The solution structure of the regulatory domain of tyrosine hydroxylase

Abstract

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine in the biosynthesis of the catecholamine neurotransmitters. The activity of the enzyme is regulated by phosphorylation of serine residues in a regulatory domain and by binding of catecholamines to the active site. Available structures of TyrH lack the regulatory domain, limiting the understanding of the effect of regulation on structure. We report the use of NMR spectroscopy to analyze the solution structure of the isolated regulatory domain of rat TyrH. The protein is composed of a largely unstructured N-terminal region (residues 1-71) and a well-folded C-terminal portion (residues 72-159). The structure of a truncated version of the regulatory domain containing residues 65-159 has been determined and establishes that it is an ACT domain. The isolated domain is a homodimer in solution, with the structure of each monomer very similar to that of the core of the regulatory domain of phenylalanine hydroxylase. Two TyrH regulatory domain monomers form an ACT domain dimer composed of a sheet of eight strands with four α-helices on one side of the sheet. Backbone dynamic analyses were carried out to characterize the conformational flexibility of TyrH65-159. The results provide molecular details critical for understanding the regulatory mechanism of TyrH.

Keywords: ACT domain; NMR spectroscopy; regulation; solution structure; tyrosine hydroxylase.

Figure 1

NMR spectra of the isolated regulatory domain of tyrosine hydroxylase. (a) Overlay of the 2D ¹H-¹⁵N HSQC spectra of RDTyrH (red) and RDTyrH_65–159 (blue). (b) 2D ¹H-¹⁵N HSQC spectrum of 0.8 mM RDTyrH_65–159 showing the assignments of the individual residues. Conditions: 50 mM sodium phosphate, 1 μM leupeptin, 1 μM pepstatin A and 5% D₂O (pH 7.0), at 300 K at a magnetic field strength of 14.1 T (600 MHz ¹H).

Figure 2

Structure of the regulatory domain of tyrosine hydroxylase. (a) Superposition of the backbones of the 10 lowest-energy structures of RDTyrH_65–159 in two orientations with an 180° rotation along the Y-axis. β-Strands are in green and helices in red. (b) Ribbon diagram of a representative low-energy structure in two orientations with an 180° rotation along the Y-axis indicating the location of secondary structure elements.

Figure 3

Model-free parameters for RDTyrH_65–159 backbone amides derived by the fitting of the ¹⁵N T₁, ¹⁵N T₂, and ¹H-¹⁵N NOE data. Lipari-Szabo S², S²_f, τ_e, and R_ex parameters are shown from top to bottom, respectively. Missing S², S²_f, τ_e, and R_ex data points indicate that this parameter was not included in the motional model for that residue. A representation of the RDTyrH_65–159 secondary structure is shown along the top.

Figure 4

Comparison of the structures of the regulatory domains of phenylalanine hydroxylase and tyrosine hydroxylase. (a) Overlay of the ribbon diagrams of RDTyrH_65–159 (green) and RDPheH (medium purple, residues 19–117, PDB code 2PHM). (b) Topology diagrams of RDTyrH_65–159 (top) and RDPheH (bottom); the structural elements are labeled in green and magenta, respectively. (c) Structure-based sequence alignment of the regulatory domains of the three amino acid hydroxylases. The secondary structural elements of RDTyrH_65–159 and RDPheH are labeled green and magenta, respectively. To generate the alignment, a structure-based alignment of RDTyrH_65–159 and RDPheH was first performed with Chimera. The sequence of the regulatory domain of TrpH was then added to the alignment using Clustal X.

Figure 5

The dynamic properties of the backbone of the RDTyrH_65–159 dimer: a) the generalized order parameter S² values with colors ranging from yellow to red and blue corresponding to S² values from 0.4 to 0.85, and >0.85; b) the internal motions on the ps to ns time scales with colors ranging from yellow to red and magenta corresponding to T_e values from 0 to 40 ps and >500 ps; c) residues with conformational changes (R_ex) on the microsecond to millisecond time scales colored in blue. Residues for which the respective parameter was not included in the model are colored in grey.

Figure 6

Structural comparisons between the RDTyrH_65–159 dimer (green) and three ACT domain dimers: (a) phosphoglycerate dehydrogenase (orange, PDB code 1PSD), (b) aspartate kinase from Arabidopsis (red, PDB code 2CDQ), and (c) E. coli IlvH, the regulatory subunit of acetohydroxyacid synthase (magenta, PDB code 2F1F).

Figure 7

Structural models of the combined regulatory, catalytic, and tetramerization domains of TyrH. a, b: Models of the tetramer (a) and monomer (b) derived from the structure of PheH (PDB code 2PHM) by replacing the regulatory domain of PheH with the RDTyrH_65–159 monomer (magenta) and the catalytic domain of PheH with the combined catalytic and tetramerization domains of TyrH (PDB code 1TOH, green), respectively. c, d: Models of the tetramer (c) and monomer (d) derived from the model of Jaffe et al. by replacing the dimer of the regulatory domain of PheH with the RDTyrH_65–159 dimer (red) and the catalytic and tetramerization domains of PheH with the catalytic and tetramerization domains of TyrH (green), respectively. The missing residues connecting the two domains were added with Modeller in Chimera.