First Structure of Full-Length Mammalian Phenylalanine Hydroxylase Reveals the Architecture of an Autoinhibited Tetramer

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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 between 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 human 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 site. Continuous electron density can be observed for residues 20–136 and 143–450 within each of the four protomers in the tetramer (with some variation at each termini), as well as for one iron ion per chain and two waters at the active site of each chain. The catalytic domains of the four protomers are arranged in approximate 222 symmetry, where each catalytic domain occupies one corner of a rectangle. The arrangement can be described as a dimer-of-dimers, where the dimers with the greatest buried surface area lie along the short edge of the rectangle (Fig. 1B). Although each short-edge dimer (the BD dimer or the AC dimer) is intimately assembled, the spacing between these two dimers is large, resulting in very little dimer– Fig. 1. The structure of PAH. (A) The annotated domain structure of mam- dimer interaction along the long edge of the tetramer, and hence a malian PAH. (B) The 2.9 Å PAH crystal structure in orthogonal views, colored as rectangular rather than square arrangement. The ACT domains in part A, subunit A is shown in ribbons; subunit B is as a Cα trace; subunit C is in extend above and below the plane of this rectangle (Fig. 1B, sticks; and subunit D is in transparent spheres. In cyan, the subunits are labeled Bottom). The four C-terminal multimerization domains contain near the catalytic domain (Top); in red, they are labeled near the regulatory α-helices that assemble into an antiparallel bundle in the center of domain (Bottom). The dotted black circle illustrates the autoregulatory domain this tetrameric arrangement. The catalytic domain of each pro- partially occluding the enzyme active site (iron, in orange sphere). (C)Com- tomer contains an active site that is partly occluded by the parison of the subunit structures of full-length PAH and those of the composite – autoregulatory region and a partially disordered active-site lid 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 (residues 130 150). The conformations of the catalytic and using either black or white) are aligned with the two subunits of 2PAH (cyan) regulatory domains of the four protomers are highly similar, with and the one subunit of 1PHZ (orange). The catalytic domain is in spheres, the RMS deviations of ∼0.3 Å across Cα atoms. The C-terminal he- regulatory domain is in ribbons, and the multimerization domain is as a Cα trace. lices of the four protomers adopt two distinct orientations with The arrow denotes where the ACT domain and one helix of 2PAH conflict. respect to the catalytic domain, differing by a tilt of ∼10° (Fig. 1C); protomers situated across the diagonal of the tetramer contain similarly positioned helices (Figs. 1C and 2A). This asymmetry is Numerous crystal structures are known for one- and two- unexpectedly different from the tetramer apparent in the two- domain constructs of mammalian PAH (Table S1). However, until domain human PAH structure, where similarly positioned helices now, no structure has been available for the full-length, three- are within each short-edge dimer (Fig. 2B). Because the C-terminal

Arturo et al. PNAS | March 1, 2016 | vol. 113 | no. 9 | 2395 Downloaded by guest on September 23, 2021 helices form a four-helix bundle in the center of the PAH tet- disordered on chains B and C). Interestingly, in the two-domain ramer, these differences in their positions give rise to asymmetry human PAH structure (2PAH), the C-terminal helices are fully and cause the architecture of the tetramer to twist and deviate ordered. Thus, the twist between the short-edge dimers in the significantly from the bona fide 222 symmetry found in the two- structure of full-length PAH is accompanied by a shift in the domain tyrosine hydroxylase structure (Fig. 2C). environment at the ends of C-terminal helices that no longer The short-edge dimers form extensive interfaces (buried areas favors ordered packing. of 6,512 and 7,033 Å2 for the BC and AD dimers, respectively) The C-terminal helices form an antiparallel four-helix bundle between the catalytic domain of one subunit and the regulatory that is the central point of tetramer association. These helices domain of the adjacent subunit; this interface also includes that are conformationally flexible, which is evident in the noniso- portion of the multimerization domain that precedes the C-ter- morphism we observe among multiple PAH crystals. We have minal helix. Interestingly, the short-edge dimer interface involves partially refined two additional structures for full-length PAH at 8 aa whose substitutions are disease-associated, including three lower resolution, using crystals grown under