First structure of full-length mammalian phenylalaninehydroxylase reveals the architecture of anautoinhibited tetramerEmilia 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, DrexelUniversity 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-namics, and function for the enzyme phenylalanine hydroxylase(PAH) can lead to needed new therapies for phenylketonuria, themost common inborn error of amino acid metabolism. PAH is amultidomain homo-multimeric protein whose conformation andmultimerization properties respond to allosteric activation by thesubstrate phenylalanine (Phe); the allosteric regulation is neces-sary to maintain Phe below neurotoxic levels. A recently introducedmodel for allosteric regulation of PAH involves major domainmotions and architecturally distinct PAH tetramers [Jaffe EK, Stith L,Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Bio-phys 530(2):73–82]. Herein, we present, to our knowledge, the firstX-ray crystal structure for a full-length mammalian (rat) PAH in anautoinhibited conformation. Chromatographic isolation of a mono-disperse tetrameric PAH, in the absence of Phe, facilitated determi-nation of the 2.9 Å crystal structure. The structure of full-length PAHsupersedes a composite homology model that had been used exten-sively to rationalize phenylketonuria genotype–phenotype relation-ships. 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 structuraldetail for activated PAH remains a barrier to complete understandingof phenylketonuria genotype–phenotype relationships. Nevertheless,the use of SAXS and X-ray crystallography together to inspect PAHstructure provides, to our knowledge, the first complete view of theenzyme in a tetrameric form that was not possible with prior partialcrystal structures, and facilitates interpretation of a wealth of bio-chemical and structural data that was hitherto impossible to evaluate.

phenylalanine hydroxylase | phenylketonuria | X-ray crystallography |small-angle X-ray scattering | allosteric regulation

Mammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1)is a multidomain homo-multimeric protein whose dys-

function causes the most common inborn error in amino acidmetabolism, phenylketonuria (PKU), and milder forms of hy-perphenylalaninemia (OMIM 261600) (1). PAH catalyzes thehydroxylation of phenylalanine (Phe) to tyrosine, using nonhemeiron and the cosubstrates tetrahydrobiopterin and molecular oxygen(2, 3). A detailed kinetic mechanism has recently been derived fromelegant single-turnover studies (4). PAH activity must be carefullyregulated, because although Phe is an essential amino acid, highPhe levels are neurotoxic. Thus, Phe allosterically activates PAH bybinding to a regulatory domain. Phosphorylation at Ser16 potenti-ates the effects of Phe, with phosphorylated PAH achieving fullactivation at lower Phe concentrations than the unphosphorylatedprotein (5, 6). Allosteric activation by Phe is accompanied by amajor conformational change, as evidenced by changes in proteinfluorescence and proteolytic susceptibility, and by stabilization of atetrameric conformer (3).

There are >500 disease-associated missense variants of humanPAH; the amino acid substitutions are distributed throughoutthe 452-residue protein and among all its domains (Fig. 1A)(7–9). Of those disease-associated variants that have been stud-ied in vitro (e.g., ref. 10), some confound the allosteric response,and some are interpreted as structurally unstable. We also sug-gest that the activities of some disease-associated variants may bedysregulated by an altered equilibrium among conformers havingdifferent intrinsic levels of activity, arguing by analogy to theenzyme porphobilinogen synthase (PBGS) and its porphyria-associated variants (11). Consistent with this notion, we haverecently established that PAH can assemble into architecturallydistinct tetrameric conformers (12), and propose that theseconformers differ in activity due to differences in active-site ac-cess. This idea has important implications for drug discovery, asit implies that small molecules could potentially modulate theconformational equilibrium of PAH, as has already been dem-onstrated for PBGS (e.g., ref. 13). Deciphering the relationshipamong PAH structure, dynamics, and function is a necessary firststep in testing this hypothesis.

Significance

Phenylketonuria and milder hyperphenylalaninemias consti-tute the most common inborn error of amino acid metabo-lism, usually caused by defective phenylalanine hydroxylase(PAH). Although a highly restricted diet prevents intellectualimpairment during development, additional therapies are re-quired to combat cognitive dysfunction, executive dysfunc-tion, and psychiatric disorders that arise due to dietary lapsesthroughout life. New therapies can arise from thorough un-derstanding of the conformational space available to full-length PAH, which has defied crystal structure determinationfor decades. We present the first X-ray crystal structure offull-length PAH, whose solution relevance is supported bysmall-angle X-ray scattering. The current structure is an auto-inhibited tetramer; the scattering data support the existenceof an architecturally distinct tetramer that is stabilized by theallosteric activator phenylalanine.

