The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP-importin α interactions.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Marked and rapid effects of pharmacological HIF-2α antagonism on hypoxic ventilatory control.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer, and there is great interest in applying inhibitors of HIF as anticancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors affect physiological responses to hypoxia at relevant doses. Here, we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumor growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatization and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerization with HIF-1ß, did not respond to PT2385, indicating that these effects are on-target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerization with HIF-1ß. Although PT2385 was well tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

Safety, hemodynamic effects, and detection of acute xenon inhalation: rationale for banning xenon from sport.

This study aimed to quantify the sedative effects, detection rates, and cardiovascular responses to xenon. On 3 occasions, participants breathed xenon (FiXe 30% for 20 min; FiXe 50% for 5 min; FiXe 70% for 2 min) in a nonblinded design. Sedation was monitored by a board-certified anesthesiologist. During 70% xenon, participants were also verbally instructed to operate a manual value with time-to-task failure being recorded. Beat-by-beat hemodynamics were measured continuously by ECG, photoplethysmography, and transcranial Doppler. Over 48 h postadministration, xenon was measured in blood and urine by gas chromatography-mass spectrometry. Xenon caused variable levels of sedation and restlessness. Task failure of the self-operating value occurred at 60-90 s in most individuals. Over the first minute, 50% and 70% xenon caused a substantial reduction in total peripheral resistance (P < 0.05). All dosages caused an increase in cardiac output (P < 0.05). By the end of xenon inhalation, slight hypertension was observed after all three doses (P < 0.05), with an increase in middle cerebral artery velocity (P < 0.05). Xenon was consistently detected, albeit in trace amounts, up to 3 h after all three doses of xenon inhalation in blood and urine with variable results thereafter. Xenon inhalation caused sedation incompatible with self-operation of a breathing apparatus, thus causing a potential life-threatening condition in the absence of an anesthesiologist. Yet, xenon can only be reliably detected in blood and urine up to 3 h postacute dosing.NEW & NOTEWORTHY Breathing xenon in dosages conceivable for doping purposes (FiXe 30% for 20 min; FiXe 50% for 5 min; FiXe 70% for 2 min) causes an initial rapid fall in total peripheral resistance with tachycardia and thereafter a mild hypertension with elevated middle cerebral artery velocity. These dose duration intervals cause sedation that is incompatible with operating a breathing apparatus and can only be detected in blood and urine samples with a high probability for up to ~3 h.

Effect of acute and chronic xenon inhalation on erythropoietin, hematological parameters, and athletic performance.

This study aimed to assess the efficacy of acute subanesthetic dosages of xenon inhalation to cause erythropoiesis and determine the effect of chronic xenon dosing on hematological parameters and athletic performance. To assess the acute effects, seven subjects breathed three subanesthetic concentrations of xenon: 30% fraction of inspired xenon (FiXe) for 20 min, 50% FiXe for 5 min, and 70% FiXe for 2 min. Erythropoietin (EPO) was measured at baseline, during, and after xenon inhalation. To determine the chronic effects, eight subjects breathed 70% FiXe for 2 min on 7 consecutive days, and EPO, total blood, and plasma volume were measured. Phase II involved assessment of 12 subjects for EPO, total blood volume, maximal oxygen uptake, and 3-km time before and after random assignment to 4 wk of xenon or sham gas inhalation. FiXe 50% and 70% stimulated an increase in EPO at 6 h [+2.3 mIU/mL; 95% confidence interval (CI) 0.1-4.5; P = 0.038] and at 192 h postinhalation (+2.9 mIU/mL; 95% CI 0.6-5.1; P = 0.017), respectively. Seven consecutive days of dosing significantly elevated plasma volume (+491 mL; 95% CI 194-789; P = 0.002). Phase II showed no significant effect on EPO, hemoglobin mass, plasma volume, maximal oxygen uptake, or 3-km time. Acute exposure to subanesthetic doses of xenon caused a consistent increase in EPO, and 7 consecutive days of xenon inhalation significantly expanded plasma volume. However, this physiological response appeared to be transient, and 4 wk of xenon inhalation did not stimulate increases in plasma volume or erythropoiesis, leaving cardiorespiratory fitness and athletic performance unchanged.NEW & NOTEWORTHY This is the first study to examine each element of the cascade by which xenon inhalation is purported to take effect, starting with measurement of the hypoxia-inducible factor effector, erythropoietin, to hemoglobin mass and blood volume and athletic performance. We found that acute exposure to xenon increased serum erythropoietin concentration, although major markers of erythropoiesis remained unchanged. While daily dosing significantly expanded plasma volume, no physiological or performance benefits were apparent following 4 wk of dosing.

