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Acta Crystallographica Section F: Structural Biology Communications logoLink to Acta Crystallographica Section F: Structural Biology Communications
. 2020 Apr 28;76(Pt 5):194–198. doi: 10.1107/S2053230X20004604

Structure of a single-chain H2A/H2B dimer

Christopher Warren a,‡, Jeffrey B Bonanno a, Steven C Almo a, David Shechter a,*
PMCID: PMC7193513  NIHMSID: NIHMS1591133  PMID: 32356520

A single-chain, tailless Xenopus laevis H2A/H2B dimer construct (scH2BH2A) was engineered by directly fusing the C-terminus of H2B to the N-terminus of H2A without an artificial linker sequence. A high-resolution crystal structure of scH2BH2A shows that it adopts a nearly identical fold to that of nucleosomal H2A/H2B and may be useful for future structural studies of many H2A/H2B-interacting proteins.

Keywords: histones, chromatin, H2A/H2B dimer, Xenopus laevis, crystallography

Abstract

Chromatin is the complex assembly of nucleic acids and proteins that makes up the physiological form of the eukaryotic genome. The nucleosome is the fundamental repeating unit of chromatin, and is composed of ∼147 bp of DNA wrapped around a histone octamer formed by two copies of each core histone: H2A, H2B, H3 and H4. Prior to nucleosome assembly, and during histone eviction, histones are typically assembled into soluble H2A/H2B dimers and H3/H4 dimers and tetramers. A multitude of factors interact with soluble histone dimers and tetramers, including chaperones, importins, histone-modifying enzymes and chromatin-remodeling enzymes. It is still unclear how many of these proteins recognize soluble histones; therefore, there is a need for new structural tools to study non-nucleosomal histones. Here, a single-chain, tailless Xenopus H2A/H2B dimer was created by directly fusing the C-terminus of H2B to the N-terminus of H2A. It is shown that this construct (termed scH2BH2A) is readily expressed in bacteria and can be purified under non-denaturing conditions. A 1.31 Å resolution crystal structure of scH2BH2A shows that it adopts a conformation that is nearly identical to that of nucleosomal H2A/H2B. This new tool is likely to facilitate future structural studies of many H2A/H2B-interacting proteins.

1. Introduction  

The diploid human genome is composed of over six billion base pairs (bp) of DNA, which would be approximately 2 m long if stretched end to end. This amount of genetic information needs to be condensed approximately 10 000-fold in order to be accommodated in the micrometre-scale cell nucleus. To achieve this level of compaction, eukaryotes wrap their DNA around highly basic histone proteins. The reversible, non­covalent interactions between DNA and histone proteins allow histones to be deposited on, slid along or removed from DNA to enable essential cellular functions such as DNA replication, transcription and damage repair. The assemblies of DNA, histones and other associated proteins that make up the physiological form of the genome are collectively referred to as chromatin (Kornberg & Thomas, 1974 ▸).

Core histones (H2A, H2B, H3 and H4) are small, highly basic proteins that fold into a compact domain composed of three helices connected by short loops, with intrinsically disordered N- and C-terminal tails of variable lengths. The long central helix (α2) is flanked by two shorter helices (α1 and α3) connected by loops 1 (L1) and 2 (L2), respectively (Arents & Moudrianakis, 1995 ▸). This histone fold mediates the dimerization of complementary histones via a ‘handshake’ inter­action, where histones interact in an antiparallel fashion such that L1 of one histone is adjacent to L2 of the partner histone. The heterodimers are held together largely by hydrophobic interactions between the interacting histones.

In the nucleosome, the histone octamer organizes 147 bp of DNA. The DNA is organized in ∼1.7 negative superhelical turns with pseudo-twofold symmetry, with each histone dimer pair organizing ∼30 bp of DNA (Davey et al., 2002 ▸; Luger et al., 1997 ▸). Histone proteins make largely electrostatic contacts with DNA. Specifically, both positively charged amino acids and partial positive charges from helix dipoles interact with the negatively charged phosphate groups on the DNA backbone, with very few interactions being made between the histones and bases of the DNA. Histone–histone contacts are made throughout the octameric structure, with four-helix bundles formed between H3:H3 and H2B:H4. Nucleosomes are variably spaced, with linker DNA ranging from 20 to 80 bp in length. To facilitate the compaction of chromatin fibers, linker histones such as H1 are able to bind nucleosomes and linker DNA at the DNA entry and exit points (Izzo et al., 2013 ▸; Zhou et al., 2013 ▸; Bednar et al., 2017 ▸).