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;and E.C.A., K.G., P.J.L., and E.K.J. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 5DEN).1To whom correspondence should be addressed. Email: Eileen.Jaffe@fccc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1516967113/-/DCSupplemental.

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Numerous crystal structures are known for one- and two-domain constructs of mammalian PAH (Table S1). However, untilnow, no structure has been available for the full-length, three-

domain enzyme, possibly because the presence of multiple dis-tinct conformers frustrated efforts to crystallize the full-lengthprotein. Recognition of the existence of alternate tetramericassemblies and the ability to isolate a single species has nowallowed us to generate monodisperse samples of full-length ratPAH suitable for biophysical analysis using X-ray crystallographyand small-angle X-ray scattering (SAXS). We report here, to ourknowledge, the first crystal structure for full-length PAH, at aresolution of 2.9 Å, which supersedes a composite homologymodel that has been used since 1999 to help rationalize PKU-relatedgenotype/phenotype relationships (14).

ResultsPAH Crystal Structure. This study focuses on rat PAH, which is 96%similar (92% identical) to the human enzyme. The 15 mostcommon disease-associated amino acids are fully conserved be-tween rat and human, making the rodent protein an excellentmodel system for study of PKU. Rat PAH, heterologouslyexpressed in Escherichia coli and purified via phenyl-Sepharoseaffinity chromatography (15), can be further fractionated on anion-exchange column to partially resolve two tetrameric species(faster migrating and slower migrating) and one dimeric species,as determined by native PAGE (Fig. S1). In fractionated PAHsamples, the distribution of these species is stable over long timecourses, suggesting that these fractions are good starting points forcrystallization trials. The faster-migrating tetramer is the pre-dominant component in these preparations (12), and using thefraction most enriched in this species, we were able to producewell-diffracting crystals that yielded a 2.9 Å structure (Fig. 1B andTable S2), to our knowledge, the first structure for any full-lengthmammalian PAH. The full-length structure and the previouslyused composite homology model are compared in Fig. 1C [withthe caveat that the composite model combined a two-domain ratPAH 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].The crystal asymmetric unit contains one tetramer, which

adopts an autoinhibited form in which the autoregulatory regionpartially occludes the enzyme active site. Continuous electrondensity can be observed for residues 20–136 and 143–450 withineach of the four protomers in the tetramer (with some variation ateach termini), as well as for one iron ion per chain and two watersat the active site of each chain. The catalytic domains of the fourprotomers are arranged in approximate 222 symmetry, where eachcatalytic domain occupies one corner of a rectangle. The ar-rangement can be described as a dimer-of-dimers, where the di-mers with the greatest buried surface area lie along the short edgeof the rectangle (Fig. 1B). Although each short-edge dimer (theBD dimer or the AC dimer) is intimately assembled, the spacingbetween these two dimers is large, resulting in very little dimer–dimer interaction along the long edge of the tetramer, and hence arectangular rather than square arrangement. The ACT domainsextend above and below the plane of this rectangle (Fig. 1B,Bottom). The four C-terminal multimerization domains containα-helices that assemble into an antiparallel bundle in the center ofthis tetrameric arrangement. The catalytic domain of each pro-tomer contains an active site that is partly occluded by theautoregulatory region and a partially disordered active-site lid(residues ∼130–150). The conformations of the catalytic andregulatory domains of the four protomers are highly similar, withRMS deviations of ∼0.3 Å across Cα atoms. The C-terminal he-lices of the four protomers adopt two distinct orientations withrespect to the catalytic domain, differing by a tilt of ∼10° (Fig. 1C);protomers situated across the diagonal of the tetramer containsimilarly positioned helices (Figs. 1C and 2A). This asymmetry isunexpectedly different from the tetramer apparent in the two-domain human PAH structure, where similarly positioned helicesare within each short-edge dimer (Fig. 2B). Because the C-terminal

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Fig. 1. The structure of PAH. (A) The annotated domain structure of mam-malian PAH. (B) The 2.9 Å PAH crystal structure in orthogonal views, colored asin part A, subunit A is shown in ribbons; subunit B is as a Cα trace; subunit C is insticks; and subunit D is in transparent spheres. In cyan, the subunits are labelednear the catalytic domain (Top); in red, they are labeled near the regulatorydomain (Bottom). The dotted black circle illustrates the autoregulatory domainpartially occluding the enzyme active site (iron, in orange sphere). (C) Com-parison of the subunit structures of full-length PAH and those of the compositehomology model; the subunit overlay aligns residues 144–410. The four subunitsof the full-length PAH structure (the diagonal pairs of subunits are illustratedusing 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, theregulatory domain is in ribbons, and themultimerization domain is as a Cα trace.The arrow denotes where the ACT domain and one helix of 2PAH conflict.

