</td><td>Cat# H3663; RRID: AB_262051</td></tr><tr><td>FLAG (1:4000)</td><td>Sigma-Aldrich</td><td>Cat# F1804; RRID: AB_262044</td></tr><tr><td>Vinculin (1:1000)</td><td>Bethyl</td><td>Cat# A302-535A; RRID: AB_1999080</td></tr><tr><td>NCOR1 (1:1000)</td><td>Cell Signaling Technologies</td><td>Cat# 5948S; RRID: AB_10834809</td></tr><tr><td>MAFF (1:1000)</td><td>Proteintech</td><td>Cat# 12771-1-AP; RRID: AB_2137677</td></tr><tr><td>PCNA (1:1000)</td><td>Santa Cruz Biotechnologies</td><td>Cat# sc-56; RRID: AB_628110</td></tr><tr><td>Cyclin D1 (1:1000)</td><td>Thermo Fisher Scientific</td><td>Cat# MA1-39546; RRID: AB_11000217</td></tr><tr><td>GAPDH (1:1000)</td><td>Cell Signaling</td><td>Cat# 97166; RRID: AB_2756824</td></tr><tr><td>β-actin (1:5000)</td><td>Sigma-Aldrich</td><td>Cat# A5441; RRID: AB_476744</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>E. coli NEB5α</td><td>New England Biolabs</td><td>Cat# C2992H</td></tr><tr><td>E. coli NEB Stable</td><td>New England Biolabs</td><td>Cat# C3040H</td></tr><tr><td>E. coli BL21 (DE3)</td><td>New England Biolabs</td><td>Cat# C2527I</td></tr><tr><td>E. coli DH5α</td><td>New England Biolabs</td><td>Cat# C2987I</td></tr><tr><td>E. coli DH10Bac</td><td>Life Technologies</td><td>Cat# 10361012</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>Cat# L3000150</td></tr><tr><td>Cycloheximide</td><td>Sigma-Aldrich</td><td>Cat# C7698-1G</td></tr><tr><td>MG132</td><td>Peptides International</td><td>Cat# IZL-3175v</td></tr><tr><td>MLN-4924</td><td>Active Biochem</td><td>Cat# A-1139</td></tr><tr><td>Spermine NONOate</td><td>Enzo Life Sciences</td><td>Cat# ALX-430-013-5005</td></tr><tr><td>S-1-propenyl-L-cysteine</td><td>MedChemExpress</td><td>Cat# HY-111827</td></tr><tr><td>S-Nitrosoglutathione</td><td>EMD Millipore</td><td>Cat# 487920</td></tr><tr><td>NOR3</td><td>Sigma-Aldrich</td><td>Cat# E2895</td></tr><tr><td>Hemin</td><td>Sigma-Aldrich</td><td>Cat# 51280-1G</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>S-Nitrosylated Protein Detection Kit (Biotin Switch)</td><td>Cayman Chemical</td><td>Cat# 10006518</td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>Cat# 23225</td></tr><tr><td>Mycostrip, Mycoplasma Detection Kit</td><td>Invivogen</td><td>Cat# rep-mys-50</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>FBXO22-BACH1-BTB structure</td><td>This paper</td><td>PDB: 8UA3</td></tr><tr><td>SCF<sup>FBXO22</sup>-BACH1-BTB structure</td><td>This paper</td><td>PDB: 8UA6</td></tr><tr><td>Monomeric FBXL17-BACH1-BTB structure</td><td>This paper</td><td>PDB: 8UAH</td></tr><tr><td>Monomeric SCF<sup>FBXL17</sup>-BACH1-BTB structure</td><td>This paper</td><td>PDB: 8UBT</td></tr><tr><td>Dimeric SCF<sup>FBXL17</sup>-BACH1-BTB structure</td><td>This paper</td><td>PDB: 8UBU</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Dimeric FBXL17LRR-BACH1-BTB structure</td><td>This paper</td><td>PDB: 8UBV</td></tr><tr><td>EM map of FBXO22-BACH1-BTB</td><td>This paper</td><td>EMDB: EMD-42049</td></tr><tr><td>EM map of SCF<sup>FBXO22</sup>-BACH1-BTB</td><td>This paper</td><td>EMDB: EMD-42051</td></tr><tr><td>EM map of monomeric FBXL17-BACH1-BTB</td><td>This paper</td><td>EMDB: EMD-42064</td></tr><tr><td>EM map of monomeric SCF<sup>FBXL17</sup>-BACH1-BTB</td><td>This paper</td><td>EMDB: EMD-42102</td></tr><tr><td>EM map of dimeric SCF<sup>FBXL17</sup>-BACH1-BTB</td><td>This paper</td><td>EMDB: EMD-42105</td></tr><tr><td>close conformation</td><td></td><td></td></tr><tr><td>EM map of dimeric FBXL17LRR-BACH1-BTB</td><td>This paper</td><td>EMDB: EMD-42106</td></tr><tr><td>EM map of dimeric SCF<sup>FBXL17</sup>-BACH1-BTB open conformation</td><td>This paper</td><td>EMDB: EMD-42115</td></tr><tr><td>HDX-MS data</td><td>This paper</td><td>ProteomeXchange: PXD053923</td></tr><tr><td>Original images of western blot data</td><td>This study; Mendeley Data</td><td>https://dx.doi.org/10.17632/vf73rr8p5n.1</td></tr><tr><td>Proteomic data</td><td>This study</td><td>ProteomeXchange: PXD056513</td></tr></tbody></table>

Experimental models: Cell lines

<table><tr><td>HEK293T (female)</td><td>ATCC</td><td>Cat# CRL-3216; RRID: CVCL_0063</td></tr><tr><td>HCT-116 (male)</td><td>ATCC</td><td>Cat# CCL-247; RRID: CVCL_0291</td></tr><tr><td>FBXO22 <sup>−/−</sup> HCT-116 (male)</td><td>This paper</td><td>N/A</td></tr><tr><td>FBXL17 <sup>−/−</sup> HCT-116 (male)</td><td>This paper</td><td>N/A</td></tr><tr><td>FBXO22 <sup>−/−</sup>; FBXL17 <sup>−/−</sup> HCT-116 (male)</td><td>This paper</td><td>N/A</td></tr><tr><td>Spodoptera frugiperda Sf9</td><td>Life Technologies</td><td>Cat# B825-01</td></tr><tr><td>Trichoplusia ni Hi5</td><td>Life Technologies</td><td>Cat# B85502</td></tr></table>

Oligonucleotides

<table><tr><td>Full list of oligos</td><td>See Table S2</td><td>N/A</td></tr></table>

