Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Representative STED image of NETs stained with anti-MPO and the DNA dye YOYO-1. n = 3 independent experiments. b , Representative STORM image of NETs stained with anti-MPO antibody. n = 10 independent experiments. c , Single-colour immunofluorescence and autocorrelation of MPO or nucleosomes (nuc) on the thinnest NET DNA filaments acquired using STED microscopy. n = 3 independent experiments (8,059 NET filaments analysed). d , Single-colour immunofluorescence and autocorrelation of MPO or nucleosomes on the thinnest NET DNA filaments acquired using STORM. n = 10 independent MPO autocorrelation experiments (1,143 NET filament fragments analysed) and n = 5 independent experiments (773 NET filaments analysed). e , Dual-colour immunofluorescence analysis of MPO and nucleosome NET staining acquired by STED microscopy. n = 3 independent experiments. f , Cross-correlation of MPO and nucleosomes quantified from a . n = 3 independent experiments (8,059 filaments analysed). a.u., arbitrary units. g , NET nucleosome fractionation. Neutrophils were stimulated with PMA for 4 h to induce NETs. NETs were digested into mononucleosomes using micrococcal nuclease and fractionated over a 10–30% sucrose gradient. The fractions (fract.) were analysed using western blotting with antibodies against MPO and histones, and DNA was electrophoresed in agarose to identify 140 bp mononucleosome DNA. n = 4 independent experiments. h , Co-immunoprecipitation of MPO–nucleosome complexes from PMA- or nigericin-induced NETs. MPO immunoprecipitates were blotted for histones and MPO. DNase-I-digested nucleosomes were used as a control. Inputs were calculated from DNA concentrations of each experimental condition and monitored by agarose gel DNA electrophoresis (bottom). n = 5 independent experiments using PMA and nigericin (nig.) stimulation. i , Native gel shift assay. HeLa mononucleosomes and rMPO, MPO or catalase (catal.) were incubated 10 min at room temperature with MPO inhibitors (inhib.) azide or ABAH and subsequently subjected to native gel shift assay to monitor MPO–nucleosome interactions. SDS–PAGE gels demonstrate inputs. n = 3 independent experiments. Uncropped gels and blots of g – i are shown in Supplementary Fig. . Scale bars, 3 μm ( a and b ) and 1 μm ( e ).

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Staining, Immunofluorescence, Microscopy, Fractionation, Western Blot, Immunoprecipitation, Control, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Gel Shift, Incubation, Inhibition, SDS Page

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Representative SIM image of NETs stained with anti-MPO and DNA dye YOYO-1. n = 5 independent experiments. b , Single colour immunofluorescence and autocorrelation of MPO or nucleosomes on the thinnest NET DNA filaments acquired using SIM. n = 5 independent MPO and nucleosome autocorrelation experiments (71,992 NET filament fragments analysed for MPO and nucleosome). c , Representative STED image of NETs stained with anti-MPO and anti-DNA. n = 3 independent experiments. d , Representative dual colour immunofluorescence of MPO and nucleosome on NETs acquired by SIM. n = 5 independent experiments. e , Cross-correlation of MPO and nucleosome quantified from panel d . n = 5 independent experiments (71,992 NET filament fragments analysed). f , MPO and 3D9 co-localize in SIM microscopy. Neutrophils were stimulated with PMA for 4 h and labelled with antibodies to MPO (green) and 3D9 (magenta), an antibody that recognizes an epitope generated by the proteolytic processing of histone H3 and is NET specific. These data reproduce previous staining with the nucleosomal marker (PL2.3). Scale bar = 3 µm, boxes 2 × 2 µm. n = 4 independent experiments. g , MPO colocalises with cleaved histone H3. Cross-correlation (same method as in Fig. ) of 3D9 and PL-2.3 demonstrating that these two antibodies are highly colocalized and of MPO with 3D9 demonstrating colocalization as well. n = 4 independent experiments.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Staining, Immunofluorescence, Microscopy, Generated, Marker

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: Neutrophils were stimulated with PMA or Panton Valentine Leukotoxin (PVL) and stained for MPO (green) and nucleosomes (PL2.3, magenta). Both labels are periodic and are similarly distributed in both stimuli. Boxed zoom-ins show co-localization of MPO and nucleosomes. Scale bar: 2 μm, boxes 2 × 2 μm. n = 3 independent experiments.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Staining