essentially identical of the most common PKU-associated variants, I65T, R68S, and conditions. In these structures, the position of the C-terminal R413P. The twist between the short-edge dimers means that the helix varies, changing the relative positions of the two dimers other dimers (long-face and diagonal) interact predominantly within the tetramer (Fig. 2E). If one superposes all B subunits, through the C-terminal helices and are characterized by signifi- the BD short-edge dimers align well, but different twists about cantly smaller buried interfaces [the long-edge (AB and CD) and the C-terminal helices cause positions of the AC dimers to vary ∼ the diagonal (BC and AD) dimers bury 1,412, 766, 435, and by as much as 7 Å. This unusual level of flexibility in the PAH ’ 258 Å2, respectively]. This twist also causes the distance between four-helix bundle may be responsible for some of the enzyme s the ACT domains to differ on the two faces of the tetramer; the unusual kinetic characteristics, which caused us to initially ends of the C-terminal helices extend toward this space between identify it as a putative morpheein (17). the ACT domains. None of the chains are ordered all of the way PAH Active-Site Access. Two structural elements govern PAH ac- to their most C-terminal residue, but the helices on the side of tive-site access, the autoregulatory region and the active-site lid. the tetramer with the closer ACT spacing contain more order In our PAH structure, residues 20–25 of the autoregulatory re- (the C-terminal three and four residues are disordered on chains gion lie across the opening to the active site; we have postulated A and D, respectively, whereas the C-terminal six residues are that this occlusion is relieved by a Phe-modulated formation of an ACT domain dimer (Fig. S2A) (12). The insight that regulatory domain positioning may govern active-site access sheds ACB new light on deciphering the order of catalytic events, because previous mechanistic studies have uniformly used PAH that lacks the regulatory domain (e.g., refs. 2 and 4). The other structural element governing active-site access is an active-site lid or loop (approximately residues 130–150) (18). In all 15 structures of the isolated human PAH catalytic domain, the lid is fully ordered and exists in one of two different conformations (“open” vs. “closed”), dependent upon active-site occupancy (color coded in Table S1, illustrated in Fig. 3 A–C). The lid is open unless three different ligands are all present: iron, a Phe analog, and a pterin. In contrast, in all structures of multidomain rat and human PAH Two-domain (including our structure of the full-length enzyme), the lid is Full length PAH Two-domain PAH tyrosine hydroxylase partially disordered, specifically at amino acids ∼137–142 (e.g., DE Fig. 3D), which is the portion of the lid that differs most between D C the open and closed conformations. For these multidomain structures, most of the portions of the lid that can be seen align best with the open-lid conformation. However, at one point (immediately N-terminal to the disordered portion of the lid), the backbone in the multidomain structures is more closed-like than open (Fig. 3E, Left). In the single-domain structures where the lid is fully ordered, B factors for lid residues are higher than those for the rest of the structure (Fig. S3), consistent with the B A notion that this region is a mobile element that can change conformation—opening and closing the active site—as part of the en- Fig. 2. Overall architectures of PAH and related structures. (A) Structure of zyme’s catalytic function, perhaps in response to regulatory signals. full-length PAH showing that subunits with similar structures (white like A notable difference between the structure of full-length rat white, black like black) are positioned across the diagonal of the tetramer, when viewed from the perspective of the catalytic domains (Top). The reg- PAH and those of the isolated human PAH catalytic domain is ulatory domains of these similarly structured subunits are on the same side the conformation at Phe131 (Fig. 3E, Right, and Fig. S4A), which of the tetramer (Bottom). These are the subunits for which the predicted is poised to act as a hinge for the active-site lid; differences in ACT domain dimerization would occur in formation of the Phe-stabilized Phe131 conformation can help rationalize the lid disorder in the allosterically activated tetramer. (B) The two-domain PAH tetramer (2PAH, full-length enzyme. For all of the single-domain structures, the dimer in the asymmetric unit, one white, one black), is assembled with backbone at Phe131 adopts one of two conformations, depend- identical subunits adjacent along the short edge of the tetramer (from the ing on whether the active-site lid is open or closed; however, the perspective of the catalytic domains). (C) The two-domain structure of ty- side-chain position remains more or less