PAS Domain-Mediated Dimerization of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) in the Hypoxia Response Pathway



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PAS (Per-ARNT-SIM) domains are versatile protein-protein interaction domains that are often used as regulatory modules in a variety of important biological pathways. Although their importance in several of these pathways has been well established, only sparse structural data exists that could help elucidate a general mode of PAS-PAS interaction. As such, more examples of these domains must be studied using a variety of techniques to understand how these domains carry out viable functions within the cell.The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) is a constituent of heterodimeric transcriptional activation complexes used in several important biochemical pathways. One such complex is the hypoxia inducible transcription factor (HIF), which allows mammalian cells to respond to changes in oxygen availability. In HIF, ARNT dimerizes with HIFa to upregulate genes involved in the hypoxia response. The dimerization of ARNT and HIFa involves interactions between PAS domains in both proteins, but details regarding how these domains interact in this and other heterodimeric complexes have not previously been well characterized. To investigate these interactions within the context of the HIF/ARNT heterodimer, we expressed the C-terminal PAS domains from both proteins and characterized the complex using NMR-based methods. Solution structures of each domain are presented, as well as a model of the ARNT/HIF heterodimeric PAS complex. This model was used to identify of key interfacial residues, and the roles of these were tested in a variety of ways by sitedirected mutagenesis. In addition, extended constructs from ARNT that include other components of the full-length protein were investigated to establish the validity of a reductionist approach in the study of individual PAS domains from this system. In many biological systems, PAS domains bind small molecules to regulate proteinprotein interactions. With this in mind, we also subjected PAS domains from HIFa and ARNT to an NMR-based screen against an in-house library of 800 compounds to determine the potential of these domains to bind small molecules. With have used this approach to identify compounds that can disrupt PAS-PAS interactions in the hypoxia pathway in order to help elucidate some of the molecular details of PAS domain-mediated signaling.

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