Recombinant DNA

<table><tr><td>pcDNA3.1 2xFLAG-2xSTREP-BACH1</td><td>Lignitto et al.<sup>30</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-BACH1</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP-BACH2</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP-FBXO22</td><td>Lignitto et al.<sup>30</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-FBXO22</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 FLAG-FBXL17</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-FBXL17</td><td>This paper</td><td>N/A</td></tr><tr><td>pSpCas9(BB)-2A-GFP (px458)</td><td>Addgene</td><td>Plasmid# 48138</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (D366A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (D366R)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (I368A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (N372A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (F373D)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (K377A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (K377D)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (L375A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP FBXO22 (L375D)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP-BACH1 (F9Y)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP-BACH1 (α6-BACH2)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 2xFLAG-2xSTREP-BACH2 (α6-BACH1)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-BACH1 (E12A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-BACH1 (C122E)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-BACH1 (F125A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-BACH1 (F125D)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-BACH1 (F128A)</td><td>This paper</td><td>N/A</td></tr></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>pcDNA3.1 HA-BACH1 (F128D)</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-BACH1 BTB (K129*), *: stop codon</td><td>This paper</td><td>N/A</td></tr><tr><td>pcDNA3.1 HA-FBXL17 ΔCTH (T675*), *: stop codon</td><td>This paper</td><td>N/A</td></tr><tr><td>pFB-GST-FBXO22</td><td>This paper</td><td>N/A</td></tr><tr><td>pFB-His-SKP1</td><td>This paper</td><td>N/A</td></tr><tr><td>pACE-His-MBP-FBXL17</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-GST-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-GST-Avi-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-Avi-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-Avi-BACH1-BTB (C34A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-Avi-BACH1-BTB (C55A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-Avi-BACH1-BTB (C107A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-Avi-BACH1-BTB (C109A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-Avi-BACH1-BTB (C122A)</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-Avi-BACH1-BTB (C122E)</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-His-Avi-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-His-Avi-BACH1-BTB (S17C/V20I/V32I/S50A/H60W/S61Q)</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-MBP-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-MBP-BACH1-BTB (S17C/V20I/V32I/S50A/H60W/S61Q)</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-GST-Avi-BACH2-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-GST-BACH2-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-BACH2-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-MBP-BACH2-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-MSB-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pACE-His-Venus-BACH1-FL</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-MSB-FLAG-BACH1-BTB</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-GST-MAF</td><td>This paper</td><td>N/A</td></tr><tr><td>pAL-His-MBP-BACH1-bZIP</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-GST-NCOR1</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-GST-GLMN</td><td>This paper</td><td>N/A</td></tr><tr><td>pET-His-RBX1-His-CUL1</td><td>This paper</td><td>N/A</td></tr></tbody></table>

Software and algorithms

<table><tr><td>ImageJ Software 2.0</td><td>NIH</td><td>https://imagej.net/software/imagej2/</td></tr><tr><td>GraphPad Prism v9.5.1</td><td>Graphpad Software</td><td>https://www.graphpad.com</td></tr><tr><td>MotionCor2</td><td>Zheng et al.<sup>72</sup></td><td>https://emcore.ucsf.edu/ucsf-software</td></tr><tr><td>AlphaFold-Multimer in Google ColabFold</td><td>Mirdita et al.<sup>73</sup></td><td>https://colab.research.google.com.github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb</td></tr><tr><td>UCSF Chimera</td><td>Pettersen et al.<sup>74</sup></td><td>https://www.cgl.ucsf.edu/chimera/</td></tr><tr><td>CryoSPARC</td><td>Structura Biotechnology Inc.</td><td>https://cryosparc.com</td></tr><tr><td>Coot</td><td>Emsley et al.<sup>75</sup></td><td>https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td></tr><tr><td>PHENIX</td><td>Adams et al.<sup>76</sup><br>Afonine et al.<sup>77</sup></td><td>https://phenix-online.org</td></tr><tr><td>PyMOL</td><td>Schrödinger</td><td>https://www.pymol.org</td></tr><tr><td>HDExaminer v3</td><td>Sierra Analytics</td><td>https://massspec.com/hdexaminer/</td></tr><tr><td>HXExpress v2</td><td>Guttman et al.<sup>78</sup></td><td>https://www.hxms.com/HXExpress/</td></tr></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Other</td><td></td><td></td></tr><tr><td>HiTrap Q HP</td><td>GE Healthcare</td><td>Cat# 17115401</td></tr><tr><td>Superdex 200 Increase 10/300 GL</td><td>GE Healthcare</td><td>Cat# 28990944</td></tr><tr><td>Quantifoil R1.2/1.3 200 mesh copper grid</td><td>Electron Microscopy Sciences</td><td>Cat# Q250-CR1.3</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Cell lines

Cell lines were purchased from ATCC. HCT-116 (male, ATCC CCL-247) cells were propagated in McCoy's 5A medium (Gibco). HEK293T (female, ATCC CRL-3216) cells were propagated in Dulbecco's modified Eagle's medium (DMEM) (Gibco). Tissue culture media were supplemented with 10% fetal bovine serum (FBS) (Corning Life Sciences) and 1% penicillin-streptomycin (Corning Life Sciences). Cells were maintained at 37 °C and 5% CO₂ in a humidified atmosphere. Cells were routinely monitored for *Mycoplasma* contamination using the MycoStrip™ - Mycoplasma Detection Kit (Invivogen). No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. Specific details about cell lines used are provided in the key resources table.

### Source organism

For DNA extraction, *E. coli* DH5α, NEB5α, and NEB stable was used. For bacmid production, *E. coli* DH10Bac was used. For baculovirus production and amplification, Sf9 insect cells were used. For protein expression, both *E. coli* BL21 (DE3) and Hi5 insect cells were used. *E. coli* grew at 37 °C in Luria-Bertani medium (Fisher). Protein expression was induced by 0.2-0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C overnight. Insect cells grew at 27 °C in Grace's Insect Medium (Gibco) supplemented with 10% fetal bovine serum (Cytiva) and 1% penicillin-streptomycin-glutamine (Gibco).

