eros nox2 p22 phox complex  (Thermo Fisher)

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 EROS-NOX2-p22 phox -7G5 Fab complex was concentrated and applied onto a Superose 6 Increase 10/300 GL column (GE HealthCare, Chicago, IL) in Buffer D (20 mM Tris pH 8.0, 250 mM NaCl, 0.06% digitonin) for size-exclusion chromatography (SEC).

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 EROS-NOX2-p22 phox -7G5 Fab complex was concentrated and applied onto a Superose 6 Increase 10/300 GL column (GE HealthCare, Chicago, IL) in Buffer D (20 mM Tris pH 8.0, 250 mM NaCl, 0.06% digitonin) for size-exclusion chromatography (SEC).

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 EROS-NOX2-p22 phox -7G5 Fab complex was concentrated and applied onto a Superose 6 Increase 10/300 GL column (GE HealthCare, Chicago, IL) in Buffer D (20 mM Tris pH 8.0, 250 mM NaCl, 0.06% digitonin) for size-exclusion chromatography (SEC).

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 EROS-NOX2-p22 phox -7G5 Fab complex was concentrated and applied onto a Superose 6 Increase 10/300 GL column (GE HealthCare, Chicago, IL) in Buffer D (20 mM Tris pH 8.0, 250 mM NaCl, 0.06% digitonin) for size-exclusion chromatography (SEC).

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 EROS-NOX2-p22 phox -7G5 Fab complex was concentrated and applied onto a Superose 6 Increase 10/300 GL column (GE HealthCare, Chicago, IL) in Buffer D (20 mM Tris pH 8.0, 250 mM NaCl, 0.06% digitonin) for size-exclusion chromatography (SEC).

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 EROS-NOX2-p22 phox -7G5 Fab complex was concentrated and applied onto a Superose 6 Increase 10/300 GL column (GE HealthCare, Chicago, IL) in Buffer D (20 mM Tris pH 8.0, 250 mM NaCl, 0.06% digitonin) for size-exclusion chromatography (SEC).

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 EROS-NOX2-p22 phox -7G5 Fab complex was concentrated and applied onto a Superose 6 Increase 10/300 GL column (GE HealthCare, Chicago, IL) in Buffer D (20 mM Tris pH 8.0, 250 mM NaCl, 0.06% digitonin) for size-exclusion chromatography (SEC).

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: 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: The cryoSPARC v3.3.1 software (Structura Biotechnology, Toronto, Canada) was utilized to perform single particle analysis of the EROS-NOX2-p22 phox -7G5 complex.

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 cryoSPARC v3.3.1 software (Structura Biotechnology, Toronto, Canada) was utilized to perform single particle analysis of the EROS-NOX2-p22 phox -7G5 complex.

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 cryoSPARC v3.3.1 software (Structura Biotechnology, Toronto, Canada) was utilized to perform single particle analysis of the EROS-NOX2-p22 phox -7G5 complex.

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 cryoSPARC v3.3.1 software (Structura Biotechnology, Toronto, Canada) was utilized to perform single particle analysis of the EROS-NOX2-p22 phox -7G5 complex.

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 cryoSPARC v3.3.1 software (Structura Biotechnology, Toronto, Canada) was utilized to perform single particle analysis of the EROS-NOX2-p22 phox -7G5 complex.

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 cryoSPARC v3.3.1 software (Structura Biotechnology, Toronto, Canada) was utilized to perform single particle analysis of the EROS-NOX2-p22 phox -7G5 complex.

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 cryoSPARC v3.3.1 software (Structura Biotechnology, Toronto, Canada) was utilized to perform single particle analysis of the EROS-NOX2-p22 phox -7G5 complex.

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: To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22 phox heterotrimeric complex.

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: To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22 phox heterotrimeric complex.

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: To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22 phox heterotrimeric complex.

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: To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22 phox heterotrimeric complex.

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: To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22 phox heterotrimeric complex.

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: To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22 phox heterotrimeric complex.

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: To delineate how EROS interacts with NOX2, we solved the cryo-EM structure of the EROS-NOX2-p22 phox heterotrimeric complex.

Techniques: Binding Assay