Histones are heavily post-translationally modified, mostly on their tails, by methylation, acetylation, phosphorylation, ubiquitination and sumolyation. These chemical modifications, along with modifications to the DNA itself, are ‘written’ and ‘erased’ by various enzymes and can recruit different ‘reader’ proteins, which in turn can recruit other regulators of gene expression. These chemical modifications are heritable, as they can be propagated through cell division and possibly across generations in eukaryotic organisms (Hansen et al., 2008 ▸; Greer et al., 2011 ▸; Heard & Martienssen, 2014 ▸). While writer, eraser and reader proteins are central for establishing and maintaining epigenetic states, other proteins known as histone chaperones and ATP-dependent chromatin re­modelers are necessary to deposit and position histones on DNA (Tyler, 2002 ▸).

Histone proteins therefore have a large interaction network both on and off DNA. While there are many structures of nucleosomes and nucleosome-containing protein complexes, there are far fewer reported structures of individual histone dimers and tetramers and their associated complexes. Many of the structures of proteins bound to H2A/H2B dimers have utilized either tailless or covalently linked constructs (Hondele et al., 2013 ▸; Obri et al., 2014 ▸; Latrick et al., 2016 ▸; Zhou et al., 2008 ▸; Huang et al., 2020 ▸). These constructs are likely to be necessary to stabilize the non-nucleosomal H2A/H2B dimer and limit flexibility for crystallographic or NMR analysis. Here, we describe a new single-chain construct derived from the Xenopus laevis H2A/H2B dimer, in which the histone tails are truncated and the C-terminus of H2B is directly fused to the N-terminus of H2A without an artificial linker sequence. This construct, termed single-chain H2BH2A (scH2BH2A), is readily expressed in bacteria, is easily purified under nondenaturing conditions and reproducibly yields crystals that diffract to high resolution. The structure of scH2BH2A shows that it folds into a nearly identical conformation to that observed in nucleosomal H2A/H2B, indicating that this tool may be useful in future structural studies of many H2A/H2B-binding proteins.

2. Methods  

2.1. Cloning, expression and purification of scH2BH2A  

Overlapping PCR methods were used to remove the N-terminal tail of Xenopus H2B and directly fuse the C-terminus of H2B to the N-terminal helix of Xenopus H2A without an artificial linker sequence, while also removing the C-terminal tail and much of the N-terminal tail of H2A (H2B residues 34–126 fused to H2A residues 14–105). This PCR product was cloned into the pRUTH5 vector containing an N-terminal His6 tag followed by a Tobacco etch virus (TEV) protease site and a glycine residue (His6-TEV-Gly-scH2BH2A). This construct was transformed into Escherichia coli BL21(DE3) Rosetta 2 cells, which were grown in LB medium to an OD600 of 0.7. Protein expression was induced by adding 1 mM isopropyl β-d-1-thiogalactopyranoside to the culture and shaking for 3 h at 37°C. The cells were pelleted and lysed by sonication in lysis buffer (25 mM Tris pH 8.0, 1 M sodium chloride, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 mM β-mercapto­ethanol). The lysate was clarified by centrifugation at 14 000 rev min−1 and the supernatant was incubated with Ni–NTA resin while rotating for 1 h at 4°C and then passed over a gravity column. The resin was extensively washed with lysis buffer including 10 mM imidazole and was eluted with lysis buffer including 360 mM imidazole. Imidazole was removed by dialysis into lysis buffer and the His6 tag was cleaved by overnight incubation with TEV protease. Subtractive Ni–NTA chromatography was used to remove the cleaved tag, any uncleaved target protein and His6-tagged TEV protease. The flowthrough was concentrated and purified by size-exclusion chromatography on a Superdex 75 column equilibrated in 25 mM Tris pH 8.0, 1 M NaCl, 1 mM EDTA. scH2BH2A eluted from the column as a single peak and was >95% pure by SDS–PAGE analysis (Fig. 1 ▸ a). scH2BH2A was aliquoted, flash-frozen and stored at −80°C.

Figure 1.

Figure 1

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Purification and crystallization of scH2BH2A. (a) 0.5 µl purified and concentrated scH2BH2A was run on 15% SDS–PAGE and stained with Coomassie Brilliant Blue. Molecular-mass markers are labeled in kDa. (b) Representative crystals of scH2BH2A. (c) Representative X-ray diffraction from an scH2BH2A crystal.