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helices form a four-helix bundle in the center of the PAH tet-ramer, these differences in their positions give rise to asymmetryand cause the architecture of the tetramer to twist and deviatesignificantly from the bona fide 222 symmetry found in the two-domain tyrosine hydroxylase structure (Fig. 2C).The short-edge dimers form extensive interfaces (buried areas

of 6,512 and 7,033 Å2 for the BC and AD dimers, respectively)between the catalytic domain of one subunit and the regulatorydomain of the adjacent subunit; this interface also includes thatportion of the multimerization domain that precedes the C-ter-minal helix. Interestingly, the short-edge dimer interface involves8 aa whose substitutions are disease-associated, including threeof the most common PKU-associated variants, I65T, R68S, andR413P. The twist between the short-edge dimers means that theother dimers (long-face and diagonal) interact predominantlythrough the C-terminal helices and are characterized by signifi-cantly smaller buried interfaces [the long-edge (AB and CD) andthe diagonal (BC and AD) dimers bury 1,412, 766, 435, and258 Å2, respectively]. This twist also causes the distance betweenthe ACT domains to differ on the two faces of the tetramer; theends of the C-terminal helices extend toward this space betweenthe ACT domains. None of the chains are ordered all of the wayto their most C-terminal residue, but the helices on the side ofthe tetramer with the closer ACT spacing contain more order(the C-terminal three and four residues are disordered on chainsA and D, respectively, whereas the C-terminal six residues are

disordered on chains B and C). Interestingly, in the two-domainhuman PAH structure (2PAH), the C-terminal helices are fullyordered. Thus, the twist between the short-edge dimers in thestructure of full-length PAH is accompanied by a shift in theenvironment at the ends of C-terminal helices that no longerfavors ordered packing.The C-terminal helices form an antiparallel four-helix bundle

that is the central point of tetramer association. These helicesare conformationally flexible, which is evident in the noniso-morphism we observe among multiple PAH crystals. We havepartially refined two additional structures for full-length PAH atlower resolution, using crystals grown under essentially identicalconditions. In these structures, the position of the C-terminalhelix varies, changing the relative positions of the two dimerswithin the tetramer (Fig. 2E). If one superposes all B subunits,the BD short-edge dimers align well, but different twists aboutthe C-terminal helices cause positions of the AC dimers to varyby as much as ∼7 Å. This unusual level of flexibility in the PAHfour-helix bundle may be responsible for some of the enzyme’sunusual kinetic characteristics, which caused us to initiallyidentify it as a putative morpheein (17).

PAH Active-Site Access. Two structural elements govern PAH ac-tive-site access, the autoregulatory region and the active-site lid.In our PAH structure, residues 20–25 of the autoregulatory re-gion lie across the opening to the active site; we have postulatedthat this occlusion is relieved by a Phe-modulated formation ofan ACT domain dimer (Fig. S2A) (12). The insight that regu-latory domain positioning may govern active-site access shedsnew light on deciphering the order of catalytic events, becauseprevious mechanistic studies have uniformly used PAH that lacksthe regulatory domain (e.g., refs. 2 and 4). The other structuralelement governing active-site access is an active-site lid or loop(approximately residues 130–150) (18). In all 15 structures of theisolated human PAH catalytic domain, the lid is fully orderedand exists in one of two different conformations (“open” vs.“closed”), dependent upon active-site occupancy (color coded inTable S1, illustrated in Fig. 3 A–C). The lid is open unless threedifferent ligands are all present: iron, a Phe analog, and a pterin.In contrast, in all structures of multidomain rat and human PAH(including our structure of the full-length enzyme), the lid ispartially disordered, specifically at amino acids ∼137–142 (e.g.,Fig. 3D), which is the portion of the lid that differs most betweenthe open and closed conformations. For these multidomainstructures, most of the portions of the lid that can be seen alignbest 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-likethan open (Fig. 3E, Left). In the single-domain structures wherethe lid is fully ordered, B factors for lid residues are higher thanthose for the rest of the structure (Fig. S3), consistent with thenotion that this region is a mobile element that can change con-formation—opening and closing the active site—as part of the en-zyme’s catalytic function, perhaps in response to regulatory signals.A notable difference between the structure of full-length rat

