Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: VEO-IBD NOX1 mutations abolish catalytic activity and prevent peroxynitrite formation. (A) Superoxide generation of CHO-NOXO1-NOXA1-p22 phox cells (control) or cells co-expressing NOX1 WT, NOX1 N122H or NOX1 T497A in the presence or absence of PMA (in RLU and as % of WT). (B) Immunoblot of cell lines in (A). (C) Detection of peroxynitrite with FIBA in the absence or presence of 10 μM l -NAME (in RFU). Cell lines in (A) co-expressing iNOS as indicated were stimulated with PMA. (D) Nitrate quantification in supernatants of indicated cell lines after PMA stimulation (1 h). (E) Immunoblot of cell lines in (C). A two-way ANOVA with multiple comparisons test was performed (A) or a Kruskal-Wallis test with multiple comparisons (D). (non-significant (NS) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Article Snippet: The cryo-EM structure of the NOX2-p22 phox complex has recently been solved [ , ].

Techniques: Activity Assay, Expressing, Western Blot

Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: Conserved asparagine in the HxxxHxxN motif is essential for the catalytic activity of NOX/DUOX. (A) NOX1-5 and DUOX1-2 sequence alignments with asparagine (blue) and histidines (yellow), and with threonine (blue) and GRP sequence (yellow). (B) CHO-NOXO1-NOXA1-p22 phox cell lines were transfected with NOX1 WT and mutants, followed by PMA stimulation. (C) COS7-p22 phox cells were transfected with p47 phox , p67 phox , and NOX2 WT or mutants, followed by PMA stimulation. (D) COS7-p22 phox cells were transfected with NOX4 WT or mutants, constitutive H 2 O 2 generation was measured. (E) H661-DUOXA2 cells were transfected with DUOX2 or mutants, PMA/thapsigargin stimulated H 2 O 2 generation was measured. (F) Percentage of NOX4 or DUOX2 positive cells by cell surface staining, transfections as above. A one-way ANOVA with multiple comparisons (NOX1,2) or Kruskal-Wallis test with multiple comparisons (NOX4, DUOX2) was performed (non-significant (NS) P > 0.05, *P ≤ 0.05, ****P ≤ 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cryo-EM structure of the NOX2-p22 phox complex has recently been solved [ , ].

Techniques: Activity Assay, Sequencing, Transfection, Staining

Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: NOX1 modelling reveals structural features involved in the oxygen reduction center. (A) Cartoon representation of a NOX1/p22 phox heterodimer model inside view (left) or eclipsed (p22 phox behind, center). On the right, eclipsed view in surface mode revealing lateral access to the distal heme (zoom), NOX1 (teal blue), p22 phox (orange). (B) Zoom, on a vertical section within NOX1, on the distal heme region in NOX1 with cavities highlighted in surface mode. An additional internal pocket at the distal heme site is revealed. The side chains of the heme-chelating histidines as well as N122 and H119, delimiting the internal pocket, are represented by sticks. The vertical section within NOX1 allows to see only TM3, TM4 and partly TM5, while the others have been eliminated from front for clarity. (C) Zoom in NOX1 structure of the N122 environment at the interface between TM3 and TM2 observed from the outside (left) or the inside of NOX1 (right). (D) Modelling the structural impact of mutations at position N122. Modification performed by the use of the mutagenesis function within Pymol software. From left to right, the N122H, N122Q, N122L and N122T mutations. For the first two, several possible rotamers have been represented simultaneously for the corresponding mutated side chain. In the case of N122L and N122T modeled mutations another rotamer of the facing T49 side chain has been considered as possible adaptation to the mutation of N122. In (C) and (D), dotted yellow line represented H-bond and dotted green line possible hydrophobic interaction. (E) CHO-NOXO1-NOXA1-p22 phox cells were transfected with NOX1 WT and mutants, followed by PMA stimulation. Immunoblots indicate equal protein expression. One-way ANOVA with repeated measures (non-significant (NS) P > 0.05, *P ≤ 0.05, ****P ≤ 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cryo-EM structure of the NOX2-p22 phox complex has recently been solved [ , ].