## METHOD DETAILS

### Protein preparation

Human FBXO22 (N-terminally GST-tagged) and SKP1 (N-terminally His-tagged) were co-expressed and purified from Hi5 insect cells using glutathione affinity chromatography followed by TEV cleavage to remove the tags if necessary. The tag-free FBXO22-SKP1 complex was further purified by anion exchange and size exclusion chromatography. The wild-type and mutant BACH1-BTB domain (amino acids 7-128) or BACH2-BTB domain (amino acids 9-131) constructs were expressed with an N-terminal His- or GST-tag in *E. coli* BL21. The full-length BACH1 protein was constructed with an N-terminal His-Venus tag and expressed in Hi5 insect cells. His or GST-tagged BACH1 or BACH2 was purified by Ni-NTA or glutathione resin with subsequent TEV protease treatment to remove the tags if necessary. BACH1 or BACH2 was further purified by anion exchange and/or size exclusion chromatography. To prepare biotin-tagged BACH1-BTB or BACH2-BTB, an additional Avi-tag was designed after the TEV cleavage site. After TEV cleavage of the His- or GST-tag, Avi-BACH1-BTB or Avi-BACH2-BTB was generated and subsequently biotinylated in a biotin ligase BirA-catalyzed biotinylation reaction. Biotinylated protein was further purified by size exclusion chromatography. To make the BACH1-BTB-BACH2-BTB heterodimer, BACH1-BTB and BACH2-BTB with different tags were co-expressed in *E. coli* BL21 and isolated by tandem purification. For example, MBP-tagged BACH2-BTB/MSB-tagged BACH1-BTB were purified by amylose resin (for MBP-tagged protein) and anion exchange (for MSB-tagged protein, eluted at high salt ~450 mM NaCl). In another case, GST-Avi-BACH1-BTB-His-BACH2-BTB were tandemly purified, biotinylated and TEV treated to generate the biotin-BACH1-BTB-tag-free BACH2-BTB heterodimer. The FBXL17-binding defective BACH1-BTB<sup>MT</sup> (S17C/V20I/V32I/S50A/H60W/S61Q) was used to prepare the BACH1-BTB<sup>WT</sup>-BACH1-BTB<sup>MT</sup> heterodimer. MBP-tagged BACH1-BTB<sup>MT</sup> and His-Avi-tagged BACH1-BTB<sup>WT</sup> were co-expressed in *E. coli* BL21, purified by Ni-NTA resin and subject to biotinylation. Amylose resin was subsequently used to purify the MBP-BACH1-BTB<sup>MT</sup>-His-biotin-BACH1-BTB<sup>WT</sup> heterodimer and remove extra biotin. The MBP-BACH1-BTB<sup>WT</sup>-His-biotin-BACH1-BTB<sup>WT</sup> heterodimer was made by the same method. GST-tagged NCOR1 was expressed in *E. coli* BL21 and purified by glutathione resin and size exclusion chromatography. GST-MAF (254-361) and MBP-BACH1-bZIP (510-658) were co-expressed in *E. coli* BL21 and subject to tandem affinity purification. GLMN with an N-terminal GST tag was expressed and purified from *E. coli* BL21 using glutathione affinity chromatography followed by TEV cleavage to remove the tag. Tag-free GLMN was further purified by anion exchange and size exclusion chromatography. RBX1 (amino acids 16-108) and CUL1 with two short unstructured segments (amino acids 1-12 and 58-81) truncated were constructed with an N-terminal His-tag followed by a TEV cleavage site. After co-expression in *E. coli* BL21(DE3), the CUL1-RBX1 complex was purified by a Ni-NTA affinity column followed by TEV cleavage to remove the tag. The tag-free CUL1-RBX1 was further purified by gel filtration. Separately purified BACH1-BTB, FBXO22-SKP1, CUL1-RBX1, and GLMN were mixed at the molar ratio of 3:1:1:1.5 with BACH1 and GLMN at extra to assemble and purify the GLMN-SCF<sup>FBXO22-BACH1</sup> complex on size exclusion chromatography for cryo-EM analysis. FBXL17 (amino acids 310-701) with an
N-terminal His-MBP-tag and SKP1 with an N-terminal His-tag were co-expressed and purified from Hi5 insect cells using Ni-NTA affinity chromatography followed by TEV cleavage to remove the tags if necessary. GST-BACH1-BTB without or with NOR3-S1PC treatment and FBXL17-SKP1 were incubated for 16 hours or at various times, respectively, and purified by a glutathione affinity column. Thereafter, CUL1-RBX1 and GLMN were loaded on the above column to form the GLMN- SCF<sup>FBXL17-BACH1-BTB</sup> complex. After on-column cleavage by TEV, the tag-free complex was collected in the flow-through of the glutathione affinity column and was further purified by size exclusion chromatography for cryo-EM analysis.

## Affinity pull-down assay

The GST, Ni²⁺, and biotin pull-down assays were performed using ~15-80 µg of purified GST-, His-, or biotin-tagged protein as the bait. Their binding partners were applied in excess. BACH1-BTB was pre-treated with the saturating amount of NOR3 and S1PC at 1.8 mM overnight at 4°C if indicated. Reaction mixtures with various incubation times were incubated with 20 µL glutathione, Ni²⁺ or Strep-Tactin beads at 4°C with gentle shaking. After extensive wash with the buffer containing 20 mM Tris, pH 8.0, 150 mM NaCl, 0.25 mM TCEP (additional 20 mM imidazole for the Ni²⁺ pull-down assay), the protein complexes on the beads were eluted by 40 µl 20 mM glutathione, 300 mM imidazole, or 50 mM biotin. The eluted samples were resolved by SDS-PAGE and analyzed by Coomassie staining.

## BioLayer interferometry

The binding affinity between FBXO22 (or FBXL17) and BACH1-BTB was measured using the Octet Red 96 (ForteBio, Pall Life Sciences) following the manufacturer's procedures. The optical probes were coated with streptavidin, loaded with 200 nM biotinylated BACH1-BTB (without or with NOR3-S1PC treatment), or anti-GST probes loaded with 200 nM GST-FBXO22-SKP1. Subsequently, the probes were quenched with 0.5 mM biocytin or 1 µM GST protein prior to kinetic binding analysis. The reactions were carried out in black 96 well plates maintained at 30°C. The reaction volume was 200 µL in each well. The binding buffer contained 25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM TCEP, 0.1% Tween-20, and 0.05 mg/mL Bovine Serum Albumin. As the analyte, BACH1-BTB or FBXL17-SKP1 was tested at various concentrations. There was no binding of the analyte to the unloaded probes. Binding kinetics of the analyte at different concentrations were measured simultaneously. The data were analyzed by the Octet data analysis software. The dissociation constant (K_d) was determined from the steady-state equilibrium measurements. All BLI experiments have been repeated at least 2-3 times.

## AlphaLISA

Amplified luminescent proximity homogeneous assays for measuring protein-protein interactions were performed using EnSpire reader (PerkinElmer). Biotinylated BACH1-BTB was immobilized to streptavidin-coated AlphaScreen donor beads. His-SKP1-FBXO22 was bound to anti-His acceptor beads. The acceptor and donor beads were brought into proximity by the interactions between the E3 and its substrate. Excitation of the donor beads by a laser beam of 680 nm promotes the formation of singlet oxygen. When an acceptor bead is in close proximity, the singlet oxygen reacts with thioxene derivatives in the acceptor beads and causes the emission of 520-620 nm photons, which are detected as the binding signal. If the beads are not in close proximity to each other, the oxygen will return to its ground state and the acceptor beads will not emit light. Competition assays were performed by titrating GST-tagged NCOR1 fragments or GST alone at various concentrations. To establish the binding signal, the concentration of tagged E3 and biotinylated BACH1-BTB were first titrated with fixed amount of donor and acceptor beads. The concentration of each component was selected based on the rising AlphaScreen signal before the beads became saturated. 25 nM His-SKP1-FBXO22 and 8.3 nM biotinylated BACH1-BTB were used. The binding assay buffer contained 25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM TCEP, 0.1% Tween-20, and 0.05 mg/mL Bovine Serum Albumin. 5 µg/mL donor and acceptor beads were used in the assays. The experiments were performed in three or four replicates. IC₅₀ was determined using non-linear curve fitting of the dose response curves generated with Prism (GraphPad).

## SEC-MALS

Size-exclusion chromatography coupled with light scattering, refractive index, and ultraviolet absorption (SEC-LS-RI-UV) was done using a SEC-MALS system consisting of an Agilent HPLC pump with a 1260 Infinity lamp, a Dawn light scattering instrument (Wyatt), an OptiLab refractive index instrument (Wyatt), and the Superdex 200 Increase 10/300 GL (GE Healthcare). The column was equilibrated with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.25 mM TCEP. Tag-free FBXO22-SKP1, His-BACH1-BTB and their mixture were analyzed at room temperature. Similarly, His-MBP-FBXL17-SKP1, NOR3-S1PC-treated His-BACH1-BTB and their mixture with different incubation times were analyzed.

## Differential scanning fluorimetry (DSF)

The Applied Biosystems StepOnePlus Real-Time PCR machine was used to perform the differential scanning fluorimetry thermostability assay. 5 µg BACH1 protein was pre-treated with the saturating amount of NOR3 and S1PC at 100 µM overnight at 4°C. For negative control, 5 µg BACH1 was treated with the same amount of the compound solvent (DMSO). 10 µl protein was mixed with 10 µl 0.25% Protein Thermal Shift™ Dye (Applied Biosystems) in a MicroAmp Fast 96-well reaction plate (0.1 ml). The temperature increment was 0.3 °C, recording fluorescence from 30 to 90°C. To compare WT BACH1-BTB and the BACH1-BTB C122E mutant,
<figure><img src="image_44.png" /></figure>

2.5 µg of each protein was mixed with Protein Thermal Shift™ Dye for analysis. The temperature increment was 0.3 °C, recording fluorescence from 35°C to 90°C. The data was analyzed by Protein Thermal Shift software 1.4. The melting temperature (Tm) was determined based on the peak of the first derivative of the fluorescence vs temperature plots. The experiments were performed in three replicates.

