Structural Plasticity in Human Heterochromatin Protein 1β

<div><p>As essential components of the molecular machine assembling heterochromatin in eukaryotes, HP1 (Heterochromatin Protein 1) proteins are key regulators of genome function. While several high-resolution structures of the two globular regions of HP1, chromo and chromoshadow domains, in their free form or in complex with recognition-motif peptides are available, less is known about the conformational behavior of the full-length protein. Here, we used NMR spectroscopy in combination with small angle X-ray scattering and dynamic light scattering to characterize the dynamic and structural properties of full-length human HP1β (hHP1β) in solution. We show that the hinge region is highly flexible and enables a largely unrestricted spatial search by the two globular domains for their binding partners. In addition, the binding pockets within the chromo and chromoshadow domains experience internal dynamics that can be useful for the versatile recognition of different binding partners. In particular, we provide evidence for the presence of a distinct structural propensity in free hHP1β that prepares a binding-competent interface for the formation of the intermolecular β-sheet with methylated histone H3. The structural plasticity of hHP1β supports its ability to bind and connect a wide variety of binding partners in epigenetic processes.</p> </div

Rotational diffusion tensor of CD and CSD within full-length hHP1β.

<p>Diffusion tensor parameters for the different tumbling models, obtained from experimental ¹⁵N spin-relaxation data through ROTDIF, are listed: the overall rotational correlation time τ_c; D_xx, D_yy and D_zz are the principal values of the diffusion tensor; Q is the quality factor defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060887#pone.0060887-Walker1" target="_blank">[27]</a>; P defines the probability that an improvement in the fit when a more complex model is applied has occurred by chance. The best model for CD and CSD is marked in bold. The little improvement in the fit with the more complex fully-anisotropic model was not statistically significant for CD.</p

Internal dynamics.

<p><b>A. </b>¹⁵N linewidths of perdeuterated hHP1β as a function of residue number, measured from a TROSY-HSQC recorded at 700 MHz and 303 K. <b>B.</b> R_ex values of CD in full-length hHP1β as a function of residue number. <b>C.</b> Comparison of steady-state ¹H-¹⁵N heteronuclear NOE values of CD in full-length hHP1β in the free state (black circles) with those of CD in full-length hHP1β in complex with the H3K_C9me3 peptide (1–15) at a molar ratio of 1∶4 (white circles). Both measurements were performed at 298 K, 600 MHz proton Larmor frequency, 5 s recycle delay, on a 0.3 mM ¹⁵N-perdeuterated hHP1β sample.</p

hHP1β populates an extended ensemble.

<p><b>A.</b> Dynamic light scattering of hHP1β. The histogram plot shows the experimental data from one measurement consisting of 20 acquisitions. The diffusion coefficient value (D) reported is an average of five measurements done at identical conditions. <b>B.</b> PFG-NMR based diffusion plot of hHP1β. The natural logarithm of the intensity ratio I/I₀ linearly correlates (R = 0.98) with Q, a combined parameter dependent on the gradients strength and delays as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060887#pone.0060887-Zheng1" target="_blank">[51]</a>. For the intensity ratio, four integrated signals in the 2.4–0.7 ppm region were measured from 32 spectra recorded with increasing gradient strength from 5–75% of the maximum value. Diffusion coefficient values (D) from NMR and DLS experiments were converted into hydrodynamic radius (R_h) values based on the Stokes-Einstein’s equation. <b>C.</b> Small angle X-ray scattering profile of hHP1β. The plot displays the decimal logarithm of the scattering intensity as a function of momentum transfer, s. The distance distribution function is displayed in the inset. <b>D. </b><i>R_g</i> distributions from EOM for hHP1β: initial random pool (continuous line) and selected ensembles averaged over 50 independent EOM runs (dashed line).</p

Backbone dynamics probed by ¹⁵N- relaxation rates.

<p><b>A, B, C. </b>¹⁵N spin-relaxation rates for ¹⁵N-perdeuterated hHP1β measured at a proton Larmor frequency of 600 MHz at 298 K. Residues with severe peak overlap or insufficient signal-to-noise ratio were excluded. R₁ (A), R₂ (B) and steady-state ¹H-¹⁵N heteronuclear NOE (C) are shown along the protein sequence. The R₂ rates were derived from R_1ρ measurements upon correction for the off-resonance tilted field as described in the Methods. <b>D.</b> Graphical analysis of reduced spectral density mapping. The solid line represents the theoretical function of J(ω_N) versus J(0) assuming a rigid single Lorentzian motion. Experimental values are plotted as points with labels specific for each hHP1β domain. <b>E.</b> The rotational correlation time τ_c, determined for each residue from the R₂/R₁ ratio, is shown as a function of residue number. <b>F.</b> Illustration of the diffusion tensor of CD (left, axially symmetric) and CSD (right, fully anisotropic) in full-length hHP1β. The x- and y-axes are shown as half-axes only for the fully anisotropic tensor. Pictures were prepared using MolMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060887#pone.0060887-Koradi1" target="_blank">[57]</a>.</p

Analysis of Phosphorylation-Dependent Protein–Protein Interactions of Histone H3

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails

Analysis of Phosphorylation-Dependent Protein–Protein Interactions of Histone H3

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p