Techniques: Modification, Mutagenesis, Software, Transfection, Western Blot, Expressing

Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: VEO-IBD NOX1 mutations abolish catalytic activity and prevent peroxynitrite formation. (A) Superoxide generation of CHO-NOXO1-NOXA1-p22 phox cells (control) or cells co-expressing NOX1 WT, NOX1 N122H or NOX1 T497A in the presence or absence of PMA (in RLU and as % of WT). (B) Immunoblot of cell lines in (A). (C) Detection of peroxynitrite with FIBA in the absence or presence of 10 μM l -NAME (in RFU). Cell lines in (A) co-expressing iNOS as indicated were stimulated with PMA. (D) Nitrate quantification in supernatants of indicated cell lines after PMA stimulation (1 h). (E) Immunoblot of cell lines in (C). A two-way ANOVA with multiple comparisons test was performed (A) or a Kruskal-Wallis test with multiple comparisons (D). (non-significant (NS) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Article Snippet: The p22 phox structure used for the heterodimer NOX1/p22 phox model building came from the cryo-EM solved structure of the NOX2-p22 phox core complex [ ].

Techniques: Activity Assay, Expressing, Western Blot

Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: Conserved asparagine in the HxxxHxxN motif is essential for the catalytic activity of NOX/DUOX. (A) NOX1-5 and DUOX1-2 sequence alignments with asparagine (blue) and histidines (yellow), and with threonine (blue) and GRP sequence (yellow). (B) CHO-NOXO1-NOXA1-p22 phox cell lines were transfected with NOX1 WT and mutants, followed by PMA stimulation. (C) COS7-p22 phox cells were transfected with p47 phox , p67 phox , and NOX2 WT or mutants, followed by PMA stimulation. (D) COS7-p22 phox cells were transfected with NOX4 WT or mutants, constitutive H 2 O 2 generation was measured. (E) H661-DUOXA2 cells were transfected with DUOX2 or mutants, PMA/thapsigargin stimulated H 2 O 2 generation was measured. (F) Percentage of NOX4 or DUOX2 positive cells by cell surface staining, transfections as above. A one-way ANOVA with multiple comparisons (NOX1,2) or Kruskal-Wallis test with multiple comparisons (NOX4, DUOX2) was performed (non-significant (NS) P > 0.05, *P ≤ 0.05, ****P ≤ 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The p22 phox structure used for the heterodimer NOX1/p22 phox model building came from the cryo-EM solved structure of the NOX2-p22 phox core complex [ ].

Techniques: Activity Assay, Sequencing, Transfection, Staining

Journal: Redox Biology

Article Title: VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity

doi: 10.1016/j.redox.2023.102905

Figure Lengend Snippet: NOX1 modelling reveals structural features involved in the oxygen reduction center. (A) Cartoon representation of a NOX1/p22 phox heterodimer model inside view (left) or eclipsed (p22 phox behind, center). On the right, eclipsed view in surface mode revealing lateral access to the distal heme (zoom), NOX1 (teal blue), p22 phox (orange). (B) Zoom, on a vertical section within NOX1, on the distal heme region in NOX1 with cavities highlighted in surface mode. An additional internal pocket at the distal heme site is revealed. The side chains of the heme-chelating histidines as well as N122 and H119, delimiting the internal pocket, are represented by sticks. The vertical section within NOX1 allows to see only TM3, TM4 and partly TM5, while the others have been eliminated from front for clarity. (C) Zoom in NOX1 structure of the N122 environment at the interface between TM3 and TM2 observed from the outside (left) or the inside of NOX1 (right). (D) Modelling the structural impact of mutations at position N122. Modification performed by the use of the mutagenesis function within Pymol software. From left to right, the N122H, N122Q, N122L and N122T mutations. For the first two, several possible rotamers have been represented simultaneously for the corresponding mutated side chain. In the case of N122L and N122T modeled mutations another rotamer of the facing T49 side chain has been considered as possible adaptation to the mutation of N122. In (C) and (D), dotted yellow line represented H-bond and dotted green line possible hydrophobic interaction. (E) CHO-NOXO1-NOXA1-p22 phox cells were transfected with NOX1 WT and mutants, followed by PMA stimulation. Immunoblots indicate equal protein expression. One-way ANOVA with repeated measures (non-significant (NS) P > 0.05, *P ≤ 0.05, ****P ≤ 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The p22 phox structure used for the heterodimer NOX1/p22 phox model building came from the cryo-EM solved structure of the NOX2-p22 phox core complex [ ].

