A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds

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.