Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

Abstract

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

Figure 1. Interaction between PAH-RD and Phenylalanine.

(A) Schematic of the regulatory domain (brown), catalytic domain (green) and tetramerization domain (pink) of the human PAH polypeptide. (B) DSF of the unliganded (grey line; Tm = 54.5 °C ± 0.01 SD) and Phe-bound (pink line; Tm = 75.2 °C ± 0.2 SD) hPAH-RD^1–118. (C) DSF of the unliganded (grey line; Tm = 62.1 °C ± 0.02 SD) and Phe-bound (pink line; Tm = 73.3 °C ± 0.1 SD) hPAH-RD^19–118.

Figure 2. Crystal structure of hPAH-RD.

(A) Ribbon representation of hPAH-RD dimeric structure, coloured grey and yellow for the two subunits. Phe is shown in sticks. (Inset) 2F_o-F_c electron density map showing the bound Phe ligand. (B) Left: Topology of the ACT fold in hPAH-RD. Right: Structural superposition of hPAH-RD (yellow) with other ACT folds (grey) including PDB codes 2 mda, 2dt9 and 2f1f. (C) Structural superposition of hPAH-RD dimer (grey and yellow subunits) with hTH-RD dimer (white subunits). The two subunits of a dimer are annotated as A and B. (D) Structural superposition of hPAH-RD (yellow) with the rat counterpart (rPAH) extracted from its RD + CD structure (purple). (E) Alignment of the RD sequences between hPAH (this study) and rat PAH (PDB code 1 phz). Secondary structures from our hPAH-RD data are shown. Red line denotes the region covered in the hPAH-RD structure. (F) Phe binding site at the dimer interface of hPAH-RD^19–118. The two subunits and Phe are colored as in A.

Figure 3. Phenylalanine stabilizes the dimeric conformation of PAH-RD.

(A) SEC-MALS of unliganded (grey line) and Phe-bound (pink line) of hPAH-RD^1–118. (B) SEC-MALS of unliganded (grey line) and Phe-bound (pink line) of hPAH-RD^19–118. (C) SAXS profiles for hPAH-RD^19–118 are plotted for the unliganded (grey line), Phe-bound (pink line) and crystal-structure-simulated (blue line, calculated using CRYSOL) data. Guinier plots (left) and real space P(r) distributions (right) are shown as inset. (D) Ab initio model of Phe-bound hPAH-RD^19–118 derived from experimental SAXS data (using a D_max estimate of 60 Å), superimposed with the crystallographic dimer of hPAH-RD. (E) Ab initio model of unliganded hPAH-RD^19–118 derived from experimental SAXS data (using a D_max estimate of 83 Å), revealing a rod-like shape as the dominant species, potentially accommodating four hPAH-RD crystallographic dimers as modelled. (F) R_G and D_max values calculated for the unliganded, Phe-bound and crystal-structure-simulated SAXS data of hPAH-RD.

Figure 4. A phenylalanine binding mutation disrupts the propensity of hPAH-RD to homodimerize.

(A) DSF of the unliganded (grey line; Tm = 49.8 °C ± 0.2 SD)) and Phe-bound (pink line; Tm = 53.0 °C ± 0.03 SD) hPAH-RD p.E76A. (B) SEC-MALS of unliganded (grey line) and Phe-bound (pink line) of PAH-RD p.E76A.

Figure 5. Proposed model of PAH activation by Phenylalanine.

In the basal, unliganded state, as represented by the full-length rat PAH structure (PDB code 5DEN), the four RDs of a PAH tetramer do not interact with each other. Each RD (yellow) interacts with its corresponding CD domain (green) via the N-terminal loop region, resulting in steric hindrance of the CD active site. Phe binding relieves the steric hindrance due to homodimerization of RD, supported in this study, hence allowing unhindered access of substrates into the active sites.