Techniques: Modification, Mutagenesis, Software, Transfection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: Sequence alignment of EROS and the YCF4 gene product EROS is an ortholog of the plant protein encoded by YCF4 , which is necessary for the expression of proteins belonging to the photosynthetic photosystem I complex. The Homo sapiens CYBC1 (UniProtKB: Q9BQA9) and Arabidopsis thaliana YCF4 (UniProtKB: P56788) sequences were aligned. Conserved residues are highlighted in pink and gray (pink: fully conserved, gray: highly conserved). Secondary structures are depicted as light blue cylinders (α helices), arrows (β sheets), and lines (loops). Dashed lines indicate unmodeled residues. The residues involved in NOX2 interactions are colored in blue.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques:

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Interactions between H1 and H2 of EROS and TM2 and TM6 of NOX2. Of note, EROS residues R22 and N62 form hydrogen bonds with NOX2 residues F78 and R80, respectively. Additionally, the EROS residue Y51 is involved in hydrogen bonding with NOX2 residue W270. Residues at this interface (EROS: R22, L26, A37, S41, p43, F50, Y51, F57, N62; NOX2: L76, F78, L79, R80, M268, W270, I273, Y280). The residues of EROS and NOX2 are depicted as blue and green sticks, respectively; the hydrogen bonds are represented by dark dash lines. B. Interaction of the inner heme with EROS. Loop 2 protrudes from EROS and forms a hydrogen bond between Y119 and the lower heme (maroon). Two other EROS residues, E115 and Q140, form hydrogen bonds with NOX2 residues C86 and C85 on Loop B, respectively. In addition, there are hydrophobic interactions between R118/Y119 of EROS and W206/H210 of NOX2 in TM5. C. The TM domains are removed to show more clearly pocket-like interactions between NOX2 and three clusters of EROS including four polar interactions (R22, N62, E115, Q140 on EROS). D. The EROS-NOX2 interface in the FAD-binding domain of NOX2. A total of 20 amino acids, 10 each from EROS and NOX2, participate in this interaction and form a tight zipper that prevents FAD access to the DH domain of NOX2. The hydrogen bonds are shown as dark dashed lines. E and F. Cartoon and surface representation of FAD binding to NOX2 (green) in the presence of EROS (blue in E ) and in its absence ( F , NOX2 is shown in gray). G. The EROS-NOX2 interface in the NADPH-binding domain of NOX2. Ten residues, five from EROS (R108, R129, A131, T132, G133) and the other five from NOX2 (G412, P415, G538, E568, F570) are involved in this interaction. H. Schematic representation of the electron transfer path showing the relative positions and distances between FAD and the two hemes in the absence (FAD in orange) and presence (FAD in blue) of EROS. The edge-to-edge distance between the inner heme and FAD in resting NOX2 (6.1 Å) is shorter than that in EROS-NOX2 (26.4 Å). The ferric ions are shown as orange spheres.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Schematic representation of NanoLuc complementation assay. The two components, LgBiT and SmBiT, were fused to EROS and NOX2 respectively, as detailed in Methods . These engineered constructs (EROS-N-LgBiT, NOX2-C-SmBiT) were cotransfected into COS-7 cells together with the p47 phox and p67 phox expression plasmids. The changes in luminescence intensity were measured after addition of the substrate coelenterazine H (10 μM) and PMA (200 ng/ml), with a 1 min interval recording at 460 nm. Hypothetical dissociation of EROS from NOX2 is marked by an arrowhead. B. Data from 10 to 60 min after PMA addition are shown and quantified in the right panel. C. Superoxide production in reconstituted COS-7 cells (COS 91/22 ) cotransfected with expression plasmids of p47 phox , p67 phox , with or without an EROS expressing plasmid. The PMA-induced superoxide production was measured in real time for 60 min using isoluminol, and data were quantified and presented in the right panel. Data shown are mean ± SEM based on multiple independent experiments. *, p < 0.05, ***, p <0.001.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Construct, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. NOX2 in the inactive state. The association of EROS protects nascent NOX2 against proteosomal degradation and, at the same time, prevents FAD and NADPH from binding to the DH domain. B. NOX2 in the primed state. Priming signals induce dissociation of EROS from NOX2, allowing FAD access to NOX2. C. NOX2 in the active state. Docking of the cytosolic NOX activators and Rac-GTP changes the conformation of NOX2, permitting NADPH binding and electron transfer.