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Co-immunoprecipitation of MPO/nucleosome complexes from MSU crystal-induced NETs. MPO immunoprecipitates were blotted for histones and MPO. DNase-I-digested nucleosomes were used as a control. Inputs were calculated from DNA concentrations of each experimental condition and were monitored by agarose gel DNA electrophoresis (bottom panel). n = 3 independent experiments using MSU crystals as a NET stimulus. b , Native gel shift assay using Hela derived nucleosomes and rMPO or catalase at 50–150 mM NaCl concentrations. Nucleosome shifting was monitored by staining gels with ethidium bromide. DNase-I-digested nucleosome was used as a control and abolished shifted bands and acted as a proxy for nucleosome identification. 10 µl from each reaction was subjected to SDS-PAGE to monitor inputs. n = 5 independent experiments. c , Native gel shift assay using HeLa derived nucleosomes and native MPO or catalase at 0–150 mM NaCl concentrations. Native gels were stained with ethidium bromide or InstaBlue to monitor band shifts after 10 min of incubation at room temperature. DNase I was used as a control. A small aliquot of each 10 min reaction was monitored using SDS-PAGE. n = 3 independent experiments. Uncropped gels and blots of panels a-c are shown in Supplementary Fig. . d , Mass photometry measurements and quantification of purified rMPO and MPO samples at 10 nM final concentration. e , Representative example of enzymatic activity of MPO bound versus unbound mononucleosomes. NETs were digested into mononucleosomes using micrococcal nuclease, fractionated and then harvested for enzymatic activity in the presence or absence of DNase I. Error bars represent one standard deviation.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Immunoprecipitation, Control, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Gel Shift, Derivative Assay, Staining, SDS Page, Incubation, Purification, Concentration Assay, Activity Assay, Standard Deviation

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Cryo-EM reconstruction of rMPO–nucleosome complex shown in two orientations, which are related by a turn of almost 180°. b , Molecular model of the rMPO–nucleosome complex in the same orientations as in a . The nucleosome consists of histones H2A, H2B, H3 and H4 (two copies of each), and the Widom-601 143 bp DNA sequence. c , A view of the surface of the nucleosome from the top through rMPO (most of the rMPO molecule is omitted to allow an unobstructed view) reveals that MPO binds predominantly to the acidic patch (demarcated in green) and also to a smaller auxiliary interface (indicated in purple). d , The acidic patch interface is dominated by two arginine anchors, Arg473 and Arg653 of MPO, which bind tightly into acidic pockets generated by residues from histones H2A (light blue) and H2B (light purple). Hydrogen bonds are indicated as green dotted lines. e , Magnified view of arginine Arg473’s canonical acidic patch anchoring interactions with Glu61, AspD90 and Glu92 of histone H2A. f , Magnified view of the second arginine anchor, Arg653, which interacts with H2A Glu56. g , The auxiliary interface is created by MPO residues Met688, Arg691 and Gln692 interacting with histone H3.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Cryo-EM Sample Prep, Sequencing, Generated

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Structure of the single-chain antibody fragment PL2-6 bound to a nucleosome (PDB 6DZT) . b , Close-up of PL2-6 binding to the acidic patch by using two arginine anchors (R124 and R126) that bind in similar positions as the arginine anchors of MPO (Fig. ). c , Acidic patch binding interface of the chromatin-associated protein RCC1 which is essential to recruit Ran to chromatin (PDB 3MVD) . RCC1 (brown) uses two arginine anchors (R223 and R216) that bind in similar positions as the arginine anchors of MPO (Fig. ). d , Acidic patch binding interface of the BAH domain of Sir3 which is part of the chromatin silencing complex SIR (PDB 3TU4) . Sir3 (green) uses two arginine anchors (R29 and R28) that bind in similar positions as the arginine anchors of MPO (Fig. ). e , Acidic patch binding interface of the histone H3 K79 methyltransferase Dot1L (PDB 6O96) . Dot1L (white) uses one arginine anchor (R282) binding to a position similar to MPO arginine anchor 2 (Fig. ). f , Acidic patch binding interface of the LANA peptide of Kaposi’s sarcoma herpesvirus (KSHV; PDB 1ZLA) . The LANA peptide (olive) which is essential for episome persistence uses one arginine anchor (R9) binding to a position very similar to MPO arginine anchor 1 (Fig. ). g , Superposition of position 1 arginine anchors of MPO (R473, orange), RCC1 (R223, brown), Sir3 (R29, green) and the KSHV LANA peptide (R9, olive). For clarity, only the MPO-bound nucleosome is shown as a semi-transparent surface, cartoons and side chains of selected residues. h , Superposition of position 2 arginine anchors of MPO (R653, orange), RCC1 (R216, brown), Sir3 (R26, green) and Dot1L (R282, white). For clarity, only the MPO-bound nucleosome is shown as a semi-transparent surface, cartoons and side chains of selected residues.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Binding Assay