### In vitro ubiquitination assay

A reaction mixture containing 0.5-4 µM His-Venus-BACH1 (full length) or 1µM MSB-Flag-BACH1-BTB, 1-3 µM FBXO22-SKP1 or FBXL17-SKP1, 0.5-1 µM NEDD8~CUL1-RBX1, 0.5-1 µM E2(s) (UBCH5 alone or together with CDC34), 0.4 µM UBE1 and 50-100 µM ubiquitin and 2 mM ATP/10 mM MgCl₂ was incubated at 37 °C for 0.5-1.5 h with multiple controls, each lacking one component of the reaction. 100 µM NCOR1 was used and pre-mixed with BACH1 for 10 mins if indicated. To monitor the effect of NO treatment on ubiquitination, BACH1 was pre-treated with 1.8 mM NOR3 and S1PC. The reaction mixtures were resolved by a 10% SDS-PAGE gel. The BACH1 signal was monitored by detecting the Venus-fluorescence absorbance at 488 nm or anti-FLAG-HRP western blots. The absorbance at 647 nm was used to detect the protein markers.

### Electrophoretic mobility shift assay (EMSA)

1-3 µM GST-MAF or GST-MAF/MBP-BACH1-bZIP was incubated with 100 nM HS2-700 DNA probe (5'-5IRD700/AGCACAG CAGTGCTGAGTCATGCTGAGTCATGCTGAGGCTT-3') annealed with unlabeled complementary DNA strand in sub-stoichiometric amount. The MAF/BACH1 complex was pre-treated with NOR3 and S1PC if indicated. To overcome the effect of 250 µM TCEP used in the protein buffer, NOR3-S1PC was titrated at a concentration of 66 µM, 200 µM, 600 µM, and 1.8 mM. Samples were separated using 5% Mini-PROTEAN® TBE Gels (Bio-Rad) with running buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.2). The TBE gels were run in the cold room (4 °C). The absorbance at 680 nm was used to detect the DNA probes.

### Cryo-EM sample preparation and data collection

The GLMN-SCF<sup>FBXO22-BACH1-BTB</sup> complex and the GLMN-SCF<sup>FBXL17-BACH1-BTB</sup> complex (without or with NOR3-S1PC treatment with different incubation times) were concentrated to ~2.0 mg/ml for cryo-EM sample preparation. For each complex, ~3 µL sample was applied to a glow-discharged Quantifoil R1.2/1.3 200 mesh copper grid. The grid was subsequently blotted for ~3 s (blotting force at -2, temperature at 10 °C, and relative humidity at 100%), plunged and flash frozen into liquid ethane using a Vitrobot Mark IV system (Thermo Fisher Scientific), and stored in liquid nitrogen for data collection. The grids were inspected and screened with Talos Glacios equipped with a K3 camera. For the GLMN-SCF<sup>FBXO22-BACH1-BTB</sup> sample, data collection was carried out on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV at the University of Washington. The automation scheme was implemented using the Leginon software<sup>79</sup> at a nominal magnification of 105 K, resulting in a physical pixel size of 0.84 Å. The zero-loss-energy images were acquired on a Gatan K3 direct detector operated in super-resolution counting mode (pixel size: 0.42 Å) with the slit width of post-column Gatan BioQuantum GIF energy filter set to be 20 eV. The dose rate was adjusted to 13.9 e-/Å<sup>2</sup>S, and a total dose of 59 e-/Å<sup>2</sup> for each image fractionated into 50 frames. The images were recorded at a defocus range of -0.8~3.5 µm. For the chemical-treated or untreated GLMN-SCF<sup>FBXL17-BACH1-BTB</sup> complex, data collection was carried out on a 300 kV FEI Titan Krios transmission electron microscope operated at 300 kV at the HHMI Janelia Research Campus. The automated data collection was carried out with SerialEM software<sup>80</sup> using beam-image shift<sup>81</sup> at a nominal magnification of 165 K. The images were acquired on Falcon 4i direct electron detector operated in electron counting mode (pixel size: 0.743 Å) with the slit width of Selectris X (Thermo Fisher Scientific) energy filter set to be 6 eV. The dose rate was set to 15.39 e-/Å<sup>2</sup>S, and a total dose of 60 e-/Å<sup>2</sup> for each image fractionated into EER (Electron-event representation) frames.<sup>82</sup> The images were recorded at a defocus range of 0.8~1.5 µm.

### Image processing and 3D reconstruction

For the GLMN-SCF<sup>FBXO22-BACH1-BTB</sup> sample, 6,131 movies were acquired. The beam-induced motion of each micrograph stack was corrected by MotionCor2.<sup>72</sup> The motion-corrected micrographs were imported into CryoSPARC for the following processing.<sup>83</sup> The defocus parameter of each motion-corrected micrograph was determined by Gctf.<sup>84</sup> 4,991 micrographs were kept after filtering the micrographs with CTF parameters and visual inspection. An *ab-initio* reconstruction from a previous data collection was used to generate 2D projections as templates for particle picking. 2,888,035 particles were picked, extracted, and subjected to 2D classification. After two rounds of 2D classification, 243,712 particles were selected as training particles for Topaz particle picking.<sup>85</sup> 3,645,675 particles were picked with Topaz. After data cleaning by 2D classification, 949,569 particles were kept and subjected to *ab-initio* reconstruction. Subsequently, all the particles were used for heterogeneous refinement. 413,179 particles from good reconstruction were selected for non-uniform refinement<sup>86</sup> to generate a reconstruction with an overall resolution of 3.9 Å. To better resolve the interface between the substrate and its receptor, a soft mask focused on BACH1-BTB and FBXO22-SKP1 was applied to 3D Variability Analysis.<sup>87</sup> 171,378 particles were chosen for local refinement. The BACH1-BTB-FBXO22-SKP1 subcomplex was refined to 3.8 Å. More details about the data processing can be found in Figure S1.

For the native GLMN-SCF<sup>FBXL17-BACH1-BTB</sup> sample, 12,618 movies were collected. The beam-induced motion of micrographs was corrected by Patch Motion Correction in CryoSPARC. The defocus of motion-corrected micrographs was estimated by CTFFIND.<sup>88</sup> 12,423 micrographs were kept while the rest were rejected by CTF parameters. These micrographs were subjected to Blob particle
picker and two rounds of 2D classification sequentially. 497,102 out of 3,087,124 particles were picked for heterogeneous refinement using reconstructions from the previous dataset as templates. 267,523 good particles from the refinement were selected and further cleaned by 2D classification. 84,154 particles were chosen as training particles for Topaz particle picking. 7,309,475 particles were picked with Topaz and subjected to two rounds of 2D classification. 1,099,238 particles were selected for *ab-initio* reconstruction and heterogeneous refinement sequentially. 364,786 particles from one good class were applied to non-uniform refinement. The overall resolution of the complex in the monomeric form was refined to 3.1 Å. To improve the local density of FBXL17-BACH1-BTB, all particles were subjected to 3D Variability. 193,908 particles were selected for local refinement to generate the SKP1-FBXL17-BACH1-BTB subcomplex with a resolution of 3.2 Å. In addition to the above complex in the monomeric form, the dimeric GLMN-SCF<sup>FBXL17-BACH1-BTB</sup> complex was detected as well. 30,123 particles of the dimer were selected as training particles for Topaz particle picking. 244,189 out of 2,969,728 particles were chosen for heterogeneous refinement. Two good classes representing different conformations, named dSCF<sup>BTB-I</sup> and dSCF<sup>BTB-II</sup>, were kept with 54,546 particles and 47,336 particles, respectively. The C2 symmetry was imposed for non-uniform refinement. The overall resolution of the two classes reached 4.4 Å and 4.5 Å, respectively. To overcome the pseudo-symmetry of the dimeric reconstructions, all particles of dSCF<sup>BTB-I</sup> were subjected to symmetry expansion in CryoSPARC. Afterward, a soft mask focused on the leucine-rich repeat domain of FBXL17 and BACH1-BTB was applied to subtract the signal from raw particles. These subtracted particles were used for local refinement. The density of the FBXL17-LRR-BACH1-BTB structure was refined to 4.1 Å. See Figure S7 for more details.