invariant. In the full- rosine hydroxylase (1TOH, monomer in the asymmetric unit) is symmetric. (D) Representation of full-length PAH, similar to part A, showing selected length protein, the backbone position of Phe131 is more like the distances to illustrate the asymmetry of the tetramer. (E) The 2.9 Å structure closed-lid conformation, but the side chain is in a position not (cyan), overlaid on two other, lower resolution, structures of full-length PAH seen in any of the single-domain structures. This side-chain [resolutions, 3.1 Å (black) and 3.9 Å (magenta)]. The B subunits of the three position is stabilized by a cation–π interaction between Phe131 structures were superposed. and Arg111 (Arg111 is not ordered in any of the single-domain

2396 | www.pnas.org/cgi/doi/10.1073/pnas.1516967113 Arturo et al. Downloaded by guest on September 23, 2021 A B C D (1PHZ and 2PHM), although they were not explicitly discussed (6). All of these interdomain interactions are dependent on the position of the regulatory domain, and we predict they will be forfeited in the transition to an allosterically activated tetramer containing dimerized ACT domains.

SAXS Correlates the PAH Crystal Structure with the Solution Structure in the Absence of Phe and Confirms the Existence of an Architecturally E Distinct Tetramer in the Presence of Phe. To examine the relationship between the full-length crystal structure and the oligomers of PAH that exist in solution in the absence of Phe, we performed SAXS analysis on the PAH preparations used for crystallography using size-exclusion chromatography in-line with synchrotron SAXS (20). The experimental radius of gyration (Rg), maximum interatomic distance (Dmax)(Tables S3 and S4), and pairwise shape distribution (Fig. 4A) closely match those calculated from the tetrameric crystal structure. Modeling of missing amino acids as beads on each chain was necessary for the best correlations in the higher scattering angles (χCRYSOL = 1.1–1.3 for each of 10 independent CORAL calculations) (Fig. 4A and B). These results allow us to conclude that the crystal structure is consistent with the solution structure of the PAH tet- Fig. 3. Insight into what controls the configuration of the PAH active-site ramer in the absence of Phe. However, the resolution of this ap- – lid. (A C) Space filling images of the catalytic domains of PAH structures proach does not preclude the existence of other, equally consistent, colored as in Fig. 1A, with the active site (within 10 Å of the iron ion, shown as an orange sphere) colored white and active-site ligands in sticks colored tetramer conformations under these conditions. by element. In all open structures, the RMS deviation between Cα positions In the presence of 1 mM Phe (sufficient for full allosteric in this lid is 0.3 Å; the corresponding value for closed structures is 0.2 Å. The activation of PAH), the shape of tetrameric PAH changes sig-

highest resolution examples are used for illustration. (A) 1PAH contains only nificantly (Fig. 4C, Fig. S6, and Tables S3 and S4). Invariant BIOCHEMISTRY

iron in the active site; (B) 1J8U contains iron and BH4;(C) 1MMT contains analyses (Fig. S6 and Tables S3 and S4) suggest that the Phe- – iron, BH4, and norleucine. (D) Positioned and colored as per parts A C is the stabilized conformation is not due to significant differences in current crystal structure subunit D, which contains only iron in the active site. flexibility and disorder, but rather differences in the configura- (E) An overlay of 1J8U (open, green) and 1MMT (closed, magenta) on resi- tions of structural domains. A discrete peak feature appears in – − dues 144 410 of subunit D of the current structure (disordered, gray) helps the primary data at q ∼ 0.1 Å 1 (Fig. 4C) upon Phe addition. illustrate the various lid conformations. The coloring on part E corresponds ∼ to the coloring used in Table S1. This would correlate to a 60 Å length scale, which would be expected to strongly correlate with changes in interatomic distances between globular domains. By P(r) analysis, these differ- structures). Interestingly, the Phe131 side chain adopts this same ences coincide with increases in Rg and a redistribution of position in the two-domain (regulatory plus catalytic) structures interatomic vectors to greater values (Fig. 4D and Tables S3 and 1PHZ and 2PHM. Careful scrutiny of the iterative-build com- S4). However, Dmax between the two states does not differ. In- Δ posite omit electron density (19) for our full-length enzyme spection of the difference P(r) [ P(r)] plot between these two structure suggests that a minor alternate conformation might be states (Fig. 4D, Lower) shows a redistribution from vectors at ∼ ∼ ∼ present for Phe131, wherein the side chain occupies the same 30 and 90 Å to vectors at 60 Å. The longest dimensions of position seen in the single-domain structures (Fig. S4C). The the full-length PAH tetramer are defined by the