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Scheme showing the reconstitution of complexes comprising native MPO and nucleosomes by mixing (1) and incubation (2), followed by purification using size exclusion chromatography via a Superdex 200 5/150 column (3) and subsequent analysis by cryo-EM (4). b , Representative micrograph of a sample prepared via the reconstitution route shown in panel a . The sample contains a large amount of free, unbound DNA (thin, elongated filaments) which indicates nucleosome disassembly. Selected 2D classes resemble dimeric MPO bound to a DNA filament. c , Native gel shift assay using recombinant nucleosomes and the indicated molar equivalents (eq) of rMPO or MPO, respectively. After 10 min incubation, an aliquot from each reaction was run in native gels, stained with ethidium bromide to visualize DNA and then subsequently silver stained to visualize protein. In addition, reduced SDS-PAGE gels of the same samples were run to visualize total protein and DNA levels in each reaction. n = 4 independent experiments. d , Representative negative stain micrograph of MPO reconstituted with Widom 601 DNA (without histone proteins). MPO binds to the DNA. e , MPO-DNA binding assay. Recombinant or native MPO was incubated with dsDNA (either synthetic Widom-601 DNA or DNA derived from HeLa nucleosomes) and subjected to DNA agarose electrophoresis after 30 min of incubation at room temperature. DNA was visualized by ethidium bromide staining. n = 3 independent experiments. Uncropped gels of panels c,e are shown in Supplementary Fig. .

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Incubation, Purification, Size-exclusion Chromatography, Cryo-EM Sample Prep, Gel Shift, Recombinant, Staining, SDS Page, DNA Binding Assay, Derivative Assay, Electrophoresis

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Streptavidin pull-down of biotinylated nucleosomes incubated with indicated molar equivalents of rMPO or MPO, respectively. Nucleosomal DNA was stained on agarose gels using EtBr, while proteins in the pull-down and supernatant fractions were blotted and visualized using the indicated antibodies. Streptavidin beads alone, rMPO alone, MPO alone, DNase-I-treated nucleosomes and catalase co-incubated with nucleosomes were used as controls. n = 3 independent experiments. Uncropped gels and blots are shown in Supplementary Fig. . b , Cryo-EM reconstruction of MPO monomers (left) and dimers (right) bound to nucleosomes as observed during a time-course experiment. The binding position of MPO monomers and dimers on the acidic patch is almost identical, also to rMPO. c , The cryo-EM structure of MPO dimer–nucleosome complex reveals that, in addition to acidic-patch binding through the first MPO protomer, the second protomer contacts the DNA. d , The acidic patch interface in native MPO is highly similar to that of rMPO. Arg473 and Arg653 of MPO bind tightly into acidic pockets generated by residues from histones H2A (light blue) and H2B (light purple). e , Magnified view of the interface between the second MPO protomer and the DNA in the MPO dimer–nucleosome complex. The interface is characterized by charge and shape complementarity without prominent, strong contacts.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Incubation, Staining, Cryo-EM Sample Prep, Binding Assay, Generated

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Scheme showing the time course experiment for reconstitution of complexes comprising native MPO and nucleosomes. Both components are mixed (1) and incubated for periods between 15 s and 20 min as indicated (2). Then, instead of purifying via size exclusion chromatography, samples were directly subjected to analysis by cryo-EM (3). b , Cryo-EM reconstructions derived from the dataset corresponding to the 5 min time point of the time course experiment shown in panel a . The dataset is representative also for 2, 10 and 20 min incubations and contains free nucleosomes (left) and MPO monomer/nucleosome (middle) and MPO dimer/nucleosome complexes (right). Details of processing of this dataset are shown in Supplementary Fig. and in Supplementary Table . b , Molecular models corresponding to the reconstructions shown in panel b .