For the compound-treated GLMN-SCF<sup>FBXL17-BACH1-BTB</sup> sample, the collected data was processed with CryoSPARC in a similar manner. The data processing workflow was shown in Figure S6. Briefly, 22,720 EER movies were collected and subjected to patch motion correction and patch CTF estimation sequentially. After filtering with CTF parameters, 20,028 micrographs were kept for the subsequent blob picker and 2D classification. Representative particles were selected as the training data for Topaz particle picking. After heterogeneous refinement, a monomeric SCF<sup>FBXL17-BACH1</sup> class yielded a 3D reconstruction at 5.7 Å resolution. A dimeric SCF<sup>FBXL17-BACH1</sup> with 461 particles was selected and employed for Topaz particle picking. After 2D classification and heterogeneous refinement, two good classes of dimeric SCF<sup>FBXL17-BACH1</sup> were kept. 10,173 particles and 7,944 particles from dimeric reconstructions were subjected to non-uniform refinement with C2 symmetry imposed. After refinement, the overall resolution of two classes both reached 7.8 Å.

## Model building and refinement

The initial structural model of FBXO22-SKP1-BACH1-BTB was predicted with AlphaFold-Multimer in Google ColabFold<sup>73</sup> and fitted into the 3.8 Å Cryo-EM map by using UCSF Chimera.<sup>74</sup> Subsequently, the model was inspected and trimmed in Coot<sup>75</sup> based on the protein sequences and the EM density. The model was further improved by real-space refinement in PHENIX<sup>76,77</sup> and manual rebuilding in Coot. To generate an initial model of the monomeric SCF<sup>FBXL17-BACH1-BTB</sup> complex, BACH1-BTB (PDB: 2IHC) was applied to superimpose with and replace the KEAP1-BTB subunit of the SCF<sup>FBXL17-KEAP1-BTB</sup> complex (PDB: 6WCQ) in PyMOL. This model was fitted into the 3.2 Å cryo-EM map using UCSF Chimera. The BACH1-BTB domain was manually adjusted to fit the density in Coot. The model was further improved by using real-space refinement in PHENIX and manual rebuilding in Coot. For the SCF<sup>FBXL17-BACH1</sup> dimer, two copies of the monomeric complexes were fitted into the 4.4 Å cryo-EM maps and subsequently trimmed and adjusted in Coot. The dimeric interface of BACH1-BTB in the SCF complex was rebuilt in Coot since it is completely different from that of the isolated BACH1-BTB dimer. The model of dimeric FBXL17-BACH1-BTB was refined in PHENIX by real-space refinement. Cryo-EM data collection and refinement statistics for all structures in this paper were summarized in Table S1.

## Plasmids and antibodies

*Homo sapiens* BACH1, FBXO22, and FBXL17 cDNAs were amplified by PCR using Q5 High Fidelity DNA Polymerase (New England Biolabs) and sub-cloned into modified pcDNA3.1 vectors containing a variety of N-terminal tags, including 2xFLAG tag-2x Strep tag and HA tag. Specific details will be provided upon request. Site-directed mutagenesis was performed using Q5 High-Fidelity DNA polymerase (New England Biolabs) with primers generated using either QuikChange Primer Design (Agilent) or NEBaseChanger (New England Biolabs) and were described in Table S2. Plasmids and antibodies used in the paper were listed in the key resources table.

## Drug Treatment Procedures

Where indicated, cells were treated with 2 µM MLN-4924 (Active Biochem) for 4 hours or 10 µM MG-132 (Peptides International) for 4 hours, spermine NONOate (sNONO) (Cayman Biochem) was prepared in 0.01N NaOH and used at 100 µM for the indicated times. S-nitrosoglutathione (GSNO) (Sigma-Aldrich) was prepared in DMSO and used at 100 µM for the times indicated. S-1-propenyl-L-cysteine (S1PC) (MedChemExpress) was prepared in DMSO and used at 25 µM for the times indicated. Cycloheximide (Sigma-Aldrich) was prepared in DMSO and used at 100 µM for the times indicated.

## Co-immunoprecipitation assay

HEK293T cells were transiently transfected using polyethyleneimine (Polysciences); HCT-116 cells were transiently transfected with Lipofectamine 3000 (Thermo Fisher Scientific). Where indicated, 48 h after transfection, cells were treated with 2.5 µM MLN-4924 for 4 h before further treatment with either vehicle (DMSO) or the indicated NO donor. Cells were collected by scraping and washed with
PBS prior to lysis in IP lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl₂, 0.2% NP-40) supplemented with protease inhibitor (Complete ULTRA, Roche) and phosphatase inhibitor (Phosphatase Inhibitor Cocktail 2, Sigma-Aldrich) immediately before the experiment. The supernatant was clarified by centrifugation at 21, 000 x g for 10 minutes at 4°C. For FLAG-tagged proteins, the supernatant was incubated with FLAG-M2 agarose beads (Sigma-Aldrich) for 2h at 4°C. For HA-tagged proteins, the supernatant was incubated with Pierce Anti-HA magnetic beads (Thermo Fisher Scientific) for 2h at 4°C. The beads were then washed 5 times in lysis buffer before incubating the beads in NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) supplemented with β-mercaptoethanol (Sigma-Aldrich) at 95°C for 10 minutes. For Western blotting, samples of the whole cell extract and immunoprecipitates were separated by SDS-PAGE and transferred onto 0.45 µm Immobilon-P PVDF membranes (Millipore Sigma). Membranes were then blocked in 5% nonfat dried milk / PBST for 30 minutes at room temperature and incubated with the indicated primary antibodies at 4°C overnight. After washing the membranes, secondary antibodies conjugated with horse-radish peroxidase were applied. Immunoreactive bands were visualized via application of enhanced chemiluminescence (ECL) re-agent (Thermo Fisher Scientific). Each Western blot was performed no less than three times to ensure results exhibit a high degree of reproducibility. Representative Western blots have been selected for each figure.

## Cell-based protein stability assay

HCT-116 cells were treated with 100 µM spermine NONOate (sNONO) with or without 100 µg/mL cycloheximide (CHX) for the times indicated. Cells were then washed with ice-cold PBS supplemented with 100 µM phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich) before they were collected by scraping. Cell pellets were lysed in RIPA buffer (Pierce) supplemented with protease inhibitor (Complete ULTRA, Roche) and phosphatase inhibitor (Phosphatase Inhibitor Cocktail 2, Sigma-Aldrich) immediately before the experiment. The supernatant was clarified by centrifugation at 21, 000 x g for 10 minutes at 4°C. Protein concentrations in cell lysates were then normalized with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturers' instructions. Samples of the cell lysates were then prepared with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) supplemented with β-mercaptoethanol (Sigma-Aldrich) and changes in protein levels were visualized via Western blot. All experiments were performed at least 3 times. Quantification of Western blots were done using ImageJ and graphs were plotted using Prism 9 (Graphpad).

