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Graphical Abstract
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Figure 1. NPAC Linker Sustains Productive Nucleosome Recognition by LSD2
(A) Domain organization of LSD2 and NPAC. Disordered regions are in wavy lines. LSD2 (PDB: 4hsu) is colored according to its multidomain architecture, with the flavin adenine dinucleotide (FAD) in gold and the zinc ions in light blue.
(B) Our semisynthetic nucleosomes form a covalent adduct with the FAD of LSD2 (absorbance peak at 400 nm rather than at 458 nm, as for the oxidized enzyme).
(C) Elution profile (WTC-030S5 column, Wyatt) of semisynthetic NCP (10 μM) and LSD2 (20 μM) after 1 h of incubation with or without the NPAC linker (100 μM). Respectively, 2:2:1 and 1:1:1 stand for (LSD2/NPAC-linker)2/NCP and LSD2/NPAC-linker/NCP complexes. See also Figure S1.
(D) Elution profile of the LSD2/NPAC-linker/NCP sample used for cryo-EM (dashed lines; three 10/300 columns of Superdex 200 in series, GE Healthcare). Protein, DNA, and the flavin-H3 covalent adduct were detected by monitoring the absorbance at 280, 260, and 400 nm, respectively.
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Figure 2. Single-Particle Cryo-EM Reveals that Multiple Conformations and Few Interactions Characterize the LSD2/NPAC-Linker/NCP Complex
The quality of the density maps can be best appreciated at the left, whereas model fitting can be best visualized at the right. H2A, H2B, H3, and H4 are shown in dark gray, light gray, purple, and pink, respectively. LSD2 (PDB: 4hsu) is in light blue. The NCP structure used for map fitting was obtained from PDB: 6esf. The DNA molecule is entirely visible in all maps. The first and the last three base pairs were modified to match our 601 sequence exactly. See also Figures S3 and S4.
(A) Overview of the three classes of the LSD2/NPAC-linker/NCP complex. For class 4, LSD2 is tentatively fitted at the left, simply as a reference.
(B) Class 1 represents the intact NCP, whereas class 5 is a partly unfolded NCP: a H2A-H2B dimer is missing, and the N-terminal helix of H3 is unstructured.
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Figure 3. Close-Up Views of LSD2/NCP Interfaces
(A) Class 2 features two contact points between the LSD2 and the nucleosome, depicted in the close-up views.
(B) Class 3 displays a single contact point.
(C) Contact region (in cyan) of class 4 involves DNA super-helical location ± 6 and two short segments of H2A and H2B. Colors are the same as in Figure 2, with the NPAC linker in violet. The density maps are depicted as light gray mesh. Reference residues are labeled, and their Cα atoms are shown as spheres. Dashed lines highlight possible pathways of H3 tails connecting the NCP to the LSD2 catalytic site. See also Figures S3 and S4.
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Figure 4. LSD2 Engages the DNA Template via Electrostatic Interactions
(A) The electrostatic surface of LSD2 exposed toward the nucleosome. Positively charged patches characterize the zinc-finger domain and part of the area surrounding the H31–16-binding pocket. The bar indicates the electrostatic potential in kcal/mol∗e. Red represents negative electrostatic potential while blue represents positive charge potential.
(B) Close-up views of the LSD2-nucleosome interfaces in class 2 (middle) and class 3 (right), highlighting the LSD2 residues subjected to mutagenesis (red spheres at Cα atoms).
(C) Effect of ionic strength on the binding affinities of full-length and Δ30 LSD2 to DNA. Changes in the fluorescence polarization were measured in millipolarization (mP) units and plotted against LSD2 concentrations. Error bars refer to experiments carried out in triplicate. Data are represented as mean ± SEM. See also Table S1.
(D) Qualitative evaluation of LSD2 mutations on LSD2/NPAC-linker/NCP complex yield. The histogram shows the ratio of the LSD2/NPAC-linker/NCP peak to the absorbance of the free NCP peak (both recorded at 260 nm). The ratios are reported as percentages with reference to LSD2 wild type, which was given the 100% value.
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Figure 5. NPAC Dehydrogenase Domain Is Catalytically Impaired and Forms a Stable Tetramer
(A) NPAC homolog sequences are aligned with two representative members of the short-chain alcohol-dehydrogenase family from Geobacter metallireducens and G. sulfurreducens. The mutation of the catalytic lysine to methionine or asparagine is highlighted within a blue box.
(B) Crystal structure of the NPAC dehydrogenase domain (residues 261–553) shows a tetrameric assembly (PDB: 2uyy). The NADPH is visible in two subunits and is in green. See also Figures S6 and S7 and Table S3. The inset shows the comparison between the active-site regions of the NPAC dehydrogenase (in salmon) and those of the γ-hydroxybutyrate dehydrogenase from G. sulfurreducens (PDB: 3pdu; gray).
(C) Elution profiles of wild-type, M437N, and M437K NPAC dehydrogenase domains (5/150 column; Superdex 200, GE Healthcare).
