First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer Emilia C. Arturoa,b, Kushol Guptac, Annie Hérouxd, Linda Stitha, Penelope J. Crosse,f,g, Emily J. Parkere,f,g, Patrick J. Lollb, and Eileen K. Jaffea,1 aMolecular Therapeutics, Fox Chase Cancer Center, Temple University Health Systems, Philadelphia, PA 19111; bBiochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102; cBiochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; dEnergy Sciences Directorate/Photon Science Division, Brookhaven National Laboratory, Upton, NY 11973; eBiomolecular Interaction Centre, University of Canterbury, Christchurch 8041, New Zealand; fDepartment of Chemistry, University of Canterbury, Christchurch 8041, New Zealand; and gMaurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1142, New Zealand Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved January 21, 2016 (received for review August 27, 2015) Improved understanding of the relationship among structure, dy- There are >500 disease-associated missense variants of human namics, and function for the enzyme phenylalanine hydroxylase PAH; the amino acid substitutions are distributed throughout (PAH) can lead to needed new therapies for phenylketonuria, the the 452-residue protein and among all its domains (Fig. 1A) most common inborn error of amino acid metabolism. PAH is a (7–9). Of those disease-associated variants that have been stud- multidomain homo-multimeric protein whose conformation and ied in vitro (e.g., ref. 10), some confound the allosteric response, multimerization properties respond to allosteric activation by the and some are interpreted as structurally unstable. We also sug- substrate phenylalanine (Phe); the allosteric regulation is neces- gest that the activities of some disease-associated variants may be sary to maintain Phe below neurotoxic levels. A recently introduced dysregulated by an altered equilibrium among conformers having model for allosteric regulation of PAH involves major domain different intrinsic levels of activity, arguing by analogy to the motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, enzyme porphobilinogen synthase (PBGS) and its porphyria- Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Bio- associated variants (11). Consistent with this notion, we have phys 530(2):73–82]. Herein, we present, to our knowledge, the first recently established that PAH can assemble into architecturally X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a mono- distinct tetrameric conformers (12), and propose that these disperse tetrameric PAH, in the absence of Phe, facilitated determi- conformers differ in activity due to differences in active-site ac- nation of the 2.9 Å crystal structure. The structure of full-length PAH cess. This idea has important implications for drug discovery, as supersedes a composite homology model that had been used exten- it implies that small molecules could potentially modulate the sively to rationalize phenylketonuria genotype–phenotype relation- conformational equilibrium of PAH, as has already been dem- ships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, onstrated for PBGS (e.g., ref. 13). Deciphering the relationship which dominates in the absence of Phe, is different from a Phe- among PAH structure, dynamics, and function is a necessary first stabilized allosterically activated PAH tetramer. The lack of structural step in testing this hypothesis. detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotype relationships. Nevertheless, Significance the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the Phenylketonuria and milder hyperphenylalaninemias consti- enzyme in a tetrameric form that was not possible with prior partial tute the most common inborn error of amino acid metabo- crystal structures, and facilitates interpretation of a wealth of bio- lism, usually caused by defective phenylalanine hydroxylase chemical and structural data that was hitherto impossible to evaluate. (PAH). Although a highly restricted diet prevents intellectual impairment during development, additional therapies are re- phenylalanine hydroxylase | phenylketonuria | X-ray crystallography | quired to combat cognitive dysfunction, executive dysfunc- small-angle X-ray scattering | allosteric regulation tion, and psychiatric disorders that arise due to dietary lapses throughout life. New therapies can arise from thorough un- ammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) derstanding of the conformational space available to full- Mis a multidomain homo-multimeric protein whose dys- length PAH, which has defied crystal structure determination function causes the most common inborn error in amino acid for decades. We present the first X-ray crystal structure of metabolism, phenylketonuria (PKU), and milder forms of hy- full-length PAH, whose solution relevance is supported by perphenylalaninemia (OMIM 261600) (1). PAH catalyzes the small-angle X-ray scattering. The current structure is an auto- hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme inhibited tetramer; the scattering data support the existence iron and the cosubstrates tetrahydrobiopterin and molecular oxygen of an architecturally distinct tetramer that is stabilized by the (2, 3). A detailed kinetic mechanism has recently been derived from allosteric activator phenylalanine. elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Author contributions: K.G. and E.K.J. designed research; E.C.A., K.G., A.H., L.S., P.J.C., E.J.P., and P.J.L. performed research; E.C.A., K.G., A.H., P.J.C., E.J.P., P.J.L., and E.K.J. analyzed data; Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by and E.C.A., K.G., P.J.L., and E.K.J. wrote the paper. binding to a regulatory domain. Phosphorylation at Ser16 potenti- The authors declare no conflict of interest. ates the effects of Phe, with phosphorylated PAH achieving full This article is a PNAS Direct Submission. activation at lower Phe concentrations than the unphosphorylated Data deposition: The atomic coordinates and structure factors have been deposited in the protein (5, 6). Allosteric activation by Phe is accompanied by a Protein Data Bank, www.pdb.org (PDB ID code 5DEN). major conformational change, as evidenced by changes in protein 1To whom correspondence should be addressed. Email: [email protected]. fluorescence and proteolytic susceptibility, and by stabilization of a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tetrameric conformer (3). 1073/pnas.1516967113/-/DCSupplemental. 2394–2399 | PNAS | March 1, 2016 | vol. 113 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1516967113 Downloaded by guest on September 23, 2021 domain enzyme, possibly because the presence of multiple dis- A tinct conformers frustrated efforts to crystallize the full-length protein. Recognition of the existence of alternate tetrameric assemblies and the ability to isolate a single species has now allowed us to generate monodisperse samples of full-length rat PAH suitable for biophysical analysis using X-ray crystallography and small-angle X-ray scattering (SAXS). We report here, to our knowledge, the first crystal structure for full-length PAH, at a resolution of 2.9 Å, which supersedes a composite homology Long edge model that has been used since 1999 to help rationalize PKU-related B genotype/phenotype relationships (14). D C Results PAH Crystal Structure. This study focuses on rat PAH, which is 96% similar (92% identical) to the human enzyme. The 15 most common disease-associated amino acids are fully conserved be- tween rat and human, making the rodent protein an excellent model system for study of PKU. Rat PAH, heterologously expressed in Escherichia coli and purified via phenyl-Sepharose affinity chromatography (15), can be further fractionated on an B A ion-exchange column to partially resolve two tetrameric species (faster migrating and slower migrating) and one dimeric species, Short edge Short as determined by native PAGE (Fig. S1). In fractionated PAH samples, the distribution of these species is stable over long time 90° courses, suggesting that these fractions are good starting points for crystallization trials. The faster-migrating tetramer is the pre- A dominant component in these preparations (12), and using the D fraction most enriched in this species, we were able to produce BIOCHEMISTRY well-diffracting crystals that yielded a 2.9 Å structure (Fig. 1B and Table S2), to our knowledge, the first structure for any full-length mammalian PAH. The full-length structure and the previously used composite homology model are compared in Fig. 1C [with the caveat that the composite model combined a two-domain rat PAH structure (regulatory plus catalytic) with a two-domain hu- man PAH structure (catalytic plus multimerization) (6, 14, 16), whereas the three-domain crystal structure is rat PAH]. BCThe crystal asymmetric unit contains one tetramer, which C adopts an autoinhibited form in which the autoregulatory region partially occludes the enzyme active