New protein structures provide an updated understanding of phenylketonuria

Abstract

Phenylketonuria (PKU) and less severe hyperphenylalaninemia (HPA) constitute the most common inborn error of amino acid metabolism, and is most often caused by defects in phenylalanine hydroxylase (PAH) function resulting in accumulation of Phe to neurotoxic levels. Despite the success of dietary intervention in preventing permanent neurological damage, individuals living with PKU clamor for additional non-dietary therapies. The bulk of disease-associated mutations are PAH missense variants, which occur throughout the entire 452 amino acid human PAH protein. While some disease-associated mutations affect protein structure (e.g. truncations) and others encode catalytically dead variants, most have been viewed as defective in protein folding/stability. Here we refine this view to address how PKU-associated missense variants can perturb the equilibrium among alternate native PAH structures (resting-state PAH and activated PAH), thus shifting the tipping point of this equilibrium to a neurotoxic Phe concentration. This refined view of PKU introduces opportunities for the design or discovery of therapeutic pharmacological chaperones that can help restore the tipping point to healthy Phe levels and how such a therapeutic might work with or without the inhibitory pharmacological chaperone BH4. Dysregulation of an equilibrium of architecturally distinct native PAH structures departs from the concept of “misfolding”, provides an updated understanding of PKU, and presents an enhanced foundation for understanding genotype/phenotype relationships.

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

“…Specifically, we performed biophysical calculations for all possible single‐site amino acid substitutions in human PAH using the recently published full‐length structure of rat PAH in its autoinhibited tetrameric state. In the presence of high concentrations of phenylalanine, PAH most likely undergoes dramatic conformational changes leading to a more fully accessible active site (Arturo et al., ; Jaffe, ; Patel, Kopec, Fitzpatrick, McCorvie, & Yue, ; Pey, Thorolfsson, Teigen, Ugarte, & Martinez, ; Thorolfsson, Teigen, & Martinez, ; Zhang & Fitzpatrick, ). Although different conformational states might be affected differently by mutations, structural data on a full‐length PAH are available only in the autoinhibited state, and so we were unable to assess the consequence of mutations in activated PAH.…”

Simulations of the regulatory ACT domain of human phenylalanine hydroxylase (PAH) unveil its mechanism of phenylalanine binding

“…ACT domains, which can serve in ligand sensing, were named for the first three proteins in which they were identified (1). Phe stabilization of A-PAH controls the equilibrium between RS-PAH and A-PAH and thus allows Phe to regulate PAH activity (2). The Phe-stabilized conformational change is coupled to exposure of the enzyme active site (3), thus activating the enzyme.…”

The structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin

“…BH4 also stabilizes PAH and increases the steady-state levels of the enzyme in vivo (10,11). While the activation by L-Phe induces a large conformational change (8), recently proposed to involve dimerization of RDs (12)(13)(14), BH4 binding elicits a limited conformational change proposed to be mostly constrained to the N-term tail (15,16). The importance of these N-terminal 29 residues in both the inhibitory regulation by BH4 and the activation by L-Phe is evident, as PAH lacking this tail is not regulated by either BH4 or L-Phe and is constitutively active (15).…”