Research ArticleNEUROCHEMISTRY

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

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Science Advances  08 Apr 2016:
Vol. 2, no. 4, e1500980
DOI: 10.1126/sciadv.1500980
  • 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 CuH domain in dark green, the CuM 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 CuH domain contains two disulfide bridges. The CuM 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. CuM 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 CuM 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 CuH domain to the left. (B) Viewed from the back, with the CuH 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).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/4/e1500980/DC1

    Supplementary Materials and Methods

    Fig. S1. Overall domain alignment of copper-containing hydroxylases.

    Fig. S2. Sequence alignment of copper-containing hydroxylases.

    Fig. S3. Sequence alignment of DBH from different organisms.

    Fig. S4. Size exclusion analysis of purified DBH tetramer and dimer.

    Fig. S5. Analysis of DBH tetramer conversion as a function of pH.

    Fig. S6. Analysis of DBH tetramer conversion as a function of ionic strength.

    Fig. S7. Mass spectrum of a nonseparated sample containing a mixture of dimeric and tetrameric DBH.

    Fig. S8. SDS–polyacrylamide gel electrophoresis analysis of dimeric and tetrameric DBH under nonreducing and reducing conditions.

    Fig. S9. Structure of the human DBH dimer emphasizing the integrated structure created by the C-terminal interaction with both the CuM domain and the DOMON domain.

    Fig. S10. Modeled glycosylation environments in chain A with 2FobsFcalc electron density maps contoured at σ of 1.0.

    Fig. S11. Modeled glycosylation environments in chain B with 2FobsFcalc electron density maps contoured at σ of 1.0.

    Fig. S12. Structure of the human DBH dimer with the disulfide bridges and the glycosylation sites highlighted.

    Fig. S13. Sequence alignment of DOMON domains.

    Fig. S14. The dimerization domain disulfide bridges environment with 2FobsFcalc electron density map contoured at σ of 1.0.

    Table S1. Secondary structure assignment in human DBH.

    Table S2. Domain-domain hydrogen bond contacts in chains A and B.

    Table S3. Data collection, phasing, and refinement statistics.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Overall domain alignment of copper-containing hydroxylases.
    • Fig. S2. Sequence alignment of copper-containing hydroxylases.
    • Fig. S3. Sequence alignment of DBH from different organisms.
    • Fig. S4. Size exclusion analysis of purified DBH tetramer and dimer.
    • Fig. S5. Analysis of DBH tetramer conversion as a function of pH.
    • Fig. S6. Analysis of DBH tetramer conversion as a function of ionic strength.
    • Fig. S7. Mass spectrum of a nonseparated sample containing a mixture of dimeric and tetrameric DBH.
    • Fig. S8. SDS–polyacrylamide gel electrophoresis analysis of dimeric and tetrameric DBH under nonreducing and reducing conditions.
    • Fig. S9. Structure of the human DBH dimer emphasizing the integrated structure created by the C-terminal interaction with both the CuM domain and the DOMON domain.
    • Fig. S10. Modeled glycosylation environments in chain A with 2Fobs Fcalc electron density maps contoured at σ of 1.0.
    • Fig. S11. Modeled glycosylation environments in chain B with 2Fobs Fcalc electron density maps contoured at σ of 1.0.
    • Fig. S12. Structure of the human DBH dimer with the disulfide bridges and the glycosylation sites highlighted.
    • Fig. S13. Sequence alignment of DOMON domains.
    • Fig. S14. The dimerization domain disulfide bridges environment with 2Fobs Fcalc electron density map contoured at σ of 1.0.
    • Table S1. Secondary structure assignment in human DBH.
    • Table S2. Domain-domain hydrogen bond contacts in chains A and B.
    • Table S3. Data collection, phasing, and refinement statistics.

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