arrangement of resolution of our data are not sufficient to confidently model its catalytic domains approximated by a planar rectangle. Be- minor conformers in any detail, so the map is at best suggestive. cause Dmax does not change between autoinhibited and activated states, we can conclude that the overall arrangement of catalytic However, a 2mFo-DFc map calculated for the high-resolution domains is not significantly altered, allowing us to surmise that PAH structure 1J8U also shows density for a potential alternate the observed structural changes instead correlate with discrete conformer for Phe131 (Fig. S4B), which would position the side rearrangements of the regulatory (ACT and autoregulatory) chain similarly to Phe131 in the full-length structure (Fig. 3E). domain in each state. Thus, we propose that the side chain of Phe131, regardless of the open/closed status of the active-site lid, is able to sample two Discussion positions, one of which is stabilized by an interaction with Arg111. Full-length mammalian PAH has defied determination of a Because Arg111 lies at the C terminus of the ACT domain, this crystal structure for decades, which is not uncommon for mul- suggests Phe131 may serve as a hinge or toggle controlling the tidomain multimeric allosteric proteins. In some instances, such conformation of the active-site lid, allowing the regulatory domain proteins can accommodate alternate multimeric architectures to modulate access to the active site. with varying interdomain orientations (e.g., refs. 13, 17, 21, and 22). One well-studied example, PBGS, taught us that alternate Key Interdomain Interactions. The full-length protein structure al- conformers in a slow (or metastable) equilibrium can be sepa- lows us to evaluate interdomain and intersubunit interactions es- rated on the basis of surface charge using ion exchange chro- sential to the observed PAH tetrameric assembly. Interdomain matography (23). This method was applied to PAH and allowed interactions include all three PAH domains and involve (i)an isolation of a single tetrameric species (Fig. S1), which, we be- H-bonding network between Lys113, Asp315, and Asp-27 (Fig. lieve, facilitated crystallization. The resulting structure allows S5A); (ii)cation–π interactions between both Arg123 and Arg420 the field to progress beyond a reliance on composite homology and Phe80 (Fig. S5B); (iii) an H bond between Asn30 and Gln134 models (14). (Fig. S5C); and (iv) a cation–π interaction between Arg111 and Significant differences between the structure of full-length Phe131 (Fig. 3E, Right). Many of these interactions are seen in PAH and the composite homology model are shown in Fig. 1C, the two-domain (catalytic plus regulatory) rat PAH structure which is an overlay of the four chains of the full-length structure

Arturo et al. PNAS | March 1, 2016 | vol. 113 | no. 9 | 2397 Downloaded by guest on September 23, 2021 The structure of full-length PAH raises previously unasked questions, in addition to addressing some previously outstanding ones. For example, we still lack information on the conformational space available to the first ∼20 residues. Both NMR and molecular dynamics simulations suggest that the mobile N-terminal peptide can sample two distinct conformations in the absence of Phe, but prefers one of these when phosphorylated (24, 25). Mobility in this region is lost upon addition of sufficient Phe to fully activate PAH (25), suggesting that a structure of the fully active enzyme may reveal more information about this region. Also, although our structural work reveals how conformational variability in the C terminus drives differences in tetramer ge- ometry, we have not yet deciphered the precise determinants controlling why the C-terminal helix adopts the positions it does in the two-domain PAH structure (2PAH, Fig. 2B), the tyrosine hydroxylase tetramer (1TOH, Fig. 2C) (16), or the full-length PAH structure (Fig. 2A). A related question is whether the positioning of the C-terminal helices seen in the two-domain tetramer (2PAH) reflects other tetrameric conformations of full-length PAH, for which structures are not yet known (e.g., the slower- migrating tetramer, phosphorylated PAH, and/or the allosterically activated tetramer). It is also possible that some of the conformational differences between the full-length and 2PAH structures derives from sequence differences between rat and human PAH. Fig. 4. SAXS analyses. SAXS data obtained on the PAH tetramers in the One significant difference between mammalian PAH and the absence of Phe is illustrated in parts A and B, and compared with the data in other aromatic amino acid hydroxylases is the sequence of the the presence of 1 mM Phe in parts C and D.