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Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIF␣ and ARNT. Although this complex involves protein-protein interactions mediated by basic helixloop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2␣ by NMR spectroscopy. We demonstrate that HIF-2␣ PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2␣ central ␤-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this ␤-sheet interface is widely used for both intra-and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins. C ellular responses to oxygen availability are essential for the development and homeostasis of mammalian cells, demonstrated most critically by the link between the cellular adaptation to reduced tissue oxygenation and disease progression (1, 2). In mammalian cells, these responses are mediated in part by the hypoxia-inducible factor (HIF), a heterodimeric transcription factor composed of HIF␣ and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF␤) (3). HIF activity is tightly controlled under normoxic conditions by multiple O 2 -dependent hydroxylation events of the HIF␣ subunit, which coordinately promote the ubiquitin-mediated destruction of this protein (4) and impair its ability to interact with transcriptional coactivators (5, 6) (Fig. 1a). These controls are relieved during hypoxia, allowing HIF to activate the transcription of genes that facilitate metabolic adaptation to low oxygen levels and increase local oxygen supply by angiogenesis (7).All three isoforms of HIF␣ [HIF-1␣, -2␣ (EPAS1), and -3␣] (8, 9) and ARNT belong to the basic helix-loop-helix (bHLH)-Per-ARNT-Sim (PAS) family of eukaryotic transcription factors, which contain bHLH and PAS domains (Fig. 1). The bHLH domains of these proteins serve as dimerization elements, helping determine the specificity of complex formation while providing a DNA-binding interface composed of the basic regions from each monomer (10). PAS domains are widespread components of signal transduction proteins, currently identified in Ͼ2,000 proteins from organisms in all three kingdoms of life. These domains, shown to be ...

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The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.