Warren C et al. (2017), Dynamic intramolecular regulation of the histon...

XB-ART-54383

Nat Commun 2017 Dec 20;81:2215. doi: 10.1038/s41467-017-02308-3.

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Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.

Warren C , Matsui T , Karp JM , Onikubo T , Cahill S , Brenowitz M , Cowburn D , Girvin M , Shechter D .

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Nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowed us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.

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Species referenced: Xenopus laevis
Genes referenced: h2ac21 h2bc21 npm1 ttll4

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	Fig. 1. Transient structural states of the Npm tail domain detected by NMR. a Domain layout of Xenopus laevis Npm. Core domain is colored gray, acidic stretches (A1, A2, and A3) colored red, basic NLS and C-terminus colored blue. Disorder prediction of Npm by DISOPRED3 software (0 = ordered, 1 = disordered). b Sequence of the Npm tail domain (residues 119–195). Acidic and basic residues colored red and blue, respectively. Underlined residues are assigned in the 1H-15N HSQC spectrum. c 1H-15N heteronuclear single quantum coherence (HSQC) spectrum of the Npm tail domain. d Secondary structure propensity (SSP) plot of the Npm tail domain derived from Cα, Cβ, and Hα chemical shift values. +1 indicates a stable α-helix, −1 indicates a stable β-sheet. e Circular dichroism (CD) spectrum and CONTIN secondary structure analysis of the Npm tail domain
	Fig. 2. Biophysical analyses of the conformations of the Npm tail domain. a NMR chemical shift perturbation (CSP) example spectra upon titration of NaCl and H2A/H2B. Blue to red indicates increasing amounts of NaCl or H2A/H2B (legend shown on right). b Heat map of CSP upon titration of NaCl and H2A/H2B. Yellow to red in the heat map indicates increasing CSP in the spectra and gray indicates binding-induced peak disappearance (legend at bottom right). Starred residues (Y123, W125, and A126) shown in letter D. Npm tail charge displayed using a 5aa-sliding window from −1 (red) to +1 (blue). c Analytical ultracentrifugation (AUC) sedimentation velocity profile of the Npm tail domain (15 µM) at various concentrations of NaCl. Sedimentation values normalized to s(20,w). d NMR CSP example spectra of Y123, W125, and A126 upon H2A/H2B titration. Peak disappearance indicates binding to H2A/H2B. e Graph of 15N relaxation times T1 (top) and T2 (middle), and heteronuclear 1H-15N NOE ratios (bottom) of assigned residues of the tail in the unbound (blue circles) and H2A/H2B-bound (red squares) states. Standard errors (gray bars) derived from fits to single exponential decay curves for T1 and T2 values, and from propagated signal-to-noise for 1H-15N NOE values
	Fig. 3. Paramagnetic relaxation enhancement NMR (PRE-NMR) analysis of intramolecular contacts. a PRE intensity ratio graphs (I ox/I red) of the Npm tail domain with MTSL paramagnetic spin labels at four different positions (positions indicated by arrows) and at two different NaCl concentrations (gray/red bars = 150 mM NaCl, orange points = 1 M NaCl). Error bars are inversely proportional to the propagated signal-to-noise ratio of individual resonances. Significant PRE effects (I ox/I red < 0.5) in the low-salt condition are highlighted in red. b Upper: I ox/I red graphs of the Npm tail domain bound to H2A/H2B. Two sets of intramolecular PRE effects derived from the complex (positions of spin labels indicated by arrows). Error bars are inversely proportional to the propagated signal-to-noise ratio of individual resonances. Same coloring scheme used as in a. Lower: Difference maps in I ox/I red values from the unbound and histone-bound states. Positive values indicate that residues are further from the spin label position after histone binding, negative values indicated that residues are closer to the spin label position after histone binding. c Intermolecular PRE effects measured using 1:1 molar ratio of non-isotope-labeled G132C-MTSL tail: 15N-labeled tail at 200 µM total protein concentration. Lack of strong PRE effects indicate very few or only transient intermolecular interactions. Error bars are inversely proportional to the propagated signal-to-noise ratio of individual resonances
	Fig. 4. Ensemble structural modeling and MD simulation of the Npm tail domain. a PRE Q-factor values with standard errors from the top five ensembles out of 50 with a variable number of conformers per ensemble ranging from 1 to 10. 350 ensembles corresponding to 1600 conformers were calculated in total. b Top five PRE-derived two-conformer ensemble models of the Npm tail domain. Each two-conformer ensemble is boxed. Conformers colored blue to red from N-terminus to C-terminus. c Protein Cα–Cα contact map of the tail derived from the top five two-conformer PRE models in b. Axes are residues of the tail domain from N to C-terminus from left to right and top to bottom. Colored red to yellow by average distance ranging from 0 to 50 Å, respectively. d 1 µs unrestrained all-atom molecular dynamics (MD) simulation of the Npm tail domain starting from an extended conformation. Radius of gyration (R g) measured as a function of simulation time. Representative structures show the starting, the most compact, and the most extended, and the final conformations observed during the simulation. Gray lines are the average and standard deviation of the R g measured from the PRE-NMR 2-conformer ensemble (b). e Time-averaged protein Cα–Cα contact map from the 1 µs MD simulation using an extended starting model. Same axes and coloring scale as in c