(A) The pairwise shape distri- C-terminal helix. This region of tyrosine hydroxylase and trypto- bution function [P(r)] for isolated PAH in the absence of Phe (blue circles). phan hydroxylase contains classic leucine heptad repeats, which (B) A log–log plot (log I vs. log q) is a representative fit from CORAL rigid- are absent in PAH (26); neither of which shows any propensity for body refinement (red line) vs. the experimental SAXS data (black circles) for tetramer dissociation. The lack of a classic leucine zipper may PAH in the absence of Phe. In B and C, error bars represent the combined standard uncertainty of the data collection. A representative SAXS-refined explain not only why PAH can dissociate to dimers but also the CORAL structure is shown (Inset). The fixed atomic inventory from the crystal conformational variability in the C-terminal helices (Fig. 2E). structure is gray, and the modeled inventory is as blue spheres. (C) A com- The most significant gap in our knowledge is the structure of parison of PAH before and after incubation with 1 mM Phe is shown as a the activated PAH tetramer, which can putatively be stabilized superposed log–log plot (log I vs. log q) of PAH before (blue) and after (red). by allosteric Phe binding. Because we introduced the idea that an χ2 Using a modified (34), the Fr. 15 (-Phe) SAXS data shows a discrepancy of activated tetramer contains an ACT domain dimer and that this 0.9 vs. the crystal structure, whereas in the presence of 1 mM Phe this dis- dimer interface is the site of allosteric Phe binding (12), several crepancy increases to 2.2. Using the more discerning volatility of ratio metric new studies have provided support for this concept (27–29). We [Vr, where identity is 0 and larger figures indicate higher discrepancy (34)], the Fr. 15 SAXS data show similar concordance to the structure (Vr = 4.4), suggest a model for the activated PAH tetramer (Fig. S2A) that whereas in the 1 mM Phe state this discrepancy increase significantly includes such an ACT domain dimer, similar to that of the = (Vr 10.9). Shown below is a ratio plot (green), revealing discrepancy be- regulatory domain of tyrosine hydroxylase (28). However, other tween the two profiles as a function of q; identical regions will have a ratio architectures are possible, such as that of the tyrosine-binding ∼ value of 1, whereas regions of higher discrepancy will have values that ACT domain dimer of 3-deoxyheptulosonic acid 7-phosphate deviate from unity. Errors shown represent propagated counting statistics. (D) The shape distributions determined for rat PAH in the absence (blue synthase (30). Also, as noted above, several possible geometries circles) and presence (red circles) of 1 mM Phe. In the lower panel is ΔP(r) are possible for the C-terminal four-helix bundle in the activated analysis (green); errors represent propagated errors from the initial inverse tetramer. A third unknown regarding an activated PAH structure Fourier transform. is the conformation of the autoregulatory region (amino acids 1– 32). Our SAXS data confirm that the shape of PAH changes significantly between low and high Phe concentrations, and are with the two truncated structures used to construct the composite consistent with a redistribution of the ACT domains. However, model (1PHZ and 2PAH) (6, 16). The composite homology none of the candidate models for the high-activity PAH tetramer model correctly predicts the relative orientation of the catalytic and regulatory domains, which allows the autoregulatory region to provides a satisfying correlation with the SAXS data at higher partially occlude the enzyme active site (Fig. 1B, Top). This ori- scattering angles, even when modeled using CORAL, empha- entation also allows Arg111 to modulate the position of Phe131 sizing the need for additional structural efforts. and control the conformation of the active-site lid (Fig. 3E). Our crystal structure of full-length PAH occupies an impor- However, the composite homology model incorrectly predicted tant position along the continuum of different PAH structures, the C-terminal helix positions (Fig. 1C) and the overall asymmetry and replaces a composite homology model as an improved of the tetramer (Fig. 2D). Careful attention to the homology context for understanding PKU-associated PAH variants. A full model predicted a rarely referenced steric clash between one helix understanding of genotype–phenotype relationships will likely position and Leu72 in the ACT domain (Fig. 1C, arrow). The await the details of the Phe-stabilized activated PAH tetramer crystal structure reveals no such clash. However, steric interactions structure, which we have demonstrated differs significantly from around Leu72 may still prove relevant, because Phe-modulated that of the autoinhibited tetramer. Nevertheless, the auto- activation is predicted to include the ACT domain rotating away inhibited structure identifies key interdomain interactions that from the clash point (Fig. S2B); hence this site may serve as a are likely to change in the transition between autoinhibited and conformational switch serving allosteric activation. activated PAH tetramer.

