The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1–71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol^–1). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements

Overall fold of the monomeric structure of the c-Src-SH3 domain.

<p>Overall fold of the monomeric species of the WT c-Src-SH3 domain (WT_M, PDB code 4JZ4). The AU is composed by two chains of the SH3 domain; both chains are represented as a cartoon (white). The n-Src loop residues in chains A and B are shown in red. In chain B, the poor electronic density in the difference maps does not allow to model residues 114-115. Both chains show a nickel-binding site at the N-terminal formed by the residues His83-Ser82-Gly81, with slight differences in the conformation and in the axial ligand (nickel ion is represented with a green sphere). All the figures were performed using the program Pymol 1.7 (distributed by Schrödinger).</p

Nucleation site of the WT c-Src SH3 domain.

<p>Hydrogen-bond interactions among the residues belonging to the diverging β-turn and those of the distal loop are shown in green dotted lines. WT_M (PDB code 4JZ4) chains A (panel A) and B (panel B) are shown in blue and cyan, respectively. (C) Intertwined dimer structure of the WT c-Src SH3 domain (PDB code 4JZ3), residues at chain A are shown in white sticks and those belonging to the symmetry related molecule (chain B) are in magenta sticks.</p

Hydrogen-bond distances in the water network at the distal loop and diverging β-turn.

<p>Hydrogen-bond distances in the water network at the distal loop and diverging β-turn.</p

X-ray data collection and refinement statistics.

<p>Statistics for the highest-resolution shell are shown in parentheses.</p><p>X-ray data collection and refinement statistics.</p

Thermal stability of WT and mutants of the c-Src-SH3 domain.

a<p>Errors are fitting errors to the two-state denaturation model.</p><p>Thermal stability of WT and mutants of the c-Src-SH3 domain.</p

DLS experiments.

<p>(A) Average <i>R</i>_h as a function of c-Src-SH3 domain concentration, in 0.1 M sodium acetate (pH 5.0) at 25°C. Symbols represent measured data in the presence of 5% PEG300 (open circles) and absence of PEG300 (filled circles). (B) Aggregation kinetics of c-Src-SH3 followed by DLS. The protein at a concentration of 25 mg·ml⁻¹ (3.6 mM) in 0.1 M sodium acetate pH 5.0 was incubated at 25°C (square) containing 5% PEG300 and without PEG300 at 20°C (triangles) and 25°C (circles) as a function of time.</p

Superposition of the monomeric structures of the c-Src-SH3 domain.

<p>Superposition of the crystallographic structures of the monomeric c-Src-SH3 domain (WT_M, chain A blue and B clear blue, PDB code 4JZ4; Q128E_M, chain A red and B clear red, PDB code 4OMO) with that solved by NMR (PDB code 1SRL) (grey).</p

Intertwined dimer structures of the c-Src-SH3 domain.

<p>(A) Structure of the intertwined dimer of the c-Src-SH3 domain. Open chain of the WT c-Src-SH3 domain is shown in cartoon (blue). Chain B, which generates the dimer, was obtained by symmetry (light-blue). (B) Superposition of the intertwined structures of the WT_D (blue) and Q128E_D (red), Q128K_D (yellow), Q128R_D (green) mutants. (C) Superposition of the open chain of the intertwined dimer structure of the WT c-Src-SH3 domain (blue) (PDB code 4JZ3) to chain A of the monomeric structure of the WT (PDB code 4JZ4): the overlay of the N- and C-terminal regions is shown in cyan and white, respectively.</p

Comparisons in terms of RMSD (Å).

a<p>Alignment performed taking into account only non-hydrogen atoms.</p>b<p>Alignment performed taking as reference residues 84-110 of the monomer.</p>c<p>Alignment performed taking as reference residues 117-140 of the monomer.</p><p>Comparisons in terms of RMSD (Å).</p