	Fig. 5. Mechanisms of H2A/H2B binding by the Npm tail domain. a Histone peptide array result using three different Npm truncations probed against comprehensive H2A/H2B peptides on a chip. Top = full-length Npm (1–195), middle = Npm core (1–118), bottom = GST-tagged Npm tail (119–195). Black boxes represent positions of α-helices in the histone fold. b Positions of MTSL paramagnetic spin labels on the H2A/H2B dimer structure. Histone tails removed for clarity. c I ox/I red graphs of the Npm tail domain bound to H2A/H2B at 400 µM concentration. Four sets of intermolecular PRE effects derived from the complex. Error bars are inversely proportional to the propagated signal-to-noise ratio of individual resonances. Same coloring scheme as used in Fig. 3a. d Reweighted atomic probability density maps indicating the most likely positions of A2 around the H2A/H2B dimer (gray ribbon, histone tails removed for clarity). Transparent red contoured at 10%, and solid blue contoured at 50%. Two distinct binding sites are consistent with the PRE data
	Fig. 6. Structural analysis of the pentameric Npm unbound and bound to H2A/H2B. a 1H-15N HSQC of the full-length Npm (red) overlaid with the tail alone (blue) under the same buffer and temperature conditions. Residues with significant differences in chemical shift values or those broadened in the full-length protein are labeled. b Ratio of 1H line widths of well-separated residues of the full-length protein compared to the tail domain alone. Ratio of 1 indicates peaks that are not broadened in the full-length spectrum. c SAXS curve (purple circles) of the core+A2:H2A/H2B complex. Top NMR-restrained SAXS hybrid model fit to the scattering curve (black line). d SAXS ab initio envelope of the core+A2:H2A/H2B complex (purple). Top NMR-restrained SAXS hybrid model fit into the envelope. Npm, H2A, and H2B colored gray, yellow, and red, respectively
	Fig. 7. Functional regulation of Npm by intramolecular interactions. a Npm domain map and truncations used in these experiments. Truncation names and residue numbers shown on right. b Equilibrium tryptophan fluorescence binding curves derived from the Npm tail and truncations binding to either H2A/H2B or H3/H4. Points represent average values of three replicates for each binding curve. Standard error of the points is included during the calculation of the binding curves and dissociation constants. c Dissociation constants (K D) and standard errors (±) of Npm tail and truncations derived from b. d Competitive pull-down assays using various truncations of the Npm tail and StrepII-tagged H2A/H2B dimers. Lanes 1–4, 10% inputs. Lanes 5–8, resin only controls. Lanes 9–12, standard pull-down assays. Lanes 13–18, competitive pull-down assays. Presence of Npm truncation bands in competitive pull-down lanes indicate higher affinity for H2A/H2B. e Chromatin assembly assay comparing the full-length Npm (residues 1–195) and tail domain (119–195). Concentrations of chaperone used in each indicated as molar ratio to histone octamer. f Chromatin assembly assay comparing full-length Npm (residues 1–195), ∆Cterm10 (1–185), ∆Cterm16 (1–179), and core+A2 (1–145) truncations at 11:1 molar ratio of Npm to histone octamer. g Quantification of chromatin assembly assays in f. 4–7 replicates of each shown with standard errors. One-way ANOVA and multiple group comparison of chromatin assembly activity of truncations to full-length Npm. ****p < 0.0001. h In vitro TTLL4 glutamylation assay using core+A2 truncation ± H2A/H2B. Glutamylation of Npm readout by anti-glutamylation western blot (top). Membrane stained in Direct Blue 71 (bottom). Histone binding inhibits Npm glutamylation on glutamates 127, 128, 129, and 131. i In vitro TTLL4 glutamylation assay using C-terminal truncations of Npm. Glutamylation of Npm readout by anti-glutamylation western blot (top). Membrane stained in Direct Blue 71 (bottom). The C-terminal 10 amino acids inhibit Npm glutamylation on glutamates 127, 128, 129, and 131. j Histone aggregate removal assay of linear DNA + H2A/H2B run on native TBE-PAGE with various truncations of Npm added at 2:1 molar ratio of Npm:H2A/H2B. Lower band indicates free DNA, upper bands indicate H2A/H2B:DNA aggregates. Core+A2 (1–145) is the only truncation capable of removing aggregates

References [+] :

Andrews, The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions. 2010, Pubmed, Xenbase