First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73–82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype–phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.Mammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia (OMIM 261600) (1). PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2, 3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5, 6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric conformer (3).There are >500 disease-associated missense variants of human PAH; the amino acid substitutions are distributed throughout the 452-residue protein and among all its domains (Fig. 1A) (7–9). Of those disease-associated variants that have been studied in vitro (e.g., ref. 10), some confound the allosteric response, and some are interpreted as structurally unstable. We also suggest that the activities of some disease-associated variants may be dysregulated by an altered equilibrium among conformers having different intrinsic levels of activity, arguing by analogy to the enzyme porphobilinogen synthase (PBGS) and its porphyria-associated variants (11). Consistent with this notion, we have recently established that PAH can assemble into architecturally distinct tetrameric conformers (12), and propose that these conformers differ in activity due to differences in active-site access. This idea has important implications for drug discovery, as it implies that small molecules could potentially modulate the conformational equilibrium of PAH, as has already been demonstrated for PBGS (e.g., ref. 13). Deciphering the relationship among PAH structure, dynamics, and function is a necessary first step in testing this hypothesis.Open in a separate windowFig. 1.The structure of PAH. (A) The annotated domain structure of mammalian PAH. (B) The 2.9 Å PAH crystal structure in orthogonal views, colored as in part A, subunit A is shown in ribbons; subunit B is as a Cα trace; subunit C is in sticks; and subunit D is in transparent spheres. In cyan, the subunits are labeled near the catalytic domain (Top); in red, they are labeled near the regulatory domain (Bottom). The dotted black circle illustrates the autoregulatory domain partially occluding the enzyme active site (iron, in orange sphere). (C) Comparison of the subunit structures of full-length PAH and those of the composite homology model; the subunit overlay aligns residues 144–410. The four subunits of the full-length PAH structure (the diagonal pairs of subunits are illustrated using either black or white) are aligned with the two subunits of 2PAH (cyan) and the one subunit of 1PHZ (orange). The catalytic domain is in spheres, the regulatory domain is in ribbons, and the multimerization domain is as a Cα trace. The arrow denotes where the ACT domain and one helix of 2PAH conflict.Numerous crystal structures are known for one- and two-domain constructs of mammalian PAH (14).

Table S1.

Mammalian PAH structures available in the PDB (August 2015)Open in a separate windowEntries are colored by configuration of the active-site lid (approximately residues 130–150): “open,” “closed,” and “disordered.”^*C, catalytic domain, M, multimerization domain; R, regulatory domain.^†wwPDB Chemical Component Dictionary (49).^‡Disease-associated single-residue substituted variant (A313T).^§Phosphorylated at Ser16.