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.

Keywords: Allostery; Conformational selection; Pharmacological chaperones; Phenylalanine hydroxylase; Phenylketonuria.

Figure 1. The equilibrium of architecturally distinct tetrameric PAH assemblies, shown in terms of a double-pan balance

The transition between RS-PAH and A-PAH structures requires relocation of the PAH regulatory domain (shown in magenta); this relocation forms a multimer-specific allosteric Phe (black dot) binding site, which is distant from the active site, which is where Phe is converted to tyrosine. RS-PAH predominates at low Phe. Phe-stabilized A-PAH predominates at high Phe.

Figure 2. PAH structure and model

a) The annotated domain structure of PAH illustrates the regulatory, catalytic, and multimerization domains, including subdomains. Numbered residues correspond to the termini and hinges. Coloring is reflected in the darker subunits of the molecular illustrations. b) The first crystal structure for full length PAH (PDB id 5DEN, 2.9 Å resolution (18)) is illustrated using orthogonal views (top and bottom), and represents RS-PAH. The bulk of the protein is shown in balls, while the C-terminal helix is in cartoon. Two of the four subunits are transparent. One opaque subunit is illustrated using lighter shades of the colors in part a. The subunits are labeled in cyan near the catalytic domains (top); they are labeled in red near the regulatory domain (bottom). The dotted white circle illustrates the autoregulatory region partially occluding the enzyme active site (the active site iron is obscured by the auto-inhibitory interaction). Blocked active-site access (partial or total) results in low activity. c) A composite homology model of the A-PAH tetramer (18), depicted as in part b. A-PAH is active because the active sites are accessible (white circle in top image, active site iron is visible). A-PAH contains a close association between the ACT domains of subunits located along the diagonal of the tetramer. Formation of this subunit-subunit interface was predicted in 2013 (16) along with the resultant two allosteric Phe binding sites (location indicated by green “Phe” label on top image). The oval in the bottom image shows the proposed multimer-specific interfaces that might form the binding site for a new pharmacological chaperones that could potentiate, rather than interfere with, allosteric Phe binding. d) An overlay of one subunit of the RS-PAH structure (darker shades) with one subunit of the A-PAH model (lighter shades) illustrates the relocation of the regulatory domain relative to the rest of the subunit. The regulatory domain is in cartoon, the catalytic domain is in balls, and the multimerization domain is in coil representation. Regulatory domain relocation results from a ~90° rotation relative to the catalytic domain; displacement of the regulatory domain in this way disallows active site occlusion by the autoregulatory region.

Figure 3. Phe-bound PAH ACT domain dimer

The crystal structure of a truncated human PAH ACT domain (PDB id 5FII, containing residues 34–111 (19)) forms a dimer and shows Phe (green) binding as predicted (18). The α-helices are predicted to be solvent exposed while the 8-stranded β-sheet is predicted to face into the tetramer. Grey balls are used for the side chains of Leu48 and Ile65. These four interacting residues contribute to the hydrophobic core of the ACT-domain dimer. L48S and I65T are each predicted to disrupt this important stabilizing interaction. Leu48 and Ile65 each have van der Waals interactions with the allosteric Phe.

Figure 4. The equilibrium of PAH tetramers in the context of small molecule ligands (shown in red)

Newly synthesized PAH can be drawn into the equilibrium of folded native structures by BH₄, which reversibly binds to RS-PAH. Inhibitory BH₄ binding involves and secures the auto-inhibitory interaction (see Fig 5). Allosteric Phe binding to A-PAH stabilizes that structure and effectively competes with the inhibitory effect of BH₄. Note that there is no direct interconversion between BH₄-stabilized RS-PAH and Phe-stabilized A-PAH. By targeting multimer-specific small molecule binding sites, we foresee a new pharmacological chaperone (red asterisk) that can stabilize A-PAH without interfering with allosteric Phe binding (see Fig 2c). This is predicted to potentiate the sensitivity of A-PAH to allosteric Phe binding and restore the Phe response in a subset of disease-associated PAH variants.

Figure 5. BH₄, bound two different ways at the PAH active site provides the molecular basis for stabilization of RS-PAH

(a) An overlay of crystal structures (full length RS-PAH (white); PDB id 5DEN and PAH catalytic domain containing BH₄ (cyan/green); PDB id 1J8U) illustrates that BH₄ is positioned to make two stabilizing H-bonds to Ser23 which secure the auto-inhibitory interaction. (b) An alternate overlay (full length PAH (white); PDB id 5DEN and PAH catalytic domain containing BH₄ and norleucine (cyan/green); PDB id 1MMT) shows BH₄ sitting deeper in the active site and too far away to make the stabilizing H-bonds.