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Incubation, Size-exclusion Chromatography, Cryo-EM Sample Prep, Derivative Assay

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Superposition of dimeric MPO from the nucleosome-bound cryo-EM structure (colours as in Fig. ) and in an earlier crystal structure (grey, PDB 1MHL) . Both structures are virtually identical; RMSD 0.44 Å over 1,136 residues. b , Superposition of free nucleosome (violet) and the MPO dimer-bound nucleosome (green). Notably, one DNA end (terminal 12 base pairs) of the MPO-bound nucleosome are disordered. RMSD 0.41 Å over 1,016 residues. c , Superposition of the rMPO-bound nucleosome (grey) and the MPO dimer-bound nucleosome (green). RMSD 0.50 Å over 1,016 residues. d , Superposition of the MPO monomer-bound nucleosome (blue) and the MPO dimer-bound nucleosome (green). RMSD 0.71 Å over 1,016 residues.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Superposition of nucleosomes bound to MPO monomer (blue) or dimer (grey), respectively. Both structures are highly similar (r.m.s.d. of 0.71 Å over 1,016 residues). The major difference is the one disordered DNA end in the structure bound to the MPO dimer (highlighted in green), as visible by it protruding from the grey, transparent surface of the DNA of the nucleosome bound to MPO monomer. For clarity, the histones of the MPO monomer-bound nucleosome have been omitted. b , Magnified view of the superposition of MPO dimer–nucleosome and MPO monomer–nucleosome structures shows that the terminal 12 nucleotide pairs in the MPO dimer–nucleosome complex (green cartoon, and grey transparent surface) are disordered compared with the monomer–nucleosome complex (blue) to avoid clashing with the second MPO protomer (yellow surface patch). For clarity, the proteins of the monomer–nucleosome complex are omitted. c , Cryo-EM structure of the MPO dimer–nucleosome complex intermediate state that was found only at the 15 s timepoint. In this state, MPO interacts only with the DNA through the arginine-rich surfaces of both protomers without contacting the histones. d , Nucleosome-remodelling assay using recombinant nucleosomes with an encrypted GATC restriction site that is cut by DpnII only when nucleosome–DNA interactions are perturbed. GATC nucleosomes were used to monitor the kinetics of nucleosome disassembly by rMPO and MPO. n = 3 independent experiments. Ref., reference. e , Cryo-EM structure of the nucleosome bound by two dithiothreitol (DTT)-reduced MPO monomers. The MPO molecules bind to the acidic patches on both sides of the nucleosome in an identical manner to the non-reduced sample. f , Nucleosome remodelling assay as described in d , using either non-reduced or DTT-reduced monomerized MPO. The monomerized sample cannot displace DNA from the nucleosome. n = 3 independent experiments. Uncropped gels of d and f are shown in Supplementary Fig. .

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Cryo-EM Sample Prep, Recombinant

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Cryo-EM reconstruction (in two orientations) of the MPO monomer/nucleosome complex incubated for 30 min and then purified over SEC. On the right, the reconstruction is shown as a transparent surface with the docked MPO monomer/nucleosome model of the 5 min time point dataset. b , Cryo-EM reconstruction (in two orientations) and model of the MPO dimer/nucleosome complex in the intermediate state as observed only in the 15 s time point. c , Nucleosome remodelling assay using recombinant nucleosomes with an encrypted GATC restriction site that is cut by DpnII only when nucleosome-DNA interactions are perturbed. GATC nucleosomes were employed to monitor the kinetics of nucleosome disassembly by MPO in absence and presence of the acidic patch binder PL2-6. n = 3 independent experiments. The uncropped gel is shown in Supplementary Fig. .

