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Structures of mouse DUOX1–DUOXA1 provide mechanistic insights into enzyme activation and regulation

Abstract

DUOX1, an NADPH oxidase family member, catalyzes the production of hydrogen peroxide. DUOX1 is expressed in various tissues, including the thyroid and respiratory tract, and plays a crucial role in processes such as thyroid hormone biosynthesis and innate host defense. DUOX1 co-assembles with its maturation factor DUOXA1 to form an active enzyme complex. However, the molecular mechanisms for activation and regulation of DUOX1 remain mostly unclear. Here, I present cryo-EM structures of the mammalian DUOX1–DUOXA1 complex, in the absence and presence of substrate NADPH, as well as DUOX1–DUOXA1 in an unexpected dimer-of-dimers configuration. These structures reveal atomic details of the DUOX1-DUOXA1 interaction, a lipid-mediated NADPH-binding pocket and the electron transfer path. Furthermore, biochemical and structural analyses indicate that the dimer-of-dimers configuration represents an inactive state of DUOX1–DUOXA1, suggesting an oligomerization-dependent regulatory mechanism. Together, my work provides structural bases for DUOX1–DUOXA1 activation and regulation.

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Fig. 1: Structure of DUOX1–DUOXA1 in the absence of NADPH.
Fig. 2: Interaction between DUOX1 and DUOXA1.
Fig. 3: Heme- and FAD-binding sites.
Fig. 4: The NADPH-binding site and electron transfer path.
Fig. 5: Structure of DUOX1–DUOXA1 in the dimer-of-dimers configuration.
Fig. 6: The dimer-of-dimers conformation of DUOX1–DUOXA1 represents an inactivated state.

Data availability

Cryo-EM maps and atomic models for mouse DUOX1–DUOXA1 complexes have been deposited in the EMDB and wwPDB with the following accession numbers: EMD-21962 and PDB 6WXR (apo state); EMD-21963 and PDB 6WXU (dimer of dimers); EMD-21964 and PDB 6WXV (with NADPH).

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Acknowledgements

I thank members of the Cryo-electron Microscopy and Tomography Center of St Jude Children’s Research Hospital for help with cryo-EM data collection; P. Hixson and R. Kalathur (Protein Technology Center) for help with mammalian cell culture; A. Myasnikov, M. Halic and C. Lee for helpful discussions; Z. Luo for help with bio-illustration; and C. Kalodimos, M. Halic and M. Babu for critical reading of the manuscript. J.S. is funded by the NIH (HL143037) and American Lebanese Syrian Associated Charities (ALSAC).

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J.S. designed and performed all the experiments, analyzed the results and prepared the manuscript.

Corresponding author

Correspondence to Ji Sun.

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The author declares no competing interests.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Structure determination of the DUOX1–DUOXA1 complex.

a, Construct design of DUOX1 and DUOXA1 used for structural studies and size exclusion chromatography profile of the DUOX1–DUOXA1 complex. Fractions of second peak (PK2) in the red box are concentrated and used for single-particle analysis. b, Flowchart of DUOX1–DUOXA1 structure determination. The steps in blue are carried out in cryoSPARC, ones in green in RELION. In the 2D classification, dimer-of-dimer classes are indicated by red dashed cycles. c, Interaction between the S3-S4 linker and PHD of DUOX1. The out leaflet of the membrane bilayer is indicated by a gray line. d, Disulfide bonds and glycosylation sites of the DUOX1–DUOXA1 complex. Sugars and their linked Asn side chains are shown as sticks and balls. Disulfide bonds are colored in green.

Extended Data Fig. 2 Structure of the PHD of DUOX1.

a, Superimposition between LPO and PHD of DUOX1. LPO and DUOX1 are colored in gray and blue, respectively. b, Putative heme-binding site. The possible heme-binding pocket indicated by a red oval. Positions of Ser326 and Ser108 (colored in brown) are where the heme-coordinating histidines are located. c, The putative calcium-binding site and surrounding residues. Calcium is indicated by a green sphere. d, A potential ion binding site and surrounding residues. Cryo-EM density is contoured by gray meshes.