PAH and those of the isolated human PAH catalytic domain isthe conformation at Phe131 (Fig. 3E, Right, and Fig. S4A), whichis poised to act as a hinge for the active-site lid; differences inPhe131 conformation can help rationalize the lid disorder in thefull-length enzyme. For all of the single-domain structures, thebackbone at Phe131 adopts one of two conformations, depend-ing on whether the active-site lid is open or closed; however, theside-chain position remains more or less invariant. In the full-length protein, the backbone position of Phe131 is more like theclosed-lid conformation, but the side chain is in a position notseen in any of the single-domain structures. This side-chainposition is stabilized by a cation–π interaction between Phe131and Arg111 (Arg111 is not ordered in any of the single-domain

Two-domain tyrosine hydroxylase

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Fig. 2. Overall architectures of PAH and related structures. (A) Structure offull-length PAH showing that subunits with similar structures (white likewhite, black like black) are positioned across the diagonal of the tetramer,when viewed from the perspective of the catalytic domains (Top). The reg-ulatory domains of these similarly structured subunits are on the same sideof the tetramer (Bottom). These are the subunits for which the predictedACT domain dimerization would occur in formation of the Phe-stabilizedallosterically activated tetramer. (B) The two-domain PAH tetramer (2PAH,dimer in the asymmetric unit, one white, one black), is assembled withidentical subunits adjacent along the short edge of the tetramer (from theperspective of the catalytic domains). (C) The two-domain structure of ty-rosine hydroxylase (1TOH, monomer in the asymmetric unit) is symmetric.(D) Representation of full-length PAH, similar to part A, showing selecteddistances to illustrate the asymmetry of the tetramer. (E) The 2.9 Å structure(cyan), overlaid on two other, lower resolution, structures of full-length PAH[resolutions, 3.1 Å (black) and 3.9 Å (magenta)]. The B subunits of the threestructures were superposed.

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structures). Interestingly, the Phe131 side chain adopts this sameposition in the two-domain (regulatory plus catalytic) structures1PHZ and 2PHM. Careful scrutiny of the iterative-build com-posite omit electron density (19) for our full-length enzymestructure suggests that a minor alternate conformation might bepresent for Phe131, wherein the side chain occupies the sameposition seen in the single-domain structures (Fig. S4C). Theresolution of our data are not sufficient to confidently modelminor conformers in any detail, so the map is at best suggestive.However, a 2mFo-DFc map calculated for the high-resolutionPAH structure 1J8U also shows density for a potential alternateconformer for Phe131 (Fig. S4B), which would position the sidechain similarly to Phe131 in the full-length structure (Fig. 3E).Thus, we propose that the side chain of Phe131, regardless of theopen/closed status of the active-site lid, is able to sample twopositions, one of which is stabilized by an interaction with Arg111.Because Arg111 lies at the C terminus of the ACT domain, thissuggests Phe131 may serve as a hinge or toggle controlling theconformation of the active-site lid, allowing the regulatory domainto modulate access to the active site.

Key Interdomain Interactions. The full-length protein structure al-lows us to evaluate interdomain and intersubunit interactions es-sential to the observed PAH tetrameric assembly. Interdomaininteractions include all three PAH domains and involve (i) anH-bonding network between Lys113, Asp315, and Asp-27 (Fig.S5A); (ii) cation–π interactions between both Arg123 and Arg420and Phe80 (Fig. S5B); (iii) an H bond between Asn30 and Gln134(Fig. S5C); and (iv) a cation–π interaction between Arg111 andPhe131 (Fig. 3E, Right). Many of these interactions are seen inthe two-domain (catalytic plus regulatory) rat PAH structure

(1PHZ and 2PHM), although they were not explicitly discussed(6). All of these interdomain interactions are dependent on theposition of the regulatory domain, and we predict they will beforfeited in the transition to an allosterically activated tetramercontaining dimerized ACT domains.