## CRISPR-Cas9 genome editing

To generate FBXO22 knockout cells, optimal single guide RNA (sgRNA) target sequences were designed using the Benchling CRISPR Genome Engineering tool (https://www.benchling.com). FBXO22 gRNA (CTTCGTGTTGAGTAACCTGG) was cloned into pSpCas9(BB)-2A-GFP (px458), a gift from F. Zhang (Addgene plasmid #48138), using the following primers: CACCGCTTC GTGTTGAGTAACCTGG and aaacCCAGGTTACTCAACACGAAGC. To generate the FBXL17 knockout and the FBXL17 / FBXO22 double knockout cells, sgRNA targeting FBXL17 (AAAACUGGAAGUCUAAACAA) designed by Synthego was used. Briefly, 5x10⁶ HCT-116 cells were seeded into a 15 cm dish before being transfected with 10 µg sgRNA-containing px458 plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). 48h after transfection, the top 5% of GFP-positive cells were sorted using the Beckman Coulter MoFlo XDP cell sorter (100 µm nozzle). 10 000 cells were plated between two 15 cm dishes (5000 cells per plate) and grown for about 10-14 days to allow single-cell clones to form colonies. The colonies were then picked and plated into individual wells of a 96-well plate. After 48 hours, the 96-well plate were split into two new 96-well plates, with one plate used for genotyping. For genotyping, genomic DNA was extracted using QuickExtract (Epicentre) and genotyping PCRs were performed with MyTaq HS Red Mix (Bioline) using the following genotyping primers for FBXO22: GCAACCCTATGCTGGCGTAA and AGATACCTCGCGGGTAGGG; and for FBXL17: GCTTTACCTTGAAATCACATACACA and AGGTGATCTCTTACGGTGACT. The resulting PCR products were then purified and sequenced to determine the presence of a CRISPR-mediated frameshift event that results in an early stop codon. Positive knockout candidates were further validated by Western blot. Five homozygous knockout clones were then pooled together to mitigate any clonal effects, and this collection of pooled knockout cells were propagated and subsequently used for experiments.

## Chromatin Fractionation Assay

Cells were treated with DMSO, 10 µM hemin, or 100 µM sNONO + 25 µM S1PC for 1h before being washed once with ice-cold PBS with PMSF, and then harvested by scraping. The cell pellet is then resuspended in CSK buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM EGTA, 0.5% Triton X-100, 1 mM DTT, phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and complete ULTRA EDTA-free protease inhibitor cocktail (Roche)). The CSK suspension is incubated on ice for 5 minutes before being spun down at 1300xg for 3 minutes at 4°C. The supernatant is transferred to a new tube ("soluble" fraction). The pellet is washed once with CSK buffer, spun down at 1300xg for 3 minutes at 4°C, before the supernatant is discarded. The pellet is then resuspended in chromatin lysis buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 2 mM MgCl₂, 2500 U benzonase nuclease (Sigma-Aldrich), phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and complete ULTRA EDTA-free protease inhibitor cocktail (Roche)) by vortexing and incubated at 4°C for 15 minutes before sonicating the sample at 4°C for 10 minutes. After sonication, the sample is then incubated once more at 4°C for 10 more minutes before the suspension is spun down at 21000xg for 10 minutes at 4°C. The supernatant is transferred to a new tube ("chromatin" fraction). The protein concentrations are normalized using the Pierce BCA Protein Assay kit (Thermo Scientific) before immunoblot for analysis.
## Biotin Switch Assay

Cells were treated with either 100 µM GSNO or 100 µM sNONO for 1h prior to collection. Cells were then subjected to the S-nitrosylation Protein Detection Kit (Cayman Chemical), according to the manufacturers' instructions. Briefly, free reactive thiol groups were blocked with N-ethylmaleimide (NEM) for 30 minutes at room temperature, then excess NEM was removed via acetone precipitation. Any S-nitrosylated cysteines were subsequently reduced and labelled with a sulfhydryl-specific biotin. Excess reducing and labelling were removed via acetone precipitation. The final protein pellet was then resuspended in IP lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl₂, 0.2% NP-40, 1 mM DTT) supplemented with protease inhibitor (Complete ULTRA, Roche) and phosphatase inhibitor (Phosphatase Inhibitor Cocktail 2, Sigma-Aldrich). The resuspended protein was incubated with streptavidin Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific) for 2h at 4°C before the beads were washed 4 times with IP lysis buffer. Samples of the resuspended protein and the streptavidin bead-bound protein were prepared in NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) supplemented with β-mercaptoethanol (Sigma-Aldrich) and proteins were assessed via Western blot.

## S-nitrosylation site mapping

10 µg of recombinantly purified BACH1 BTB was allowed to react in the presence of 100 µM NOR3 and 100 µM S1PC for 30 minutes at room temperature before excessing nitric oxide donor was removed by Zeba desalting column pre-equilibrated with 50 mM Tris-HCl (pH 8.0), 150 mM NaCl. Nitrosylated protein was subsequently treated with the S-nitrosylation Protein Detection Kit (Cayman Chemical), according to the manufacturers' instructions. Biotin switch-treated protein reduced with 2.5 µL of 0.2 M dithiothreitol (Sigma) for 1 hr at 57°C and the samples cooled before adding 2.5 µL of 0.5M iodoacetamide and incubated in the dark for 45 minutes. The sample was digested with 400 ng of trypsin overnight at room temperature and light agitation. The digestion was stopped by adding 10% trifluoroacetic acid (TFA) to a final concentration of 0.5% TFA. The resulting peptides were desalted using equilibrated Pierce™C18 Spin columns. The samples were washed 3 times with 0.1% TFA followed by a wash with 0.5% acetic acid. The desalted peptides were eluted using 80% acetonitrile in 0.5% acetic acid. The peptides were dried using a SpeedVac concentrator. Samples were reconstituted in 0.5% acetic acid and stored at -80°C until further analysis. 40 pmol of each sample was loaded onto an Acclaim PepMap trap column (2 cm x 75 µm) in line with an EASY-Spray analytical column (50 cm x 75 µm ID PepMap C18, 2 µm bead size) using the autosampler of an EASY-nLC 1200 HPLC (Thermo Fisher Scientific). Solvent A consists of 2% acetonitrile in 0.5% acetic acid, and Solvent B of 80% acetonitrile in 0.5% acetic acid. Peptides were gradient eluted with a flowrate of 200nL/min into an Orbitrap Eclipse Tribrid (Thermo Fisher Scientific) mass spectrometer. The gradient used was the following: in 5 min to 5% solvent B, 60 min to 35% solvent B, 10 min to 45% solvent B, and 10 min to 100% solvent B. High resolution full MS spectra were acquired with a resolution of 120,000, an AGC target of 4e5, with a maximum ion time of 50 ms, and a scan range of 400 to 1500 m/z. All HCD MS/MS spectra were collected with the following instrument parameters: resolution of 30,000, AGC target of 2e5, maximum ion of 200ms, one microscan, 2 m/z isolation window, and NCE of 27. The resulting MS/MS spectra were searched against BACH1 BTB and common contaminants using the search engine Byonic (Protein Metrics). Settings were as following: trypsin cleavage allowing for 2 missed cleavages, precursor, and fragment ion mass tolerance of 10 ppm, variable modifications of carbamidomethyl, N-ethylmaleimide, and 3-(N-Maleimidopropionyl)-biocytin on cysteine, deamidation on asparagine and glutamine, and oxidation on methionine. Spectra identified as containing modified cysteine residues were manually verified.