Modulation of HIF-2α PAS-B domain contributes to physiological responses.

Hypoxia-inducible factors (HIFs) are transcription factors in the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) protein family that contain internal hydrophobic cavities within their PAS-A and PAS-B domains. Among HIFs, the HIF-2α PAS-B domain contains a relatively large cavity exploited for the development of specific artificial ligands such as PT2399. Administration of PT2399 could suppress HIF-2α target gene expression without affecting HIF-1 activity in mice under hypoxia conditions. A single mutation (S305M) within the HIF-2α PAS-B domain suppressed HIF-2α activity while conferring resistance to PT2399 in vivo, indicating the vital role of PAS-B domain in HIF-2α hypoxia response. In contrast, the mutant mice did not phenocopy PT2399 intervention in wild-type mice under metabolic stress. Under a high-fat diet (HFD), the mutant mice exert enhanced adipogenesis and obtain larger adipose mass and body weight gain compared to wild type. However, administration of PT2399 along with HFD feeding sufficiently suppressed HFD-induced body weight and adipose mass increase through suppression of adipogenesis and lipogenesis. The accompanying decreased lipid accumulation in the liver and improved glucose tolerance in wild-type mice were not observed in the mutant mice indicating negative regulation of HIF-2α on obesity and a complex role for the PAS-B domain in metabolic regulation. Notably, short-term administration of PT2399 to obese mice decreased adipose mass and improved metabolic condition. These results indicate a regulatory role for HIF-2α in obesity progression and suggest a therapeutic opportunity for PT2399 in obesity and associated metabolic disorders.

A new paradigm for GERD pathogenesis. Not acid injury, but cytokine-mediated inflammation driven by HIF-2α: a potential role for targeting HIF-2α to prevent and treat reflux esophagitis.

Traditionally, reflux esophagitis was assumed to develop as a caustic, chemical injury inflicted by refluxed acid. Recently, however, studies in rats and humans suggest that reflux esophagitis develops as a cytokine-mediated inflammatory injury, with hypoxia inducible factor (HIF)-2α playing a major role. In response to the reflux of acid and bile, HIF-2α in esophageal epithelial cells becomes stabilized, thereby increasing production of pro-inflammatory cytokines that attract T lymphocytes and other inflammatory cells to damage the esophagus. Recent studies have identified small molecule inhibitors of HIF-2α that demonstrate exquisite isoform selectivity, and clinical trials for treatment of HIF-2α-driven kidney cancers are ongoing. It is conceivable that a HIF-2α-directed therapy might be a novel approach to prevention and treatment of reflux esophagitis.

Hypoxia-inducible factor-2α plays a role in mediating oesophagitis in GORD.