SAXS Correlates the PAH Crystal Structure with the Solution Structurein the Absence of Phe and Confirms the Existence of an ArchitecturallyDistinct Tetramer in the Presence of Phe. To examine the relationshipbetween the full-length crystal structure and the oligomers ofPAH that exist in solution in the absence of Phe, we performedSAXS analysis on the PAH preparations used for crystallog-raphy using size-exclusion chromatography in-line with syn-chrotron SAXS (20). The experimental radius of gyration (Rg),maximum interatomic distance (Dmax) (Tables S3 and S4), andpairwise shape distribution (Fig. 4A) closely match those cal-culated from the tetrameric crystal structure. Modeling ofmissing amino acids as beads on each chain was necessary forthe 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 crystalstructure is consistent with the solution structure of the PAH tet-ramer in the absence of Phe. However, the resolution of this ap-proach does not preclude the existence of other, equally consistent,tetramer conformations under these conditions.In the presence of 1 mM Phe (sufficient for full allosteric

activation of PAH), the shape of tetrameric PAH changes sig-nificantly (Fig. 4C, Fig. S6, and Tables S3 and S4). Invariantanalyses (Fig. S6 and Tables S3 and S4) suggest that the Phe-stabilized conformation is not due to significant differences inflexibility and disorder, but rather differences in the configura-tions of structural domains. A discrete peak feature appears inthe primary data at q ∼ 0.1 Å−1 (Fig. 4C) upon Phe addition.This would correlate to a ∼60 Å length scale, which would beexpected to strongly correlate with changes in interatomic dis-tances between globular domains. By P(r) analysis, these differ-ences coincide with increases in Rg and a redistribution ofinteratomic vectors to greater values (Fig. 4D and Tables S3 andS4). However, Dmax between the two states does not differ. In-spection of the difference P(r) [ΔP(r)] plot between these twostates (Fig. 4D, Lower) shows a redistribution from vectors at∼30 and ∼90 Å to vectors at ∼60 Å. The longest dimensions ofthe full-length PAH tetramer are defined by the arrangement ofits catalytic domains approximated by a planar rectangle. Be-cause Dmax does not change between autoinhibited and activatedstates, we can conclude that the overall arrangement of catalyticdomains is not significantly altered, allowing us to surmise thatthe observed structural changes instead correlate with discreterearrangements of the regulatory (ACT and autoregulatory)domain in each state.

DiscussionFull-length mammalian PAH has defied determination of acrystal structure for decades, which is not uncommon for mul-tidomain multimeric allosteric proteins. In some instances, suchproteins can accommodate alternate multimeric architectureswith varying interdomain orientations (e.g., refs. 13, 17, 21, and22). One well-studied example, PBGS, taught us that alternateconformers in a slow (or metastable) equilibrium can be sepa-rated on the basis of surface charge using ion exchange chro-matography (23). This method was applied to PAH and allowedisolation of a single tetrameric species (Fig. S1), which, we be-lieve, facilitated crystallization. The resulting structure allowsthe field to progress beyond a reliance on composite homologymodels (14).Significant differences between the structure of full-length

PAH and the composite homology model are shown in Fig. 1C,which is an overlay of the four chains of the full-length structure

A

E

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Fig. 3. Insight into what controls the configuration of the PAH active-sitelid. (A–C) Space filling images of the catalytic domains of PAH structurescolored as in Fig. 1A, with the active site (within 10 Å of the iron ion, shownas an orange sphere) colored white and active-site ligands in sticks coloredby element. In all open structures, the RMS deviation between Cα positionsin this lid is 0.3 Å; the corresponding value for closed structures is 0.2 Å. Thehighest resolution examples are used for illustration. (A) 1PAH contains onlyiron in the active site; (B) 1J8U contains iron and BH4; (C) 1MMT containsiron, BH4, and norleucine. (D) Positioned and colored as per parts A–C is thecurrent crystal structure subunit D, which contains only iron in the active site.(E) An overlay of 1J8U (open, green) and 1MMT (closed, magenta) on resi-dues 144–410 of subunit D of the current structure (disordered, gray) helpsillustrate the various lid conformations. The coloring on part E correspondsto the coloring used in Table S1.