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Figure 6. NPAC Tetramer Binds Multiple Copies of LSD2/Nucleosome
(A) NCP, LSD2, and NPACΔ205 (residues 206–553, containing the linker sequence and the dehydrogenase domain; see Figure 1A) were incubated at different molar ratios as shown above each panel (NPACΔ205 concentrations refer to the tetramer, molecular weight [MW] = 151 kDa) (Table S3). Decreasing relative amounts of NPACΔ205 with respect to nucleosome and LSD2 favors the formation of species at a higher molecular weight with greater DNA content (peak C), as calculated from the A214/A260 ratio, which reflects the protein/DNA content. Peak A elutes at the same volume of the LSD2/NCP complex containing the NPAC linker only (Ve = 2.35 mL). This peak, occasionally present in the chromatograms, is a species that has lost an intact NPACΔ205 (likely by proteolysis). The experiments were performed on a WTC-030N5 column. See also Figure S7 and Table S3.
(B) Dehydrogenase domain of NPAC forms a stable tetramer decorated by flexible N-terminal arms, which comprise a PWWP domain, an AT-hook region predicted to bind DNA, and the LSD2-activating segment.
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Figure S1 related to Figure 1. Formation of the complex between LSD2 and the nucleosome
core particle is dependent on NPAC-linker and independent on ionic strength. A-B) The ionic
strength does not affect the efficiency of LSD2/NPAC-linker/NCP complex formation, either in
absence (A) or presence (B) of the NPAC-linker (WTC-030S5 column). C) Elution profiles of a
semi-synthetic NCP carrying a double mutation on H3 (K23M K27M), incubated with LSD2 with
and without NPAC-linker (WTC-030N5 column). Protein concentrations as in the main Figure 1C.
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Figure S2 related to Table 1. Histone tails do not interfere with H3-K4 demethylase activity of
LSD2. Apparent kcat values measured in absence and presence (100 M) of the histone tail peptides
listed above each plot. Very low or no inhibition was detected. The data were fit to the MichaelisMenten curve. The experiments were performed in 50 mM HEPES pH 8.5, 100 mM NaCl, 0.3 M
LSD2, 3 M NPAC-linker, 0 - 40 µM H3K4me21-40 peptide substrate.
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Figure S3 related to Figure 2. Processing of cryo-EM micrographs. A) Processing work-flow.
B) A micrograph used for data processing showing the sample embedded in ice (scale bar: 200 Å).
Examples of representative particles are outlined by white circles. C) Representative 2D class
averages. D) Ab-initio determination of NCP density map using cisTEM. E) The five final classes
used for structural analysis.
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Figure S4 related to Figure 2. The five classes revealed by cryo-EM data processing. The
electron density map of each class is shown in three orientations using a colour gradient for the
resolution. The corresponding Fourier-Shell-Correlation (FSC) curve is reported on the right. Class
1 is the isolated nucleosome (A-B), whereas classes 2, 3, and 4 are the LSD2/NPAC-linker/NCP
complexes, in which LSD2 adopts different binding modes (C-D, E-F, G-H). Class 5 is an unfolded
NCP: DNA is sliding away from the core, now composed by an H3-H4 tetramer and only one H2AH2B dimer (I-J). The map for class 1 is depicted at a higher counter-level for illustration of highresolution features of the histone core.
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Figure S5 related to Figure 5. NPAC-PWWP is a chromatin-binding module. A) SPOT-array
technology probes histone-peptide binding to the NPAC-PWWP domain (residues 1-105). Cellulose
membrane was spotted with an array of 20-aminoacid long peptides covering the sequences of the
H3.1, H3.3, and H4 N-terminal tails with single and multiple epigenetic modifications (Table S2).
Control peptides are shown in red circles. Tightest binding was consistently observed with peptides
covering residues 26-45 of H3. B) Isothermal calorimetry experiment for H3-tail binding to PWWP.
The experiment was carried out using the PWWP protein inside the sample cell and the naked
histone H3 peptide (residues 1-40) in the syringe. C) Bio-Layer Interferometry shows that Lys36
methylation state does not affect association of H328-48 to NPAC-PWWP. D) Fluorescence
polarization binding assay using a 21 bp DNA sequence labelled with TAMRA fluorescent dye
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Figure S6 related to Figure 5. NPAC dehydrogenase domain binds NADPH. Top: NPAC
dehydrogenase domain (residues 261-553) was dialysed against a solution consisting of 4 M KBr,
100 mM K2HPO4 pH 6.5, and activated charcoal to remove the natural cofactor (blue curve). The
dialyzed sample was then desalted and split into two fractions which were incubated with NADH
(green curve) and NADPH (red curve), respectively. Each sample was then loaded on a desalting
column. The Uv/Vis absorbance spectrum confirms that NPAC dehydrogenase retains NADPH but
not NADH as shown from the absorbance peak of the reduced nicotinamide at 348 nm. Bottom:
cofactor identification by mass-spec. The cofactor was extracted through denaturation of the
recombinant NPAC dehydrogenase domain, purified, and identified by HPLC coupled with a mass
spectrometer. The spectrum is fully consistent with NADPH, with no contamination by NADP+
or
NAD+
(742 and 663 Da, respectively).
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Figure S7 related to Figure 6. SAXS analysis of LSD2, nucleosomes, NPAC dehydrogenase,
NPAC205 and LSD2/NPAC205/NCP complex. For each sample: the left panel shows the
intensity chromatogram with the distribution of molecular weight (orange) along the elution peak,
the central panel shows the average scattering plot, the right panel shows the Kratky plot. The
average scattering curve and the Kratky plot for the LSD2/NPAC205/NCP refer to the region B-C
of the chromatogram shown in the main Figure 6A, corresponding to the highest peak of the
intensity chromatogram. The structural parameters derived from Guinier and P(r) analyses are
reported in Table S3.
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