OBJECTIVE: In an earlier study wherein we induced acute reflux by interrupting proton pump inhibitor (PPI) therapy in patients with reflux oesophagitis (RO) healed by PPIs, we refuted the traditional concept that RO develops as an acid burn. The present study explored our alternative hypothesis that RO results from reflux-stimulated production of pro-inflammatory molecules mediated by hypoxia-inducible factors (HIFs). DESIGN: Using oesophageal biopsies taken from patients in our earlier study at baseline and at 1 and 2 weeks off PPIs, we immunostained for HIF-1α, HIF-2α and phospho-p65, and measured pro-inflammatory molecule mRNAs. We exposed human oesophageal squamous cell lines to acidic bile salts, and evaluated effects on HIF activation, p65 function, pro-inflammatory molecule production and immune cell migration. RESULTS: In patient biopsies, increased immunostaining for HIF-2α and phospho-p65, and increased pro-inflammatory molecule mRNA levels were seen when RO redeveloped 1 or 2 weeks after stopping PPIs. In oesophageal cells, exposure to acidic bile salts increased intracellular reactive oxygen species, which decreased prolyl hydroxylase function and stabilised HIF-2α, causing a p65-dependent increase in pro-inflammatory molecules; conditioned media from these cells increased T cell migration rates. HIF-2α inhibition by small hairpin RNA or selective small molecule antagonist blocked the increases in pro-inflammatory molecule expression and T cell migration induced by acidic bile salts. CONCLUSIONS: In patients developing RO, increases in oesophageal HIF-2α correlate with increased pro-inflammatory molecule expression. In oesophageal epithelial cells, acidic bile salts stabilise HIF-2α, which mediates expression of pro-inflammatory molecules. HIF-2α appears to have a role in RO pathogenesis. TRIAL REGISTRATION NUMBER: NCT01733810; Results.

On-target efficacy of a HIF-2α antagonist in preclinical kidney cancer models.

Clear cell renal cell carcinoma, the most common form of kidney cancer, is usually linked to inactivation of the pVHL tumour suppressor protein and consequent accumulation of the HIF-2α transcription factor (also known as EPAS1). Here we show that a small molecule (PT2399) that directly inhibits HIF-2α causes tumour regression in preclinical mouse models of primary and metastatic pVHL-defective clear cell renal cell carcinoma in an on-target fashion. pVHL-defective clear cell renal cell carcinoma cell lines display unexpectedly variable sensitivity to PT2399, however, suggesting the need for predictive biomarkers to be developed to use this approach optimally in the clinic.

Targeting renal cell carcinoma with a HIF-2 antagonist.

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Secreted IGFBP5 mediates mTORC1-dependent feedback inhibition of IGF-1 signalling.

The PI(3)K-Akt-mTORC1 pathway is a highly dynamic network that is balanced and stabilized by a number of feedback inhibition loops. Specifically, activation of mTORC1 has been shown to lead to the inhibition of its upstream growth factor signalling. Activation of the growth factor receptors is triggered by the binding of their cognate ligands in the extracellular space. However, whether secreted proteins contribute to the mTORC1-dependent feedback loops remains unclear. We found that cells with hyperactive mTORC1 secrete a protein that potently inhibits the function of IGF-1. Using a large-scale, unbiased quantitative proteomic platform, we comprehensively characterized the rapamycin-sensitive secretome in TSC2(-/-) mouse embryonic fibroblasts, and identified IGFBP5 as a secreted, mTORC1 downstream effector protein. IGFBP5 is a direct transcriptional target of HIF1, which itself is a known mTORC1 target. IGFBP5 is a potent inhibitor of both the signalling and functional outputs of IGF-1. Once secreted, IGFBP5 cooperates with intracellular branches of the feedback mechanisms to block the activation of IGF-1 signalling. Finally, IGFBP5 is a potential tumour suppressor, and the proliferation of IGFBP5-mutated cancer cells is selectively blocked by IGF-1R inhibitors.

Isoform-Selective and Stereoselective Inhibition of Hypoxia Inducible Factor-2.

Hypoxia inducible factor (HIF) transcription factors reside at the center of signaling pathways used by mammalian cells to sense and respond to low oxygen levels. While essential to maintain oxygen homeostasis, misregulation of HIF protein activity correlates with tumor development and metastasis. To provide artificial routes to target misregulated HIF activity, we identified small molecule antagonists of the HIF-2 transcription factor that bind an internal cavity within the C-terminal PAS domain of the HIF-2α subunit. Here we describe a new class of chiral small molecule ligands that provide the highest affinity binding, the most effective, isoform-selective inhibition of HIF-2 in cells, and trigger the largest protein conformation changes reported to date. The current results further illuminate the molecular mechanism of HIF-2 antagonism and suggest additional routes to develop higher affinity and potency HIF-2 antagonists.

