Journal of Molecular Biology
Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 Å Resolution†
Introduction
DNA in eukaryotic organisms is organized in a nucleoprotein complex called chromatin. Genetic processes, both vital such as transcription and replication, and pathological such as cancer and viral infection, depend on DNA in the context of chromatin. The nucleosome is the fundamental repeating unit of chromatin occurring generally every 157–240 bp. The nucleosome core, the greater part of the nucleosome, comprises an octamer, containing a single histone H3–H4 tetramer and two histone H2A–H2B dimers, and 147 bp of DNA. We have determined the structure of the nucleosome core particle containing recombinant histones and a 147 bp, defined-sequence DNA (NCP147) by X-ray crystallography at 1.9 Å resolution. The significantly improved electron density for this particle compared to previous reports on the related 146 bp particle results from the increase in DNA length from 146 to 147 bp.1., 2. This has allowed location of 3130 water molecules, 14 manganese cations and four chloride anions in the refined structure. We also report here the refined structures of the original 146 bp particle (NCP146) and a second particle (NCP146b) containing a different 146 bp DNA sequence at 2.0 Å and 2.6 Å resolution, respectively. Both NCP147 and NCP146 provide significantly more complete and accurate structures of the nucleosome core particle compared to recent reports using 146 bp DNA fragments, endogenous histones or recombinant variants.3., 4., 5.
The important contribution of solvent molecules to the stability and specificity of protein–DNA interactions has been recognized through a combination of high-resolution structural results and allied thermodynamic studies.6., 7., 8., 9., 10., 11., 12. Although the X-ray and NMR structures of over 200 DNA-binding proteins have been determined, few are of adequate quality to pinpoint solvent interactions accurately.13 Those which are, have on average 12 water molecules located in positions mediating protein–DNA interactions.14 In contrast, the NCP147 structure reveals ten times this number of water molecules at the protein–DNA interface. In regard to non-specific DNA-binding proteins, water molecules are envisioned to play the role of adapters, allowing for roughly uniform affinity between protein and differing DNA sequences despite DNA-sequence-dependent conformational variability. Although general rules may emerge for non-specific, protein–DNA association from the few relevant structures available, elucidation of important mechanistic detail requires that each individual motif be examined.15
Even though nucleosomes are ubiquitous on genomic DNA, they display local, preferred-sequence positioning as well as the ability to move diffusively along the DNA.16., 17. Solvent interactions are likely to contribute substantially to positioning and mobility. Therefore, elucidation of the structural details of all the interactions accounting for histone protein and DNA association is required to understand the molecular basis of eukaryotic genetic processes. We present here for the first time a detailed description of the water molecule and ion structure in the nucleosome core particle.
Section snippets
147 bp versus 146 bp particles
Crystals of NCP146 containing Xenopus laevis core histones and a 146 bp palindromic DNA, taken from one-half of a human α-satellite sequence repeat, were used previously for the structure determination of the nucleosome core at 2.8 Å and 2.0 Å resolution.1., 2. The NCP147, NCP146 and NCP146b structures reported here at 1.9 Å, 2.0 Å and 2.6 Å, respectively (Table 1), show the same features overall, limited only by differences in resolution and influences from DNA disorder. Theses structures show that
Discussion
The nucleosome core particle containing a DNA molecule of 147 bp yields crystals in which the DNA is far better ordered than in previous 146 bp particles. It permits for the first time extensive modeling of the solvent component. As is typical for protein–DNA complexes,14 somewhat less than one-seventh of the ASA of the isolated histone octamer and DNA components is lost in the complete complex. A disproportionate part of this reduction is the consequence of the insertion of a relatively small
Crystal preparation
Nucleosome core particles were prepared from recombinant X. laevis histones and 146 and 147 bp palindromic DNA fragments derived from human α-satellite DNA, as described previously.1., 43. The 146 bp and 147 bp sequences contain, respectively an EcoRI and HinfI restriction endonuclease site at their centers. Crystals were grown by vapor diffusion over the course of one to four weeks in droplets containing 4 mg/ml of core particle, 70–85 mM MnCl2, 50–60 mM KCl, and 20 mM potassium cacodylate (pH 6.0),
Supplementary Files
Acknowledgements
We are grateful to the staffs of the ID14-4, ID9 and SNBL beamlines at the E.S.R.F. for their assistance during data collection, and to Dr I. Berger for comments on the manuscript. We appreciate the support of the Swiss National Fund.
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Cited by (0)
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We dedicate this paper to the memory of Max Perutz who was particularly inspirational and supportive to T.J.R. in the early stages of this study.
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Present address: K. Luger, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.