Browsing by Subject "Aryl Hydrocarbon Receptor Nuclear Translocator"
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Item A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds(2009-09-04) Evans, Matthew Ryan; Gardner, Kevin H.The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous basic helix-loop-helix Period/ARNT/Single-minded (bHLH-PAS) protein that controls various biological pathways by forming heterodimeric transcriptional regulator complexes with several other bHLH-PAS proteins via the beta-sheet surfaces of its two PAS domains. The beta-sheets of PAS domains are involved in many intermolecular interactions with other proteins and natural cofactors in order to detect environmental changes in sensor PAS proteins. As part of a study of the HIF-2 alpha and ARNT PAS-B heterodimer, site-directed mutagenesis was performed on the ARNT PAS-B domain. Interestingly, one point mutation on the ARNT beta-sheet surface (Y456T) resulted in a new conformation of the domain that existed in equimolar concentrations with the wild-type conformation. Subsequent studies demonstrated that the two conformations are in equilibrium and that relative populations of the two conformations can be perturbed by additional mutations. Using solution NMR spectroscopy, we solved the high resolution solution structure of a mutant ARNT PAS-B domain in the new conformation, demonstrating that it contains a three-residue slip in register and accompanying inversion of the central beta-strand. In addition, this new conformer has a greater than hundred-fold reduction in affinity for HIF-2 alpha PAS-B, disrupting the hypoxia response pathway. Solution NMR measurements of the interconversion kinetics have let us establish that these two conformations interconvert slowly (40 min at RT) with a linear Arrhenius temperature-dependence of the interconversion rate. Stopped-flow unfolding experiments using GdmHCl on Y456T, revealed a similarly slow unfolding rate (25 min at RT) and an energy barrier to unfold of approximately 13 kcal/mol, which is nearly identical to that for the interconversion process. These data indicate that the protein must undergo a global unfolding process in order to interconvert between conformations. Lastly, these relative populations of Y456T can be affected by compound preferentially binding into the core of one of these conformations. This discovery highlights the malleability of PAS beta-sheets and suggests ARNT may act as a regulatory switch to control different biological pathways. Furthermore, this system presents a great opportunity to further understand the structural and kinetic impact of beta-strand slips observed in nature.Item PAS Domain-Mediated Dimerization of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) in the Hypoxia Response Pathway(2005-12-19) Card, Paul B.; Gardner, Kevin H.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.