F-box and leucine-rich repeat protein 5 (FBXL5): sensing intracellular iron and oxygen.

Though essential for many vital biological processes, excess iron results in the formation of damaging reactive oxygen species (ROS). Therefore, iron metabolism must be tightly regulated. F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, regulates cellular and systemic iron homeostasis by facilitating iron regulatory protein 2 (IRP2) degradation. FBXL5 possesses an N-terminal hemerythrin (Hr)-like domain that mediates its own differential stability by switching between two different conformations to communicate cellular iron availability. In addition, the FBXL5-Hr domain also senses O2 availability, albeit by a distinct mechanism. Mice lacking FBXL5 fail to sense intracellular iron levels and die in utero due to iron overload and exposure to damaging levels of oxidative stress. By closely monitoring intracellular levels of iron and oxygen, FBLX5 prevents the formation of conditions that favor ROS formation. These findings suggest that FBXL5 is essential for the maintenance of iron homeostasis and is a key sensor of bioavailable iron. Here, we describe the iron and oxygen sensing mechanisms of the FBXL5 Hr-like domain and its role in mediating ROS biology.

A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth.

The pharmacological inhibition of general transcriptional regulators has the potential to block growth through targeting multiple tumorigenic signalling pathways simultaneously. Here, using an innovative cell-based screen, we identify a structurally unique small molecule (named JIB-04) that specifically inhibits the activity of the Jumonji family of histone demethylases in vitro, in cancer cells, and in tumours in vivo. Unlike known inhibitors, JIB-04 is not a competitive inhibitor of α-ketoglutarate. In cancer, but not in patient-matched normal cells, JIB-04 alters a subset of transcriptional pathways and blocks viability. In mice, JIB-04 reduces tumour burden and prolongs survival. Importantly, we find that patients with breast tumours that overexpress Jumonji demethylases have significantly lower survival. Thus, JIB-04, a novel inhibitor of Jumonji demethylases in vitro and in vivo, constitutes a unique potential therapeutic and research tool against cancer, and validates the use of unbiased cellular screens to discover chemical modulators with disease relevance.

Allosteric inhibition of hypoxia inducible factor-2 with small molecules.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of protumorigenic pathways. Though transcription factors are typically considered 'undruggable', the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high sequence identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2-driven tumors, such as renal cell carcinomas.

Development of inhibitors of the PAS-B domain of the HIF-2α transcription factor.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure-activity relationship study of small molecules designed to inhibit HIF-2α-ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor-protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α-ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action.

F-box and leucine-rich repeat protein 5 (FBXL5) is required for maintenance of cellular and systemic iron homeostasis.

Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.

Regulating the ARNT/TACC3 axis: multiple approaches to manipulating protein/protein interactions with small molecules.

For several well-documented reasons, it has been challenging to develop artificial small molecule inhibitors of protein/protein complexes. Such reagents are of particular interest for transcription factor complexes given links between their misregulation and disease. Here we report parallel approaches to identify regulators of a hypoxia signaling transcription factor complex, involving the ARNT subunit of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming Acidic Coiled Coil Containing Protein 3) coactivator. In one route, we used in vitro NMR and biochemical screening to identify small molecules that selectively bind within the ARNT PAS (Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously implicated the small molecule KHS101 as an inhibitor of TACC3 signaling. Here, we show that KHS101 works indirectly on HIF complex formation by destabilizing both TACC3 and the HIF component HIF-1α. Overall, our data identify small molecule regulators for this important complex and highlight the utility of pursuing parallel strategies to develop protein/protein inhibitors.

Hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5) communicates cellular iron and oxygen availability by distinct mechanisms.

Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells.

Protein degradation and iron homeostasis.

Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals.

Structural and molecular characterization of iron-sensing hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5).

Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.