Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor

Abstract

The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2␣ and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2␣ PAS-B domain contains a large internal cavity that accommodates ligands identified from a small-molecule screen. Binding one of these ligands to HIF2␣ PAS-B modulates the affinity of the HIF2␣:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.internal cavity ͉ NMR ͉ X-ray crystallography ͉ hypoxia ͉ protein-ligand interactions T he hypoxia-inducible factor (HIF) transcription factors are present in multicellular organisms and adopt conserved roles in maintaining cellular oxygen homeostasis. In humans, HIF misregulation correlates with aggressive solid tumor growth and poor clinical outcomes (1, 2). Transcriptionally active HIF proteins are heterodimers of the HIF␣ and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF␤) subunits (3, 4), each containing an N-terminal basic helix-loophelix (bHLH) domain for specific DNA binding, two tandem Per-ARNT-Sim (PAS) domains to facilitate heterodimerization and C-terminal regulatory regions (5-7). Three known human HIF␣ subunit isoforms share ARNT as their bHLH-PAS protein binding partner. HIF1␣ and HIF2␣ are similarly regulated, but show cell line-specific differences in expression and gene regulation patterns (8). HIF3␣ and its splicing isoforms (9, 10) lack C-terminal sequences that recruit transcriptional coactivator proteins, suggesting that these proteins act as dominant negative pathway regulators by forming regulatory-incompetent heterodimers with ARNT.Regulation of this pathway is governed in large part by posttranslational modifications that down-regulate HIF activity under adequate cellular oxygenation levels (normoxia). The best characterized of these modifications are hydroxylations of key proline and asparagine residues in the HIF␣ C-terminal region (11,12). These hydroxylated prolines recruit the von Hippel Lindau (pVHL) E3 ubiquitin ligase, which ultimately downregulates HIF␣ protein levels through proteasomal degradation, whereas the hydroxylated asparagines block HIF␣-coactivator interactions. Oxygen-insufficient conditions (hypoxia) inactivate the hydroxylases, allowing HIF␣ subunits to escape degradation, heterodimerize with ARNT, and ultimately control the levels of Ͼ100 proteins (13). Misregulation of the HIF path...

Identification of Cys255 in HIF‐1α as a novel site for development of covalent inhibitors of HIF‐1α/ARNT PasB domain protein–protein interaction

“…Structural and biophysical characterization of the HIF-1a/ARNT heterodimer Analogous to previously described mutations in the HIF-2a/ARNT complex, 17 we made the mutation R245E in the HIF-1a PasB domain to reverse a salt bridge between ARNT residue 362 (E362R mutation) resulting in stabilization of the HIF-1a/ARNT PasB complex. 24 This mutation significantly improved solution behavior compared to the wild type HIF-1a PasB domain and allowed detailed biophysical characterization of the HIF-1a/ARNT interaction.…”

“…2). 17 Alternate orientations of His291, plus sequence differences of Ala275 versus Ile, and Leu317 versus Val, create different cavity shapes and water occupancies. In short, the shape of the cavity in HIF-1a is different, and somewhat smaller than in HIF-2a.…”

“…26 A number of drugs have been shown to inhibit HIF via indirect molecular mechanisms and present an attractive case to test the incorporation of HIF inhibitors into current cancer therapies. With the exception of acriflavine which is thought to target heterodimerization, most of the agents target HIF transcription, protein synthesis, protein stabilization, DNA binding, and 17 and in this work for HIF-1a/ARNT. While it is clear that there are additional contributions to overall binding affinity of HIF to ARNT from the PasA domains as well as from the final transcription complex assembly on hypoxia response element (HRE), the PasB domain portion of the heterodimer could represent a true molecular target to modulate the hypoxia-mediated phenotype in a therapeutic setting.…”

“…17 Additionally, structural analysis revealed the presence of an irregularly shaped 290 Å 3 internal cavity within the HIF-2a domain. Gardner and coworkers identified a series of HIF-2a selective low molecular weight compounds that bind to the HIF-2a PasB internal cavity modulating the interaction between the HIF-2a and ARNT PasB domains (PDB ID 3H7W and 3H82).…”

Can we develop effective combination antiangiogenic therapy for patients with hepatocellular carcinoma?

“…Based on partial structural and functional similarities between HIF-1 α and HIF-2 α , it is possible that many of the already identified HIF-1 α inhibitors would inhibit HIF-2 α . In addition, systematic efforts are currently ongoing to identify compounds that are effective at inhibiting both HIF-1 α and HIF-2 α for cancer treatment [44, 60]. Many of these HIF inhibitors are currently in phase I and II clinical trials (Table 2 and Fig.…”