Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR

Abstract

Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.

Fig. 1.

Structure and NMR spectra of the nucleosome. (A and B) Distribution of assigned ILV methyl groups in the nucleosome (PDB ID code 2PYO) showing top (A) and side views (B). The backbone of the histones is shown with ribbons, ILV methyl groups as spheres. Color coding: H2A, orange; H2B, red; H3, blue; H4, green; DNA, gray. (C) Methyl-TROSY spectra of ILV methyl-labeled H2A, H2B, H3, and H4 in the nucleosome. (Top) The δ1 methyl groups of Ile residues. (Bottom) The Leu-δ1/δ2 and Val-γ1/γ2 methyl groups. Leu/Val methyl groups are labeled with their stereospecific assignments where available; otherwise the two methyl groups are arbitrarily assigned as “a” or “b.” Aliased Ile-δ1 resonances are shown in gray. Ile0 represents the Ile inserted after the N-terminal Met for improving the yield of protein expression.

Fig. 2.

Identification of the interactions between HMGN2 and the nucleosome by chemical shift perturbation and paramagnetic spin labeling. (A) Overlay of the spectra of free (black) and HMGN2-bound (orange or red) nucleosomes, reconstituted with either ILV-labeled H2A (Left) or H2B (Right). Methyl groups with significant chemical shift changes are labeled. (Inset) An overlay of several titration spectra for Val45γ2 in H2B. Resonances labeled with # are natural abundance signals from the disordered regions of HMGN2. (B) Chemical shift perturbation between free and bound states for H2A (Top) and H2B (Bottom). Residues with averaged, weighted CSP larger than the 10% trimmed mean + 2 SD are labeled. To identity the binding site of HMGN2, the average, weighted CSP of the histone ILV residues was calculated according to , where Δδ_i is the difference in peak position between the free and bound states in ppm for atom i(i∈{Cγ1/2,Cδ1/2,Hγ1/2,Hδ1/2}), the ¹³C chemical shift weighting factor w_i is set σ_H,i/σ_C,i (∼0.16–0.18), σ_i is the standard deviation of deposited chemical shifts in the Biological Magnetic Resonance Data Bank (41) for atom i, and the summation extends over the N methyl groups of each residue (i.e., N = 1 for Ile, and N = 2 for Leu/Val). (C) Methyl group peak intensity ratios, I(Mn²⁺)/I(Ca²⁺), of paramagnetic Mn²⁺-EDTA and diamagnetic Ca²⁺-EDTA spin-labeled HMGN2 at positions 19, 23, 38 and 44. Intensity ratios of the two individual methyl groups for Leu/Val were averaged. Errors (1 SD) are denoted by thin bars. Residues with average intensity ratio + 2 SD < 0.5 are labeled. (D), Structural summary of NMR CSP and paramagnetic spin labeling. Methyl groups with large chemical shift changes or decreases in peak intensity are labeled. Color coding as in Fig. 1. The sequence of the nucleosome-binding domain of HMGN2 and the position of the spin labels are shown (Left). Arg/Lys residues are highlighted in blue, and Ser24 and Ser28 are shown in magenta.

Fig. 3.

Binding of HMGN2 to the nucleosome (NCP) by mutation and NMR line-shape analysis. (A) ITC titration results for wild-type HMGN2 with wild-type nucleosome (black) and selected mutants (20 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4, ionic strength I = 66 mM, [Na⁺] = 50 mM). (B) Location of mutated residues in the nucleosome. The region of DNA implicated in binding the lysine-rich region of the NBD is highlighted in red. Color coding as in Fig. 1. (C) Gel-shift assay with peptide/DNA ratios of 1, 2, and 5, respectively. LANA is the Kaposi’s sarcoma-associated herpivirus latency-associated nuclear antigen peptide (15). The DNA includes 167 bp with the 601 sequence in the middle. (D and E) Experimental NMR titration data and fits to a cooperative binding model accounting for weak nonspecific binding (model 4 in SI Text. (D) One-dimensional line shapes of H2B Val45γ2 at indicated molar ratio of nucleosome: HMGN2, experimental data in red, fits in blue. (E) Experimental (points) and simulated (solid lines) chemical shift titration curves for selected methyl groups. Dashed gray lines in D and E show best fits for a noncooperative model with identical parameters for nonspecific binding (model 2, SI Materials and Methods).

Fig. 4.

A docking structure of the NBD-nucleosome complex and comparison of its acidic patch region with those of the RCC1- and LANA-nucleosome complexes. (A) The NBD structure is shown with sticks except that the side chains of the positively charged residues (Arg22, Arg23, Arg26, Lys39, Lys41, and Lys42) are indicated with blue balls. The nucleosome is presented as a surface. B, C, and D display the interactions of the two Arg residues (blue spheres) in HMGN2, RCC1, and LANA with the residues in the acidic patch (red spheres), respectively. Residues Ser24 and Ser28 in the NBD are shown with magenta balls.

Fig. 5.

HMGN2 interferes with the binding of linker histone H1 to the nucleosome as measured by ITC. Drosophila linker histone H1_37–211 was titrated into a solution (20 mM Tris-HCl, pH 7.4, 1 mM EDTA and 1 mM DTT) containing free nucleosome (blue square), the nucleosome-(HMGN2_1–43)₂ complex (magenta circle), and the nucleosome-(HMGN2)₂ complex (cyan triangle) at 25 °C, with corresponding equilibrium dissociation constants (K_Ds) of 0.10 μM, 0.10 μM, and 0.57 μM, respectively. The titration results fit to a model in which one H1 molecule binds to one nucleosome. The nucleosome is the same as that used in NMR studies.

Fig. 6.

Implications for the function of HMGNs. The NBD is shown in cylinder. The disordered terminal regions of HMGN2 are shown in lines. For clarity, only one HMGN2 is shown here (a model with two HMGN2 and one nucleosome is shown in Fig. S6). Earlier studies have shown that regions around the DNA entry/exit point and the dyad of the nucleosome are the approximate binding sites of the globular domain of H1 (gH1, dashed orange oval) (28), which are close to the disordered C-terminal region of HMGN2. Phosphorylation of Ser24 and Ser28 of the NBD induces the dissociation of HMGNs from the nucleosome due to unfavorable electrostatic repulsion with the acidic patch.