Extended Data Fig. 3 Heme- and FAD-binding sites.

a, Structural comparison of heme-binding sites between DUOX1 (blue) and csNOX5 (gray). The transmembrane helices are labeled with S1–S6. b, Structural conservation of heme coordination in ferric oxidoreductases. DUOX1, csNOX5, Cyto b561 and Dcytb are colored in blue, gray, light blue and cyan, respectively. c, FAD-binding site in 2D representation. Hydrogen bonds, hydrophobic interactions and cation-π interactions are indicated by dashes, spokes and vertical dashes, respectively. Residues from FBD, NBD, TMD are colored in light pink, gray and blue, respectively. d, The putative oxygen-binding site. Oxygen is represented by a dashed red oval. e, The possible oxygen entering and hydrogen peroxide exiting paths.

Extended Data Fig. 4 NADPH-binding site.

a, Cryo-EM density of DUOX1–DUOXA1 with and without NADPH. The potential NADPH-binding site is indicated by red dashes. b, Structure of an NADPH molecule. c, The NADPH-binding site. Residues from TMD and NBD are colored in blue and gray, respectively. The “invisible” nicotinamide group is cycled in a cyan dashed oval. d, Cartoon and structure of the NADPH-binding site. The conserved glycines on the “GXGXG” motif are shown as magenta balls. e, The lipid-binding pocket of csNOX5 and DUOX1. Lipid or alkyl chains are colored in red. f, Functional analyses of F1097 mutations. F1097 is mutated to Tyr, Ala, Ile and Val, and the activity of the mutations are normalized to the wild-type protein (Data shown are mean and s.d. of n = 4 independent experiments).

Extended Data Fig. 5 Formation of the dimer-of-dimer interface.

a, b, Structural comparison of DUOX1 and DUOXA1 in heterodimeric and dimer-of-dimer states. c, Interaction between DUOX1 and DUOXA1. Left: interactions between DUOX1 and the N-terminal loop and glycan chain linked to N109 of DUOXA1. Right: cryo-EM density and cartoon of the glycan chain on N109. d, Interactions between transmembrane domains of DUOX1 and DUOXA1 mediated by a lipid molecule. Left: interaction details. Right: density of the lipid molecule. e, PHD-PHD interactions in the dimer-of-dimer configuration. Left: interactions between PHDs of DUOX1. Right: comparison between MPO dimers and PHD dimers of DUOX1.

Extended Data Fig. 6 The interface between DUOX1–DUOXA1 heterodimers.

a, Modeling of two DUOX1–DUOXA1 dimers into the dimer-of-dimer state. Structural crashes are indicated by red arrows. b, The potential oxygen entering/hydrogen peroxide exiting paths in the DUOX1–DUOXA1 dimer of dimers. c, FSEC curves of mouse DUOX1–DUOXA1 (blue), human DUOX1–DUOXA1 (gray) and human DUOX2DUOXA2 (orange). d, FSEC curves of mouse DUOX1–DUOXA1 (gray), mouse DUOX1–DUOXA1 with NADPH (green) and mouse DUOX1–DUOXA1 with FAD (orange). e, Modeling of DUOX2DUOXA2 complex and mapping of the hypothyroidism disease mutations. f, Accessibility of the outer heme of csNOX5 to extracellular space. The heme molecule is colored in green, indicated by a red arrow. The csNOX5 is shown as gray surface. g, The positively charged environment surrounding the heme molecule (heme #1).

Supplementary information

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Supplementary Video 1

Flexibility of cytoplasmic domains of DUOX1–DUOXA1 in the dimer-of-dimers conformation.

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Sun, J. Structures of mouse DUOX1–DUOXA1 provide mechanistic insights into enzyme activation and regulation. Nat Struct Mol Biol 27, 1086–1093 (2020). https://doi.org/10.1038/s41594-020-0501-x

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