2.2. Crystallization of scH2BH2A  

scH2BH2A precipitated at lower salt concentrations. Based on the buffer conditions used for another crystal structure of a peptide from the Spt16 subunit of FACT bound to H2A/H2B, we reasoned that the inclusion of a nondetergent sulfobetaine (NDSB) in the buffer might increase the solubility at low ionic strength (Kemble et al., 2015 ▸). NDSBs are commonly used as additives in protein crystallization screens for their ability to stabilize proteins and prevent aggregation (Vuillard et al., 2001 ▸). An aliquot of scH2BH2A was thawed and rapidly diluted 20-fold in 25 mM Tris pH 7.5, 600 mM NDSB-256 (Hampton Research) to give a final buffer composition of 25 mM Tris pH 7.5, 50 mM NaCl, 0.05 mM EDTA, 570 mM NDSB-256. scH2BH2A was concentrated to 18 mg ml−1 in a 10 kDa molecular-weight cutoff Amicon centrifugal filter unit. scH2BH2A was screened for crystallization conditions using the MCSG Suite (Microlytic) by sitting-drop vapor diffusion using 0.3 µl protein solution and 0.3 µl well solution. An initial hit from MCSG-1 condition D4 produced a single crystal that was not suitable for structure determination. This condition consisted of 0.2 M sodium thiocyanate pH 6.9, 20% PEG 3350. We expanded on this initial hit using the Silver Bullets additive screen (Hampton Research). Diffraction-quality crystals grew in 48 h in hanging-drop format using 1 µl protein solution and 1 µl well solution in condition F1 (Fig. 1 ▸ b), which contained the original buffer and precipitant with the additives methylenediphosphonic acid, phytic acid, sodium pyrophosphate and sodium triphosphate.

2.3. Data collection  

Crystals were cryoprotected in well solution supplemented with 20% glycerol and were flash-cooled in liquid nitrogen. scH2BH2A crystals were screened on the LRL-CAT beamline 31-ID-D at the Advanced Photon Source (APS) at a wavelength of 0.97931 Å (Table 1 ▸, Fig. 1 ▸ c).

Table 1. Data collection and structure refinement.

Values in parentheses are for the outer shell.

Data-collection and crystal parameters
 Diffraction source 31-ID-D, APS
 Wavelength (Å) 0.97931
 Temperature (K) 100
 Detector PILATUS 6M, Dectris
 Space group C121
  a, b, c (Å) 61.793, 44.679, 66.450
 α, β, γ (°) 90.000, 117.270, 90.000
 Resolution range (Å) 26.71–1.31 (1.39–1.31)
 Completeness (%) 99.4 (99.8)
 〈I/σ(I)〉 5.6 (1.1)
  R merge 0.058 (0.892)
  R meas 0.069 (1.052)
 CC1/2 0.999 (0.619)
 Multiplicity 3.7 (3.6)
Refinement and final model parameters
 Resolution range (Å) 20.00–1.31
 No. of reflections (work) 36283
 No. of reflections (free) 1776
  R work 0.1695
  R free 0.2063
 No. of non-H atoms 1434
 Correlation coefficient (F o − F c) 0.968
 R.m.s.d., bond lengths (Å) 0.012
 R.m.s.d., bond angles (°) 1.541
 Mean B factor (Å2) 20.418
 Ramachandran preferred 174 [98.86%]
 Ramachandran allowed 1 [0.57%]
 Ramachandran outliers 1 [0.57%]
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2.4. Structure solution and refinement  

The diffraction data were processed with HKL-3000, and molecular-replacement phasing, building and refinement were performed using the CCP4 suite and Coot (Minor et al., 2006 ▸; Winn et al., 2011 ▸; Emsley et al., 2010 ▸). Diffraction extended to a resolution of 1.31 Å and was consistent with the monoclinic space group C2, with unit-cell parameters a = 61.793, b = 44.679, c = 66.450 Å, α = 90.000, β = 117.270, γ = 90.000° and one molecule of scH2BH2A per asymmetric unit. The structure was determined by molecular replacement using chains C and D (H2A and H2B) from the crystal structure of the X. laevis nucleosome with the histone tails truncated (PDB entry 1kx5; Davey et al., 2002 ▸). Iterative cycles of building and refinement procedures, including anisotropic B-factor refinement in later stages (Merritt, 2012 ▸), yielded a final model including 176 residues in a continuous chain consisting of H2B residues 36–126 followed by H2A residues 14–98 (numbering according to UniProt entries Q92130 and P06897; the N-terminal cloning artifact glycine and H2B residues 34 and 35 along with H2A C-terminal residues 99–105 were not observed, presumably owing to disorder), 43 waters of solvation and one molecule of pyrophosphate that converged with an R work and R free of 0.1695 and 0.2063, respectively (Table 1 ▸) and had excellent stereochemistry. The coordinates were deposited in the PDB with the accession code 6w4l. All figures were generated and structure comparisons with nucleosomal H2A/H2B were performed with PyMOL (DeLano, 2002 ▸).