Article Snippet: The solved cryo-EM structure of the EROS-NOX2-p22 phox complex does not contain FAD or NADPH.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: Sequence alignment of EROS and the YCF4 gene product EROS is an ortholog of the plant protein encoded by YCF4 , which is necessary for the expression of proteins belonging to the photosynthetic photosystem I complex. The Homo sapiens CYBC1 (UniProtKB: Q9BQA9) and Arabidopsis thaliana YCF4 (UniProtKB: P56788) sequences were aligned. Conserved residues are highlighted in pink and gray (pink: fully conserved, gray: highly conserved). Secondary structures are depicted as light blue cylinders (α helices), arrows (β sheets), and lines (loops). Dashed lines indicate unmodeled residues. The residues involved in NOX2 interactions are colored in blue.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques:

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Interactions between H1 and H2 of EROS and TM2 and TM6 of NOX2. Of note, EROS residues R22 and N62 form hydrogen bonds with NOX2 residues F78 and R80, respectively. Additionally, the EROS residue Y51 is involved in hydrogen bonding with NOX2 residue W270. Residues at this interface (EROS: R22, L26, A37, S41, p43, F50, Y51, F57, N62; NOX2: L76, F78, L79, R80, M268, W270, I273, Y280). The residues of EROS and NOX2 are depicted as blue and green sticks, respectively; the hydrogen bonds are represented by dark dash lines. B. Interaction of the inner heme with EROS. Loop 2 protrudes from EROS and forms a hydrogen bond between Y119 and the lower heme (maroon). Two other EROS residues, E115 and Q140, form hydrogen bonds with NOX2 residues C86 and C85 on Loop B, respectively. In addition, there are hydrophobic interactions between R118/Y119 of EROS and W206/H210 of NOX2 in TM5. C. The TM domains are removed to show more clearly pocket-like interactions between NOX2 and three clusters of EROS including four polar interactions (R22, N62, E115, Q140 on EROS). D. The EROS-NOX2 interface in the FAD-binding domain of NOX2. A total of 20 amino acids, 10 each from EROS and NOX2, participate in this interaction and form a tight zipper that prevents FAD access to the DH domain of NOX2. The hydrogen bonds are shown as dark dashed lines. E and F. Cartoon and surface representation of FAD binding to NOX2 (green) in the presence of EROS (blue in E ) and in its absence ( F , NOX2 is shown in gray). G. The EROS-NOX2 interface in the NADPH-binding domain of NOX2. Ten residues, five from EROS (R108, R129, A131, T132, G133) and the other five from NOX2 (G412, P415, G538, E568, F570) are involved in this interaction. H. Schematic representation of the electron transfer path showing the relative positions and distances between FAD and the two hemes in the absence (FAD in orange) and presence (FAD in blue) of EROS. The edge-to-edge distance between the inner heme and FAD in resting NOX2 (6.1 Å) is shorter than that in EROS-NOX2 (26.4 Å). The ferric ions are shown as orange spheres.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Schematic representation of NanoLuc complementation assay. The two components, LgBiT and SmBiT, were fused to EROS and NOX2 respectively, as detailed in Methods . These engineered constructs (EROS-N-LgBiT, NOX2-C-SmBiT) were cotransfected into COS-7 cells together with the p47 phox and p67 phox expression plasmids. The changes in luminescence intensity were measured after addition of the substrate coelenterazine H (10 μM) and PMA (200 ng/ml), with a 1 min interval recording at 460 nm. Hypothetical dissociation of EROS from NOX2 is marked by an arrowhead. B. Data from 10 to 60 min after PMA addition are shown and quantified in the right panel. C. Superoxide production in reconstituted COS-7 cells (COS 91/22 ) cotransfected with expression plasmids of p47 phox , p67 phox , with or without an EROS expressing plasmid. The PMA-induced superoxide production was measured in real time for 60 min using isoluminol, and data were quantified and presented in the right panel. Data shown are mean ± SEM based on multiple independent experiments. *, p < 0.05, ***, p <0.001.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Construct, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. NOX2 in the inactive state. The association of EROS protects nascent NOX2 against proteosomal degradation and, at the same time, prevents FAD and NADPH from binding to the DH domain. B. NOX2 in the primed state. Priming signals induce dissociation of EROS from NOX2, allowing FAD access to NOX2. C. NOX2 in the active state. Docking of the cytosolic NOX activators and Rac-GTP changes the conformation of NOX2, permitting NADPH binding and electron transfer.

