Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 Å Resolution

https://doi.org/10.1016/S0022-2836(02)00386-8Get rights and content

Abstract

Solvent binding in the nucleosome core particle containing a 147 base pair, defined-sequence DNA is characterized from the X-ray crystal structure at 1.9 Å resolution. A single-base-pair increase in DNA length over that used previously results in substantially improved clarity of the electron density and accuracy for the histone protein and DNA atomic coordinates. The reduced disorder has allowed for the first time extensive modeling of water molecules and ions.

Over 3000 water molecules and 18 ions have been identified. Water molecules acting as hydrogen-bond bridges between protein and DNA are approximately equal in number to the direct hydrogen bonds between these components. Bridging water molecules have a dual role in promoting histone–DNA association not only by providing further stability to direct protein–DNA interactions, but also by enabling formation of many additional interactions between more distantly related elements. Water molecules residing in the minor groove play an important role in facilitating insertion of arginine side-chains. Water structure at the interface of the histones and DNA provides a means of accommodating intrinsic DNA conformational variation, thus limiting the sequence dependency of nucleosome positioning while enhancing mobility.

Monovalent anions are bound near the N termini of histone α-helices that are not occluded by DNA phosphate groups. Their location in proximity to the DNA phosphodiester backbone suggests that they damp the electrostatic interaction between the histone proteins and the DNA. Divalent cations are bound at specific sites in the nucleosome core particle and contribute to histone–histone and histone–DNA interparticle interactions. These interactions may be relevant to nucleosome association in arrays.

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.

References (47)

  • I. Filesi et al.

    The main role of the sequence-dependent DNA elasticity in determining the free energy of nucleosome formation on telomeric DNAs

    Biophys. Chem.

    (2000)
  • A. Thåström et al.

    Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences

    J. Mol. Biol.

    (1999)
  • H.R. Drew

    Can one measure the free energy of binding of the histone octamer to different DNA sequences by salt-dependent reconstitution?

    J. Mol. Biol.

    (1991)
  • J. Janin

    Wet and dry interfaces: the role of solvent in protein–protein and protein–DNA recognition

    Struct. Fold. Des.

    (1999)
  • K. Luger et al.

    The histone tails of the nucleosome

    Curr. Opin. Genet. Dev.

    (1998)
  • K.D. Collins

    Charge density-dependent strength of hydration and biological structure

    Biophys. J.

    (1997)
  • D.W. Christianson

    Structural chemistry and biology of manganese metalloenzymes

    Prog. Biophys. Mol. Biol.

    (1997)
  • K. Luger et al.

    Preparation of nucleosome core particle from recombinant histones

    Methods Enzymol.

    (1999)
  • Z. Otwinowski et al.

    Processing of X-ray diffraction data collected in oscillation mode

    Methods Enzymol.

    (1997)
  • T.E. Ferrin et al.

    The MIDAS display system

    J. Mol. Graph.

    (1988)
  • K. Luger et al.

    Crystal structure of the nucleosome core particle at 2.8 Å resolution

    Nature

    (1997)
  • Luger, K., Maeder, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. (2000). The atomic structure of the...
  • J.M. Harp et al.

    Asymmetries in the nucleosome core particle at 2.5 Å resolution

    Acta Crystallog. sect. D

    (2000)
  • Cited by (0)

    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.

    Present address: K. Luger, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.

    View full text