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with the two truncated structures used to construct the compositemodel (1PHZ and 2PAH) (6, 16). The composite homologymodel correctly predicts the relative orientation of the catalyticand regulatory domains, which allows the autoregulatory region topartially occlude the enzyme active site (Fig. 1B, Top). This ori-entation also allows Arg111 to modulate the position of Phe131and control the conformation of the active-site lid (Fig. 3E).However, the composite homology model incorrectly predictedthe C-terminal helix positions (Fig. 1C) and the overall asymmetryof the tetramer (Fig. 2D). Careful attention to the homologymodel predicted a rarely referenced steric clash between one helixposition and Leu72 in the ACT domain (Fig. 1C, arrow). Thecrystal structure reveals no such clash. However, steric interactionsaround Leu72 may still prove relevant, because Phe-modulatedactivation is predicted to include the ACT domain rotating awayfrom the clash point (Fig. S2B); hence this site may serve as aconformational switch serving allosteric activation.

The structure of full-length PAH raises previously unaskedquestions, in addition to addressing some previously outstandingones. For example, we still lack information on the conformationalspace available to the first ∼20 residues. Both NMR and molec-ular dynamics simulations suggest that the mobile N-terminalpeptide can sample two distinct conformations in the absence ofPhe, but prefers one of these when phosphorylated (24, 25).Mobility in this region is lost upon addition of sufficient Phe tofully activate PAH (25), suggesting that a structure of the fullyactive enzyme may reveal more information about this region.Also, although our structural work reveals how conformationalvariability in the C terminus drives differences in tetramer ge-ometry, we have not yet deciphered the precise determinantscontrolling why the C-terminal helix adopts the positions it does inthe two-domain PAH structure (2PAH, Fig. 2B), the tyrosinehydroxylase tetramer (1TOH, Fig. 2C) (16), or the full-lengthPAH structure (Fig. 2A). A related question is whether the posi-tioning of the C-terminal helices seen in the two-domain tetramer(2PAH) reflects other tetrameric conformations of full-lengthPAH, for which structures are not yet known (e.g., the slower-migrating tetramer, phosphorylated PAH, and/or the allostericallyactivated tetramer). It is also possible that some of the confor-mational differences between the full-length and 2PAH structuresderives from sequence differences between rat and human PAH.One significant difference between mammalian PAH and the

other aromatic amino acid hydroxylases is the sequence of theC-terminal helix. This region of tyrosine hydroxylase and trypto-phan hydroxylase contains classic leucine heptad repeats, whichare absent in PAH (26); neither of which shows any propensity fortetramer dissociation. The lack of a classic leucine zipper mayexplain not only why PAH can dissociate to dimers but also theconformational variability in the C-terminal helices (Fig. 2E).The most significant gap in our knowledge is the structure of

the activated PAH tetramer, which can putatively be stabilizedby allosteric Phe binding. Because we introduced the idea that anactivated tetramer contains an ACT domain dimer and that thisdimer interface is the site of allosteric Phe binding (12), severalnew studies have provided support for this concept (27–29). Wesuggest a model for the activated PAH tetramer (Fig. S2A) thatincludes such an ACT domain dimer, similar to that of theregulatory domain of tyrosine hydroxylase (28). However, otherarchitectures are possible, such as that of the tyrosine-bindingACT domain dimer of 3-deoxyheptulosonic acid 7-phosphatesynthase (30). Also, as noted above, several possible geometriesare possible for the C-terminal four-helix bundle in the activatedtetramer. A third unknown regarding an activated PAH structureis the conformation of the autoregulatory region (amino acids 1–32). Our SAXS data confirm that the shape of PAH changessignificantly between low and high Phe concentrations, and areconsistent with a redistribution of the ACT domains. However,none of the candidate models for the high-activity PAH tetramerprovides a satisfying correlation with the SAXS data at higherscattering angles, even when modeled using CORAL, empha-sizing the need for additional structural efforts.Our crystal structure of full-length PAH occupies an impor-

tant position along the continuum of different PAH structures,and replaces a composite homology model as an improvedcontext for understanding PKU-associated PAH variants. A fullunderstanding of genotype–phenotype relationships will likelyawait the details of the Phe-stabilized activated PAH tetramerstructure, which we have demonstrated differs significantly fromthat of the autoinhibited tetramer. Nevertheless, the auto-inhibited structure identifies key interdomain interactions thatare likely to change in the transition between autoinhibited andactivated PAH tetramer.