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Cryo-EM Sample Prep, Incubation, Purification, Recombinant

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Mass photometry of DTT-reduced MPO samples. Incubation of MPO with 50 mM DTT leads to a progressive shift in particle mass from a dominant peak around 150 kDa (MPO dimers) to almost exclusively a peak around 75 kDa (MPO monomer). After 4 h, a similar monomer:dimer ratio as in the (non-reduced) rMPO sample is reached (see Supplementary Fig. . b , MPO immunoprecipitation using DTT-reduced MPO and nucleosomes. The observed interaction of MPO with H2B depends on intact nucleosomes as DNase I treatment prevents it. The uncropped blots are shown in Supplementary Fig. . c , Cryo-EM reconstructions of samples prepared using DTT-reduced MPO and nucleosomes derived from the dataset corresponding to the 2 min (nucleosome bound by one or two MPO monomers, respectively) or 5 min (nucleosome bound by one MPO monomer and one dimer) time points of the time course experiment. Details of processing of this dataset are shown in Supplementary Figs. , and in Supplementary Table . d , Molecular models corresponding to the reconstructions shown in panel c . e , Acidic patch interface in reduced MPO (structure of nucleosome bound by one MPO monomer). Interaction of MPO with histones H2A (light blue) and H2B (light purple) is very similar as in the case of rMPO or non-reduced MPO. f , N-terminus of the MPO light chain gets disordered upon DTT reduction. Before reduction, the N-terminus is fixed by a cystine bridge between C167 and C179 (blue). Upon reduction, the density of the N-terminal four amino acids gets disordered (salmon density), whereas the remaining part of the light chain stays unperturbed. The heavy chain is shown as a white surface. Several side chains of the light chain are shown for reference.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Incubation, Immunoprecipitation, Cryo-EM Sample Prep, Derivative Assay

Journal: Nature

Article Title: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

doi: 10.1038/s41586-025-09523-9

Figure Lengend Snippet: a , Representative NET segmentation of NET tomogram, showing the ultrastructural landscape of PMA-stimulated NETs. Beside broken membranes (purple), granules (pink) and unknown types of filament (blue), large protein complexes (yellow) embedded in a wide web of DNA can be seen in tomograms of PMA-stimulated NETs. b , Reconstruction from cryo-ET in two orientations (top). On one side of the nucleosome, an additional density is present that is remotely similar to MPO monomers as observed in the molecular model of the in vitro reconstituted MPO monomer–nucleosome complex (bottom). c , Co-immunoprecipitation of MPO–nucleosome complexes from PMA-induced NETs or digested mononucleosomes from the sputum of individuals with cystic fibrosis (CF). MPO immunoprecipitates were blotted for histones and MPO. DNase-I-digested nucleosomes were used as a control. Inputs were calculated from DNA concentrations of each experimental condition and were monitored by agarose gel DNA electrophoresis (bottom). n = 5 independent experiments using PMA or 3 independent sputum donors with CF from different days of sample collection. Uncropped gels and blots are shown in Supplementary Fig. . d , Mechanistic model of the dual function of MPO in the context of NETs. After MPO (which is a mixture of monomers and dimers) translocates to the nucleus (1), it initially binds to chromosomal DNA (2). Then, both monomers and dimers can bind to the nucleosome acidic patch which already leads to unstacking of nucleosomes and initial chromatin decondensation (3). As dimeric MPO clashes with one end of the nucleosomal DNA, this DNA unwraps to prevent this clash, which initiates complete disassembly of MPO dimer-bound nucleosomes (4). Monomeric MPO, on the other hand, does not clash with the DNA, does not initiate nucleosome disassembly and stays attached to decondensed chromatin in mature NETs, where it produces hypochloric acid (HOCl), which is important for the activity of NETs.

Article Snippet: HeLA mononucleosomes or recombinant biotinylated nucleosomes (Epicypher, 16-0002 (HeLa mononucleosomes), 16-0006 (recombinant wild type) and 16-0027 (recombinant tailless)) were co-incubated with either recombinant MPO (RnD systems) or native MPO (Sigma-Aldrich) at a molar ratio of 1 nucleosome to 0.5 rMPOs or 0.25 nMPOs for 5 min at room temperature in chromatin remodelling buffer (12 mM HEPES, 40 mM Tris-HCl, 0.32 mM EDTA, 3 mM MgCl 2 , 10% glycerol, 0.02% Igepal, 60 mM KCl and indicated NaCl concentrations, pH 7.4) and then directly loaded into native gels to monitor binding of MPO to nucleosome after the addition of native sample buffer (Invitrogen).

Techniques: Tomography, In Vitro, Immunoprecipitation, Control, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Activity Assay