The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution

Abstract

The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.

Keywords: Life sciences; biochemistry; biomolecules; crystal structure; dopamine; dopamine β-hydroxylase; neurochemistry; norepinephrine; structural biology.

Fig. 1. Structure of the human DBH dimer.

(A and B) Overall structure seen from two angles (90° to each other). The DOMON domain is displayed in orange, the Cu_H domain in dark green, the Cu_M domain in light green, and the dimerization domain in magenta. The interdomain regions are in gray. (C) Secondary structure organization of DBH. The black spheres represent positions of the copper ligands. The α helix marked “*” in the dimerization domain is only seen in chain A. A detailed list of secondary structure assignment is provided in table S1. C-term, C-terminal. (D) Disulfide bridge pattern in the DBH dimer. The Cu_H domain contains two disulfide bridges. The Cu_M domain contains two disulfide bridges and forms an additional one with the dimerization domain. The DOMON domain and the dimerization domain are linked via C¹⁵⁴-C⁵⁹⁶. Chain A is linked via two intermolecular disulfide bonds with chain B in the dimerization domain. Glycosylation is observed at all four predicted sites: Asn⁶⁴, Asn¹⁸⁴, Asn³⁴⁴, and Asn⁵⁶⁶. Glycosylation clearly observed in the electron density map (see figs. S10 and S11) is shown, with N-acetylglucosamine as blue rectangles and mannose as red ovals. The position of the disulfide bridges and the glycosylation on the three-dimensional structure is shown in fig. S12.

Fig. 2. The two conformations of the DBH catalytic core reveal a closed and an open active site.

(A) Same orientation as Fig. 1B with chain A to the left. (B) View from the back, with chain A to the right. (C) Closed conformation of the catalytic domain as seen in chain A. (D) Open conformation of the catalytic domain as seen in chain B. Cu_M in chain A is modeled in the structure, whereas the three other coppers are inserted manually in a position indicated by the position of the conserved active site ligands. Same color coding as in Fig. 1.

Fig. 3. Alignment of the DBH DOMON domain with the cytochrome domain of CDH.

DOMON (chain A) is shown in orange and the cytochrome domain of CDH in gray (PDB ID 1D7B). The heme group in CDH is shown in ball-and-stick. The RMSD is 2.53 Å for the backbone atoms, indicating an identical fold of the two domains.

Fig. 4. The putative metal ion–binding site in DBH DOMON.

The prealigned coordinating residues (Asp⁹⁹, Leu¹⁰⁰, Ala¹¹⁵, and Asp¹³⁰) are shown in blue. Other possible involved residues are shown in green and orange (Asp¹¹⁴, Asp¹²⁶, Asp¹⁵⁵, and Asp¹⁵⁸). On the basis of the very oxygen-rich environment, it is likely to be either a group 1 or group 2 metal ion. The six mentioned aspartic acid residues are conserved among the DOMON domains in the copper-containing hydroxylases; see fig. S13.

Fig. 5. Domain interactions in the dimerization domain.

Chain A is shown in magenta and chain B in gray. (A) Residues involved in hydrophobic interactions are shown as sticks. (B) Residues involved in hydrophilic interactions are shown as sticks, and the disulfide bridges are shown in yellow. Electron density maps for the disulfide bridges are provided in fig. S14.

Fig. 6. Binding pockets and channels in the vicinity of the closed active site seen in chain A.

CAVER identified binding pocket and channel (yellow) in the closed catalytic core (chain A). The modeled Cu_M in the structure and the manually inserted copper ions are in blue. Two different orientations are shown. (A) Same orientation as in Fig. 2C, with the Cu_H domain to the left. (B) Viewed from the back, with the Cu_H domain to the right. Same color coding as in Fig. 1. The pocket is of sufficient size to hold the substrate (dopamine).

Fig. 7. Proposed mode of action of DBH.

The closed conformation with the coupled binuclear copper site is the catalytically active site. The open conformation serves as a way for loading of new substrate, release of product, and change in copper redox state. It is envisioned that the two sites alternate between the closed catalytically active form and the open form, known as a flip-flop mechanism (45, 46).