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...

Hypoxia Inducible Factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of many 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 smallmolecule 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 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.

A comprehensive understanding of the molecular mechanisms underpinning cellular functions is dependent on a detailed characterization of the energetics of macromolecular binding, often quantified by the equilibrium dissociation constant, KD. While many biophysical methods may be used to obtain KD, the focus of this report is a relatively new method called “microscale thermophoresis” (MST). In an MST experiment, a capillary tube filled with a solution containing a dye-labeled solute is illuminated with an infrared laser, rapidly creating a temperature gradient. Molecules will migrate along this gradient, causing changes in the observed fluorescence. Because the net migration of the labeled molecules will depend on their liganded state, a binding curve can be constructed as a function of ligand concentration from MST data and analyzed to determine KD. Herein, simulations demonstrate the limits of KD that can be measured in current instrumentation. They also show that binding kinetics are a major concern when planning and executing MST experiments. Additionally, studies of two protein-protein interactions illustrate challenges encountered in acquiring and analyzing MST data. Combined, these approaches indicate a set of best practices for performing and analyzing MST experiments. Software for rigorous data analysis is also introduced.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and are required for tumor growth, metastasis and resistance to cancer treatment. The C-terminal PAS domain of HIF2α (HIF2α PAS-B) contains a preformed solvent-inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer. To better understand how small molecules bind within this domain, we examined the structures, equilibrium and transition state thermodynamics of HIF2α PAS-B with several artificial ligands using ITC, NMR exchange spectroscopy and X-ray crystallography. Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in NMR and crystal structures. Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest a binding-competent transition state characterized by increased structural disorder. Finally, molecular dynamics simulations reveal conversion between open and closed conformations of the protein and pathways of ligand entry into the binding pocket.