2398 | www.pnas.org/cgi/doi/10.1073/pnas.1516967113 Arturo et al. Downloaded by guest on September 23, 2021 Materials and Methods SAXS Data Analysis and Modeling. The details of data acquisition for SAXS Protein Expression and Purification. Full-length rat PAH was expressed in BLR- measurements are provided in SI Materials and Methods. Modeling of the DE3 cells using a 2-d expression as described (31), with the exception that full-length PAH tetramer against its solution scatter was performed using ferrous ammonium sulfate (0.2 mM) was added for the expression phase of the program CORAL (32). The known structure was fixed and inventory not the procedure. Protein was purified as previously described (12). Phenyl resolved by crystallography were modeled using beads. Ten independent Sepharose-purified protein (28 mg) was applied to a 1-mL HiTrap Q column calculations in each state were performed and yielded comparable results. The final models were assessed using the program CRYSOL (33). preequilibrated with 30 mM Tris·HCl, pH 7.4, 20 mM KCl, 15% (vol/vol) glycerol. Following a 20 column-volume wash, PAH assemblies were resolved using a linear 30 column-volume gradient to a salt concentration of 0.4 M ACKNOWLEDGMENTS. We acknowledge Thomas Scary, Ursula Ramirez, Sarah H. Lawrence, and Jinhua Wu for contributions in optimizing crystallization and KCl, keeping other buffer components the same. Two-milliliter fractions cryoprotection conditions, and Mark Andrake for constructing the PAH model were collected (Fig. S1). shown in Fig. S2A (FCCC Molecular Modeling Facility). We acknowledge SAXS data collected at the Australian Synchrotron, access provided by the New Crystal Growth and Crystal Structure Determination. The protein used for Zealand Synchrotron Group. Grant support for E.K.J. was from Developmental crystallization was taken from a HiTrap Q column fraction highly enriched in Therapeutics Program at the Fox Chase Cancer Center, National Cancer Insti- the faster-migrating tetramer (e.g., fraction 15, Fig. S1); details for crystal- tute Comprehensive Cancer Center Grant P30CA006927, and the Pennsylvania lization, model building, and refinement are provided in SI Materials and Tobacco Settlement Fund (CURE). Use of the Synchrotron at Brookhaven Na- tional Laboratory was supported by the US Department of Energy, Office of Methods. The structure was determined from the diffraction data collected Science, Office of Basic Energy Sciences under Contract DE-AC02-98CH10886. from a single crystal at 100 K at the National Synchrotron Light Source The Life-Science and Biomedical Technology Research Resource was supported beamline X25. Phases were obtained by molecular replacement using the by the US Department of Energy, Office of Biological and Environmental highest-resolution two-domain (regulatory and catalytic) structure of rat Research (Grant P41RR012408), and by the National Center for Research PAH [1PHZ (6)]. Resources of the National Institutes of Health (Grant P41GM103473).

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