## HDX-MS analyses

Stock concentrations of BACH1 (unmodified or NO-treated, 0.2 mg/mL) in Tris buffer (20 mM tris, 150 mM NaCl, 0.25 mM TCEP, pH 8.0) were diluted into 90 µL of deuterated PBS buffer (20 mM phosphate, 150 mM NaCl, 0.02% sodium azide, 1 mM EDTA pH* 7.77, 85%D final) containing 0.2 nM bradykinin and incubated for either 3 seconds, 1 minute, 30 minutes, or 20 hours at 21°C. Each starting stock also included a mixture of imidazolium compounds to serve as exchange reference standards.⁸⁹ At the desired time point the sample was rapidly mixed with an equal volume of ice cold 0.2% formic acid and 0.1% trifluoroacetic acid (TFA) for a final pH of 2.5. Samples were then immediately frozen on ethanol/dry ice and stored at -80°C until LC-MS analysis. Undeuterated samples were prepared the same way but with undeuterated buffer for each step. Maximally deuterated samples were prepared by collecting the Nepenthesin II digested peptides after they were trapped and resolved as described below. These samples were dried down, resuspended in 10 µL of undeuterated buffer, incubated in deuterated PBS for 2 hours at 21°C, then quenched and frozen as described above. Some late-eluting peptides were outside of the collection window and therefore are absent in the maximally deuterated data.

Samples were thawed at 5°C for 4.5 minutes and injected using a custom LEAP robot integrated with an LC-MS system.⁹⁰ The protein was first passed over a Nepenthesin II column (2.1 x 30 mm; AffiPro) at 400 µL/min for inline digestion with the protease column held at 20°C. Peptides were then trapped on a Waters XSelect CSH C18 trap cartridge column (2.1 x 5 mm 2.5 µm) and resolved over a CSH C18 column (1 x 50 mm 1.7 µm 130Å) using linear gradient of 5 to 35% B (A: 0.1% FA, 0.025% TFA, 5% ACN; B: ACN with 0.1% FA) over 10 minutes and analyzed on a Thermo Orbitrap Ascend mass spectrometer at a resolution setting of 120,000. A series of washes over the trap and Nepenthesin II columns was used between injections to minimize carry-over.⁹⁰ Data dependent MS/MS acquisition was performed on an undeuterated sample of either condition using rapid CID and HCD scans and processed in Byonic (Protein Metrics) with a score cutoff of 150 to identify peptides. S-nitrosylation and cysteine mono- and di-oxidation were included as potential variable modifications. Deuterium incorporation was analyzed using HDExaminer v3 (Sierra Analytics). Peptide spectra
were exported from HDExaminer and screened for multimodal mass envelopes with PyHXExpress (https://github.com/tuttlelm/pyHXExpress). Peptides of interest were then analyzed with HXExpress.⁷⁸ Data are available via ProteomeXchange with identifier PXD053923.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Resolution estimations of cryo-EM density maps are based on the Fourier Shell Correlation (FSC) curves at the gold-standard threshold of 0.143. Protein quantification was done using Bio-rad Protein Assay Dye and comparing readings against a standard curve of BSA. Quantification of protein bands in Western blots and SDS-PAGE gels were done using ImageJ. Data were analyzed and plotted by Prism (GraphPad). Unless otherwise noted, data were presented as mean ± SD of n = 3 biologically independent samples.
Supplemental figures

<figure><img src="image_45.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_46.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_49.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Figure S1. The schematic workflow of single-particle reconstruction of the SCF<sup>FBXO22-BACH1</sup> complex, related to Figure 1</figcaption></figure>

(A) A representative cryo-EM micrograph.

(B) Typical 2D averages of the cryo-EM dataset. Scale bar, 10 nm.

(legend continued on next page)
<figure><img src="image_52.png" /></figure>

CellPress

OPEN ACCESS

Cell

Article

(C) The flowchart of single particle analysis of the SCF<sup>FBXO22-BACH1</sup> complex.

(D) The angular distribution of particles used in the final reconstruction.

(E) Fourier shell correlation (FSC) curves for SCF<sup>FBXO22-BACH1</sup>. At the gold-standard threshold of 0.143, the resolution is 3.9 Å.

(F) Representative density in local refined EM map.
<figure><img src="image_53.png" /><figcaption>A</figcaption></figure>

FIST-1

FIST-2

<figure><img src="image_54.png" /></figure>

FIST-3

FIST-3

<figure><img src="image_55.png" /></figure>

β15
β16

YabJ

<figure><img src="image_56.png" /><figcaption>B</figcaption></figure>

FBXO22

<figure><img src="image_57.png" /></figure>

FBXO22
模化体

BACH1-BTB(A)

BACH1-BTB(B)

<figure><img src="image_58.png" /><figcaption>C</figcaption></figure>

Ubiquitination:

E1, E2, ATP, Ubiquitin

<figure><img src="image_59.png" /><figcaption>D</figcaption></figure>

Ubiquitination:

E1, E2, ATP, Ubiquitin

Nedd8~CUL1RBX1

+ + - + FBXO22-SKP1

+ - + + Flag-BACH1-BTB

- + + Ubiquitin

(Ub)$_{n}$~MSB-Flag-BACH1-BTB

<figure><img src="image_60.png" /></figure>

WB: Anti-Flag

MSB-Flag-BACH1-BTB

37 °C, 1.5 h

<figure><img src="image_61.png" /><figcaption>E</figcaption></figure>

BACH1-BTB(B)

BACH1-BTB(A)

<figure><img src="image_62.png" /><figcaption>F</figcaption></figure>
Figure S2. Structural and biochemical analysis of FBXO22-BACH1 interaction, related to Figures 1 and 2

(A) A comparison of the three FIST domains of FBXO22 and homologous structure of YabJ from the YigF superfamily (PDB: 1QD9).

(B) 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.

(C and D) *In vitro* ubiquitination of BACH1 and BACH1-BTB domain by SCF<sup>FBXO22</sup>.

(E) 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.

(F) 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><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>

FBXO22
wild type

representative
FBXO22
knock-out

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

FBXL17
wild type

representative
FBXL17
knock-out

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /></figure>

<figure><img src="image_74.png" /></figure>

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>
Figure S3. Nonredundant roles of FBXO22 and FBXL17 in regulating BACH1 stability, related to Figures 3 and 4

(A and B) Characterization of FBXO22 KO, FBXL17 KO, and FBXL17/FBXO22 double KO cell lines.

(C and D) Cycloheximide (CHX) chase assays monitoring the stability of BACH1 in parental, FBXL17 KO, FBXO22 KO, and FBXL17/FBXO22 double KO HCT-116 cells.

(E) Quantification of the data in (D) with each data point representing mean densities of BACH1 relative to untreated at 0 h. Data are presented as mean ± SD.

(F and G) BACH1 CHX chase assays performed with and without sNONO treatment. The data were used to create the graph in Figure 3D.