Article Snippet: In this study, we present the first high-resolution structure of EROS bound to NOX2-p22 phox , based on cryo-EM electron density maps.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: Sequence alignment of EROS and the YCF4 gene product EROS is an ortholog of the plant protein encoded by YCF4 , which is necessary for the expression of proteins belonging to the photosynthetic photosystem I complex. The Homo sapiens CYBC1 (UniProtKB: Q9BQA9) and Arabidopsis thaliana YCF4 (UniProtKB: P56788) sequences were aligned. Conserved residues are highlighted in pink and gray (pink: fully conserved, gray: highly conserved). Secondary structures are depicted as light blue cylinders (α helices), arrows (β sheets), and lines (loops). Dashed lines indicate unmodeled residues. The residues involved in NOX2 interactions are colored in blue.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques:

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Interactions between H1 and H2 of EROS and TM2 and TM6 of NOX2. Of note, EROS residues R22 and N62 form hydrogen bonds with NOX2 residues F78 and R80, respectively. Additionally, the EROS residue Y51 is involved in hydrogen bonding with NOX2 residue W270. Residues at this interface (EROS: R22, L26, A37, S41, p43, F50, Y51, F57, N62; NOX2: L76, F78, L79, R80, M268, W270, I273, Y280). The residues of EROS and NOX2 are depicted as blue and green sticks, respectively; the hydrogen bonds are represented by dark dash lines. B. Interaction of the inner heme with EROS. Loop 2 protrudes from EROS and forms a hydrogen bond between Y119 and the lower heme (maroon). Two other EROS residues, E115 and Q140, form hydrogen bonds with NOX2 residues C86 and C85 on Loop B, respectively. In addition, there are hydrophobic interactions between R118/Y119 of EROS and W206/H210 of NOX2 in TM5. C. The TM domains are removed to show more clearly pocket-like interactions between NOX2 and three clusters of EROS including four polar interactions (R22, N62, E115, Q140 on EROS). D. The EROS-NOX2 interface in the FAD-binding domain of NOX2. A total of 20 amino acids, 10 each from EROS and NOX2, participate in this interaction and form a tight zipper that prevents FAD access to the DH domain of NOX2. The hydrogen bonds are shown as dark dashed lines. E and F. Cartoon and surface representation of FAD binding to NOX2 (green) in the presence of EROS (blue in E ) and in its absence ( F , NOX2 is shown in gray). G. The EROS-NOX2 interface in the NADPH-binding domain of NOX2. Ten residues, five from EROS (R108, R129, A131, T132, G133) and the other five from NOX2 (G412, P415, G538, E568, F570) are involved in this interaction. H. Schematic representation of the electron transfer path showing the relative positions and distances between FAD and the two hemes in the absence (FAD in orange) and presence (FAD in blue) of EROS. The edge-to-edge distance between the inner heme and FAD in resting NOX2 (6.1 Å) is shorter than that in EROS-NOX2 (26.4 Å). The ferric ions are shown as orange spheres.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. Schematic representation of NanoLuc complementation assay. The two components, LgBiT and SmBiT, were fused to EROS and NOX2 respectively, as detailed in Methods . These engineered constructs (EROS-N-LgBiT, NOX2-C-SmBiT) were cotransfected into COS-7 cells together with the p47 phox and p67 phox expression plasmids. The changes in luminescence intensity were measured after addition of the substrate coelenterazine H (10 μM) and PMA (200 ng/ml), with a 1 min interval recording at 460 nm. Hypothetical dissociation of EROS from NOX2 is marked by an arrowhead. B. Data from 10 to 60 min after PMA addition are shown and quantified in the right panel. C. Superoxide production in reconstituted COS-7 cells (COS 91/22 ) cotransfected with expression plasmids of p47 phox , p67 phox , with or without an EROS expressing plasmid. The PMA-induced superoxide production was measured in real time for 60 min using isoluminol, and data were quantified and presented in the right panel. Data shown are mean ± SEM based on multiple independent experiments. *, p < 0.05, ***, p <0.001.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Construct, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

doi: 10.1101/2023.09.11.557130

Figure Lengend Snippet: A. NOX2 in the inactive state. The association of EROS protects nascent NOX2 against proteosomal degradation and, at the same time, prevents FAD and NADPH from binding to the DH domain. B. NOX2 in the primed state. Priming signals induce dissociation of EROS from NOX2, allowing FAD access to NOX2. C. NOX2 in the active state. Docking of the cytosolic NOX activators and Rac-GTP changes the conformation of NOX2, permitting NADPH binding and electron transfer.

Article Snippet: In our study, mild detergents including LMNG and CHS were used in order to obtain the EROS-NOX2-p22 phox heterotrimeric complex for cryo-EM analysis.

Techniques: Binding Assay