PROJECT SUMMARY Dysfunction of phenylalanine hydroxylase (PAH) is the most common inborn error of amino acid metabolism and the underlying cause of phenylketonuria (PKU). By converting phenylalanine (Phe) to tyrosine, PAH maintains blood Phe at levels sufficient for protein biosynthesis, but below neurotoxic levels. Regulation is accomplished by allosteric activation by Phe. Based on extensive studies of individuals living with PKU, the current medical consensus is to control blood Phe levels throughout life to achieve and maintain normal neurological function; this argues for a better understanding of PAH structure/function relationships to support both the understanding of existing pharmacological chaperones for PAH and the future development of novel non-dietary therapeutics. In 2013 we introduced an innovative conformational selection model of PAH allostery that includes a resting-state tetramer, an architecturally distinct activated tetramer, and smaller assemblies; only activated PAH contains the allosteric Phe binding site. This site is at a multimer-specific subunit-subunit interface, the details of which remain unknown. Our model includes a previously unforeseen domain rotation, which is now strongly supported by recently published biophysical studies. 2016 marks our publication of the first crystal structure for full length resting-state mammalian PAH; this is a long-awaited contribution to the field. Small angle X-ray scattering (SAXS) supports both resting state PAH and Phe-stabilized activated PAH tetramer structures, and confirms a major conformational difference between the two, which is consistent with our allosteric model. The current application builds on these achievements. In AIM 1 we address the relevance of our allosteric model to disease. We test whether specific common disease-associated PAH variants are defective in the transition between resting-state and activated PAH and thus insensitive to allosteric activation by Phe. This hypothesis is a major departure from the conventional view of PKU as a protein folding/stability disorder. In AIM 2 we determine the structure of activated PAH using X-ray crystallography and SAXS, and we extend our work with rat PAH to human PAH using a designed variant. In AIM 3 we identify substances that can modulate PAH function (negatively or positively) by stabilizing either resting-state or activated PAH. Using in vitro methods, we will screen approved drugs and environmental contaminants, exposure to which can confound PKU phenotype. We use in silico screening of libraries of drug-like molecules to provide leads for future development of new PKU therapies. All AIMS employ established biochemical and biophysical methods to assess wild-type, disease-associated, and designed PAH variants for the transition from resting to activated states. Key methods include intrinsic protein fluorescence, SAXS, analytical ultracentrifugation, crystallography, native PAGE, enzyme kinetics, and the innovative use of ion exchange chromatography to resolve conformationally distinct PAH multimers. Our broad approach will yield new and important information applicable to a better understanding of the molecular bases for PKU.

PROJECT NARRATIVE Phenylalanine hydroxylase (PAH) dysfunction causes phenylketonuria (PKU), which is the most common inborn error of amino acid metabolism (1:15,000 births). We strive to understand how PAH changes its molecular shape in order to prevent neurotoxic accumulation of phenylalanine. The basic science understanding of PAH shape changes will lead to new or improved therapies that can minimize neurobehavioral abnormalities in individuals living with PKU.