(H) An MS/MS fragmentation spectrum assigned to the S-nitrosylated peptide from BACH1-BTB containing Cys107 together with the assignments of the fragmentation series to the sequence of the modified peptide. The B and Y ions represent the two halves of the peptide formed by splitting the original peptides between different amino acids. The split sites are indicated by the red and blue “L”-shaped marks lying above and below the peptide sequence. The B ions are the products when the charge is retained on the N terminus, and the Y ions are the product when the charge is retained at the C terminus. The peaks of the products are labeled accordingly within the spectrum. The measured m/z value of the y2 ion is consistent with the modification of Cys107 by 3-(N-maleimidopropionyl)-biocytin with a monoisotopic mass of 523 Da.

(I) SEC-MALS analyses of BACH1-BTB treated with NOR3-S1PC with experimentally determined molecular weight. The theoretical molecular weight of BACH1-BTB is 17 kDa for monomer or 34 kDa for dimer.

(J) Inhibition of *in vitro* SCF<sup>FBXO22</sup>-catalyzed BACH1 ubiquitination by NO treatment.

(K) *In vitro* ubiquitination of NO-treated BACH1 by SCF<sup>FBXL17</sup>.

(L) NO treatment abrogates the DNA-binding activity of BACH1 bZIP but not sMAF as detected by electrophoresis mobility shift assay (EMSA).
<figure><img src="image_77.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_80.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_82.png" /></figure>
Figure S4. Analysis of non-treated and NO-treated BACH1-BTB, related to Figures 4 and 5

(A) BLI measurements of the binding between FBXL17 and NOR3-S1PC-treated BACH1-BTB with a 10-min association step. K<sub>d</sub>, dissociation constant.

(B and C) BLI measurements of the interaction between FBXL17 and BACH1-BTB (WT 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 and E) Heat map summary of HDX data for unmodified (D) and NO-treated (E) BACH1-BTB. Colors under the primary sequence show deuterium uptake from <10% (blue) to >90% (red) for each time point. Bars above the protein sequence show peptide coverage; green bars represent high-confidence peptides, yellow bars represent medium-confidence peptides as assigned in HDExaminer v3. Data for the NO-treated sample reflect the population of peptides containing unmodified cysteines.

(F) Bimodal and unimodal hydrogen-deuterium exchange profiles of additional BACH1-BTB peptides. As observed with peptides 79–89, both unmodified and NO-modified BACH1 exhibit bimodal exchange profiles at peptides 22–30 and 45–52. With the NO modification, the less-protected population (green) appears much earlier and is the predominant population by the longest (20 h) time point. Note for peptides 45–52: the 3 and 60 s NO-modified time points do not pass the F-test but have been plotted as two populations due to the presence of statistically significant bimodal fits for the 30 min and 20 h time points. The peptide-based maximally deuterated peptides 45–52 control for the unmodified condition eluted late and were not collected for preparation of the maximally deuterated standard. Peptides 59–72 appear unimodal with either condition throughout the HDX time course. Importantly, the maximally deuterated controls do not show any bimodal profiles, confirming that the bimodality is not a spectral artifact.
<figure><img src="image_83.png" /><figcaption>A</figcaption></figure>

¹¹³LSVHNEESC¹²²

<figure><img src="image_84.png" /><figcaption>B</figcaption></figure>

⁹⁷ILSKENVDEVCK¹⁰⁸

<figure><img src="image_85.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_86.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_87.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_88.png" /></figure>

+ - Biotin-BACH1-BTB<sup>C122E</sup>
+ + Biotin-BACH1-BTB<sup>WT</sup>
+ + GST-FBXO22-SKP1

GST-FBXO22

SKP1

Biotin-BACH1-
BTB<sup>WT/C122E</sup>

Biotin pull down

<figure><img src="image_89.png" /><figcaption>H</figcaption></figure>

HEK293T

FLAG-BACH1

CHX (h)

BACH1

(α-FLAG)

cyclin D1

actin

<figure><img src="image_90.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_91.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_92.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_93.png" /></figure>

HEK293T

IP: α-FLAG

HA-FBXL17

FBXL17
(α-HA)

F-box panel
(α-FLAG)
Figure S5. Analysis of NO-treated BACH1-BTB and C122E mutant, related to Figure 5

(A and B) Peptide mapping experiments to identify and quantify levels of S-nitrosylation. Extracted ion chromatograms are shown for the monoisotopic peak of the 2+ charge state of peptide LSVHNIEESC and ILSKENVDEVCK as unmodified (blue), nitrosylated (+29, orange), and deoxidized (+32, purple). For LSVHNIEESC, an additional species that is trioxidized (+48, cyan) was detected and is also shown. The traces are shown for both the treated protein and the untreated sample. * The signal at the unmodified m/z corresponds to some in-source loss of the nitroso group. ** The third isotopic peak from the nitrosylated (+29) peptide overlaps with the deoxidized (+32) m/z peak. *** Unrelated peptide that is present in both treated and untreated samples.

(C) The annotated MS/MS spectra identifying unmodified and nitrosylated peptides extracted from Byonic (Protein Metrics Inc). Positions of peptide fragmentation resulting in b/y ions are shown on each sequence in the inset.

(D) Thermostability of WT BACH1-BTB and the C122E mutant determined by differential scanning fluorimetry. T_m is melting temperature and is presented as mean ± SD.

(E) Affinity pull-down assay showing the loss of FBXO22 binding in BACH1-BTB C122E mutant.

(F) The time-courses of the interactions between FBXL17 and WT BACH1-BTB vs. C122E mutant as assessed by affinity pull-down assay.

(G) Co-immunoprecipitation analysis of WT BACH1 vs. C122E mutant for FBXO22 and FBXL17 binding.

(H) CHX chase assay assessing the stability of FLAG-tagged WT BACH1 vs. C122E mutant.

(I) FLAG co-immunoprecipitation assay detecting physical association between two differentially tagged FBXL17. Three unrelated F-box proteins (FBXL1, FBXL11, and FBXL15) were included as negative controls.
<figure><img src="image_94.png" /></figure>

<figure><img src="image_95.png" /></figure>

<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /><figcaption>Figure S6. Schematic workflow of single-particle reconstruction of NO-treated SCF<sup>FBXL17-BACH1</sup> complex, related to Figure 6</figcaption></figure>

(A) A representative cryo-EM micrograph for the sample containing a mixture of SCF<sup>FBXL17</sup> and BACH1-BTB treated with NOR3-S1PC.

(B) Typical 2D averages.

(C) The flowchart of single particle analysis of the sample containing a mixture of SCF<sup>FBXL17</sup> and BACH1-BTB treated with NOR3-S1PC.

(D and E) Fitting of the WT dSCF<sup>FBXL17-BACH1-I</sup> and dSCF<sup>FBXL17-BACH1-II</sup> structures in the 3D reconstructions obtained with the NO-treated BACH1-BTB sample.
<figure><img src="image_98.png" /><figcaption>Figure S7. Cryo-EM single-particle analysis workflow of native SCF<sup>FBXL17-BACH1</sup>, related to Figure 6</figcaption></figure>

(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<sup>FBXL17</sup> and BACH1-BTB.

(D) Local resolution map of the BACH1-BTB dimer flanked by the LLR domains of two FBXL17 ranging from 3 to 8 Å.

(E) The particle angular distribution and FSC curves of dSCF<sup>FBXL17-BACH1-I</sup> with local refinement.

(F) The particle angular distribution and FSC curves of dSCF<sup>FBXL17-BACH1-II</sup>.

(G) The angular distribution and FSC curves of the monomeric SCF<sup>FBXL17-BACH1</sup> complex.