3. Results and discussion  

The purified scH2BH2A yielded reproducible crystals that consistently diffracted to high resolution (Figs. 1 ▸ a–1 ▸ c). The 1.31 Å resolution structure of scH2BH2A shows that it forms the assembly expected for an H2A/H2B dimer (Figs. 2 ▸ a and 2 ▸ b). The resolution of this structure allows the unambiguous assignment of nearly all side chains along with 43 crystallo­graphic water molecules (Fig. 2 ▸ c). A density feature that was not attributable to protein or water was modeled as a pyrophosphate molecule and was presumably derived from the additive crystallization screen condition. The pyrophosphate makes hydrogen-bonding contacts with the backbone NH of Asp52 of H2B and the side-chain OH of the next amino acid in the chain, Thr53. This pyrophosphate also makes multiple ionic interactions with the side chain of Arg72 of H2A in the same scH2BH2A molecule, as well as with the side chain of Lys121 of H2B in a symmetry-related scH2BH2A molecule (Fig. 2 ▸ d). These contacts are likely to contribute to the observed propensity for crystal formation.

Figure 2.

Figure 2

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Structure of scH2BH2A. (a) STRIDE secondary-structure alignment of scH2BH2A (top) with nucleosomal H2A/H2B (PDB entry 1aoi chains C and D, bottom; Luger et al., 1997 ▸). Conserved helices are shown as bars and the covalent link in scH2BH2A is shown as a dotted line. (b) Overall structure of scH2BH2A. The sequence corresponding to H2A is colored yellow and the sequence corresponding to H2B is colored red. Helices are labeled and the linked histone tails are shown in the upper right portion of the structure. (c) Structure of scH2BH2A with the same orientation and color scheme as in (b) with 43 crystallographic water molecules (blue spheres) and a single pyrophosphate molecule (orange and red sticks) also shown. (d) Structural details of the interactions of scH2BH2A with pyrophosphate at the crystallographic interface. Two symmetry-related scH2BH2A chains are shown in green and blue. Electrostatic and hydrogen-bonding interactions between scH2BH2A and pyrophosphate are shown as dashed lines. Helices are labeled. (e) Alignment of the scH2BH2A structure with a nucleosomal H2A/H2B dimer. scH2BH2A is colored green and nucleosomal H2A/H2B is colored blue. The structure of scH2BH2A is in the same orientation as in (b) and the linked histone tails are shown in the upper right portion of the structure.

Comparison of the scH2BH2A structure with nucleosomal H2A/H2B shows excellent agreement (Fig. 2 ▸ e). The positional r.m.s.d. between the two structures is 0.54 Å based on 150 aligned Cα atoms. Each histone module in scH2BH2A exhibits the classic histone fold composed of three helices (α1, α2 and α3) connected by two short loops (L1 and L2). Also apparent in the structure are the short N-terminal and C-terminal helices of H2A (αN and αC, respectively) and the longer αC of H2B. An extensive network of hydrophobic contacts contribute to the H2A/H2B interface. Notably, the αN helix of H2A was not apparent in a recent NMR solution structure of the human H2A/H2B dimer (Moriwaki et al., 2016 ▸). In the structure of the nucleosome, the H2A αN helix makes direct contacts with the phosphate backbone of DNA and is likely to be stabilized by these contacts. The presence of this short helix in the structure of scH2BH2A is likely to indicate that it is stabilized by the adjacent linked histone tails.

Finally, many attempts were made to co-crystallize and soak in various short, acidic peptides derived from the H2A/H2B chaperone nucleoplasmin (Npm2). We have previously demonstrated that these peptides bind to H2A/H2B by trypto­phan fluorescence and NMR (Warren et al., 2017 ▸). However, no additional electron density was observed that could be assigned to these peptides in the data arising from co-crystallization experiments, and the crystals often cracked and dissolved during soaking experiments. We suspect that the tight packing and low solvent content of this crystal form makes the introduction of peptides difficult. It may be possible to introduce peptides that bind to a different surface of scH2BH2A; however, de novo co-crystallization trials are more likely to afford appropriate crystals of scH2BH2A bound to other protein and peptide ligands.

Supplementary Material

PDB reference: single-chain H2B/H2A histone chimera from Xenopus laevis, 6w4l

Acknowledgments

The Albert Einstein Crystallographic Core X-ray Diffraction Facility is supported by NIH Shared Instrumentation Grant S10 OD020068. Data collection also utilized the resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly & Co., which operates the facility.

Funding Statement

This work was funded by National Institutes of Health grants R01GM108646, R01GM135614, F31GM116536, and S10 OD020068. American Lung Association grant LCD-564723 to David Shechter.

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Associated Data

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Supplementary Materials

PDB reference: single-chain H2B/H2A histone chimera from Xenopus laevis, 6w4l

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