A new model for allosteric regulation of phenylalanine hydroxylase: implications for disease and therapeutics

Abstract

The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ∼90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the intersubunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic.

Figure 1. Mammalian PAH structure

(a) The domain structure of mammalian PAH, numbered for the human protein. Wedges denote hinge regions. (b) On the left are two orientations of the human PAH monomer model, colored as in part a. The active site Fe is shown as an orange sphere. The transparent space filling representation illustrates the N-terminal region covering the active site. The catalytic and multimerization domain structures derive from PDB id 2PAH; the regulatory domain is a homology model based on the rat PAH structure PDBid 1PHZ, which contains both the regulatory and catalytic domains. The human PAH two-domain crystal structure is a tetramer; the corresponding tetramer for the three-domain model is shown on the right. This represents the low activity tetramer designated 4mer*. Also represented is the 2mer* ⇔ 4mer* equilibrium. (c) Following the organization of part b is the PAH model proposed to represent the high activity tetramer designated 4mer. The black circle represents the 4mer-specific subunit-subunit interaction. Also illustrated is the 2mer ⇔ 4mer equilibrium. An arrow connecting parts b and c completes the illustration of the 4mer* ⇔ 2mer* ⇔ 2mer ⇔ 4mer equilibrium. (d) Surface charge representations for similar models prepared for rat PAH. The circle shows the area of increased anionic character for 4mer relative to 4mer*.

Figure 2. ACT domain dimer structures

The ACT domain dimers are illustrated as ribbons with each subunit pair in light and dark shades. Using a space filling representation for the ligands bound at the ACT:ACT interface, examples are shown for (a) the regulatory subunit of acetolactate synthase, PDB id 2F1F, which has no ligand [76]; (b) phosphoglycerate dehydrogenase, PDB id 2PC9, which shows serine bound at the subunit-subunit interface [77]; (c) one of four ACT:ACT dimers present in protein BT0572 from Bacteroides thetaiotomicron, PDB id 2F06, where each ACT:ACT dimer in this structure contains one molecule of histidine at the subunit-subunit interface; and (d) asparate kinase, PDB id 2CDQ, which has two different (tandem) ACT:ACT dimers, only one of which contains lysine bound at the subunit-subunit interface [78]. (e) The ACT domains of human PAH in the active 4mer model. Shown in space filling representation are amino acid side chains that are substituted in disease-associated human PAH variants that have been shown to obliterate allosteric Phe binding [71].

Figure 3. The proposed models are consistent with PAH behaviors

(a) Shown are spacefill representations of a portion of rat PAH 4mer* (left) and 4mer (right) colored as in Figure 1, showing Trp120 in white and the highly basic C-terminal portion of the regulatory domain in blue. (b) SDS PAGE analysis of limited proteolysis of PS purified rat PAH (0.2 mg/mL) in the presence and absence of Phe for 1 hour at room temperature. Lanes: 1) molecular weight markers; 2) PAH incubated in the absence of Phe or trypsin; 3–6) PAH incubated in the absence or presence of Phe and trypsin as indicated.

Figure 4. SDS PAGE analysis of rat and humanPAH

(a) PS purified rat PAH; lane 1 is Coomassie stained, lane 2 is a Western blot. (b) Human PAH; lane 1 is the concentrated PS pool (Coomassie stained); lane 2 is a Western blot of the concentrated PS pool; lane 3 is a Coomassie stained gel of IEC purified human PAH (pool A).

Figure 5. Order of addition effects on PAH activity

The order of addition of reaction components has a profound effect on the initial activity of rat PAH under the conditions of [Phe] = 0.3 mM; [BH₄] = 0.075 mM; PAH = 20 μg/ml. Protein was preincubated for 5 min prior to the addition of substrate(s). Activity axis directly reflects fluorescence due to formation of tyrosine.

Figure 6. Evidence for two different tetramers of rat PAH

(a) PS-purified rat PAH is shown on native and native Western PAGE. (b) Analytical SEC (loading 100 μl at ~ 1 mg/ml) shows that there are not major changes in the tetrameric size of rat PAH in the presence or absence of Phe. (c) Analytical IEC (loading ~100 μg samples) under the same conditions as part b, reflects a significant change in the surface charge of rat PAH as a function of [Phe].

Figure 7. IEC purification and analysis of human PAH

(a) Further purification of PS purified human PAH by IEC yields a high-purity high-activity pool (A), and a disperse, low-purity low-activity pool (B). (b) Native PAGE and native Western indicate that each pool contains human PAH as both tetramer and dimer. (c) IEC behavior of 100 μl aliquots of pool A in the presence and absence of Phe at both 1 mM and 50 μM (compare to Fig 6c).

Figure 8. Phe stabilization of tetrameric rat PAH

Native Western showing the effects of BH₄ and Phe on the spontaneous tetramer(s)-dimer(s) equilibration of 0.2 mg/ml rat PAH.

Figure 9. PAH multimerization schemes

(a) A schematic of the proposed morpheein model for the allosteric regulation of PAH; formation of 4mer is a closed assembly unable to further multimerize. (b) Formation of higher order aggregates can occur through the dimerization of PAH regulatory domains when the individual subunits are in the 2mer* or 4mer* assembly. Formation of a 6mer* is illustrated. These are not closed assemblies and can further multimerize to larger assemblies through interaction of the multimerization domains and regulatory domains to form 8mer*, 10mer*, 12mer*, etc.