Fig. 4. SAXS analyses. SAXS data obtained on the PAH tetramers in theabsence of Phe is illustrated in parts A and B, and compared with the data inthe presence of 1 mM Phe in parts C and D. (A) The pairwise shape distri-bution function [P(r)] for isolated PAH in the absence of Phe (blue circles).(B) A log–log plot (log I vs. log q) is a representative fit from CORAL rigid-body refinement (red line) vs. the experimental SAXS data (black circles) forPAH in the absence of Phe. In B and C, error bars represent the combinedstandard uncertainty of the data collection. A representative SAXS-refinedCORAL structure is shown (Inset). The fixed atomic inventory from the crystalstructure is gray, and the modeled inventory is as blue spheres. (C) A com-parison of PAH before and after incubation with 1 mM Phe is shown as asuperposed log–log plot (log I vs. log q) of PAH before (blue) and after (red).Using a modified χ2 (34), the Fr. 15 (-Phe) SAXS data shows a discrepancy of0.9 vs. the crystal structure, whereas in the presence of 1 mM Phe this dis-crepancy increases to 2.2. Using the more discerning volatility of ratio metric[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),whereas in the 1 mM Phe state this discrepancy increase significantly(Vr = 10.9). Shown below is a ratio plot (green), revealing discrepancy be-tween the two profiles as a function of q; identical regions will have a ratiovalue of ∼1, whereas regions of higher discrepancy will have values thatdeviate from unity. Errors shown represent propagated counting statistics.(D) The shape distributions determined for rat PAH in the absence (bluecircles) and presence (red circles) of 1 mM Phe. In the lower panel is ΔP(r)analysis (green); errors represent propagated errors from the initial inverseFourier transform.

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Materials and MethodsProtein Expression and Purification. Full-length rat PAH was expressed in BLR-DE3 cells using a 2-d expression as described (31), with the exception thatferrous ammonium sulfate (0.2 mM) was added for the expression phase ofthe procedure. Protein was purified as previously described (12). PhenylSepharose-purified protein (28 mg) was applied to a 1-mL HiTrap Q columnpreequilibrated with 30 mM Tris·HCl, pH 7.4, 20 mM KCl, 15% (vol/vol)glycerol. Following a 20 column-volume wash, PAH assemblies were resolvedusing a linear 30 column-volume gradient to a salt concentration of 0.4 MKCl, keeping other buffer components the same. Two-milliliter fractionswere collected (Fig. S1).

Crystal Growth and Crystal Structure Determination. The protein used forcrystallization was taken from a HiTrap Q column fraction highly enriched inthe faster-migrating tetramer (e.g., fraction 15, Fig. S1); details for crystal-lization, model building, and refinement are provided in SI Materials andMethods. The structure was determined from the diffraction data collectedfrom a single crystal at 100 K at the National Synchrotron Light Sourcebeamline X25. Phases were obtained by molecular replacement using thehighest-resolution two-domain (regulatory and catalytic) structure of ratPAH [1PHZ (6)].

SAXS Data Analysis and Modeling. The details of data acquisition for SAXSmeasurements are provided in SI Materials and Methods. Modeling of thefull-length PAH tetramer against its solution scatter was performed usingthe program CORAL (32). The known structure was fixed and inventory notresolved by crystallography were modeled using beads. Ten independentcalculations in each state were performed and yielded comparable results.The final models were assessed using the program CRYSOL (33).

ACKNOWLEDGMENTS.We acknowledge Thomas Scary, Ursula Ramirez, SarahH. Lawrence, and Jinhua Wu for contributions in optimizing crystallization andcryoprotection conditions, and Mark Andrake for constructing the PAH modelshown in Fig. S2A (FCCC Molecular Modeling Facility). We acknowledge SAXSdata collected at the Australian Synchrotron, access provided by the NewZealand Synchrotron Group. Grant support for E.K.J. was from DevelopmentalTherapeutics Program at the Fox Chase Cancer Center, National Cancer Insti-tute Comprehensive Cancer Center Grant P30CA006927, and the PennsylvaniaTobacco Settlement Fund (CURE). Use of the Synchrotron at Brookhaven Na-tional Laboratory was supported by the US Department of Energy, Office ofScience, Office of Basic Energy Sciences under Contract DE-AC02-98CH10886.The Life-Science and Biomedical Technology Research Resource was supportedby the US Department of Energy, Office of Biological and EnvironmentalResearch (Grant P41RR012408), and by the National Center for ResearchResources of the National Institutes of Health (Grant P41GM103473).

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