Timothy J. Richmond

Crystal structure of the nucleosome core particle at 2.8 A resolution

The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and additional, well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighbouring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.

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

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 A 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 alpha-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.

The structure of DNA in the nucleosome core

The 1.9-Å-resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein–DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fibre formation and chromatin remodelling.

X-ray structure of a tetranucleosome and its implications for the chromatin fibre

DNA in eukaryotic chromosomes is organized in arrays of nucleosomes compacted into chromatin fibres. This higher-order structure of nucleosomes is the substrate for DNA replication, recombination, transcription and repair. Although the structure of the nucleosome core is known at near-atomic resolution, even the most fundamental information about the organization of nucleosomes in the fibre is controversial. Here we report the crystal structure of an oligonucleosome (a compact tetranucleosome) at 9 Å resolution, solved by molecular replacement using the nucleosome core structure. The structure shows that linker DNA zigzags back and forth between two stacks of nucleosome cores, which form a truncated two-start helix, and does not follow a path compatible with a one-start solenoidal helix. The length of linker DNA is most probably buffered by stretching of the DNA contained in the nucleosome cores. We have built continuous fibre models by successively stacking tetranucleosomes one on another. The resulting models are nearly fully compacted and most closely resemble the previously described crossed-linker model. They suggest that the interfaces between nucleosomes along a single helix start are polymorphic.

Preparation of nucleosome core particle from recombinant histones

Publisher Summary This chapter describes the preparation method of nucleosome core particles (NCPs) from recombinant histones. The ability to make defined NCPs, or arrays of nucleosomes, from histone proteins expressed in bacteria has several advantages over previously used methods using histones isolated from natural sources. The chapter discusses protocols for the overexpression and purification of histones H2A, H2B, H3, and H4, both as full-length proteins and as corresponding trypsin-resistant globular domains. The chapter presents a method for the refolding and purification of a histone octamer from denatured recombinant histone proteins together with a protocol for the assembly and purification of a nucleosome core particle using 146 base pairs of DNA. The purity and homogeneity of the final core-particle preparation are assessed by a high-resolution gel shift assay. The chapter presents a flow chart that describes the procedures involved in the preparation of synthetic nucleosomes.

Solvent accessible surface area and excluded volume in proteins: Analytical equations for overlapping spheres and implications for the hydrophobic effect

An analytical formula has been derived for the calculation of the solvent accessible surface area of a protein molecule or equivalently the surface area exterior to an arbitrary number of overlapping spheres. The directional derivative of this function with respect to atomic co-ordinates is provided to facilitate minimization procedures used with molecular docking algorithms and energy calculations. An analytical formula for the calculation of the volume enclosed within the accessible surface, the excluded volume, is also derived. Although the area function is not specific to the structures of proteins, the derivation was motivated by the need for a computationally feasible simulation of the hydrophobic effect in proteins. A computer program using the equations for area has been tested and has had limited application to the docking of protein alpha-helices. Possible relationships of the solvent excluded volume to hydrophobic interaction free energy and transfer free energy of solute molecules are derived from the statistical mechanics of solution.

The histone tails of the nucleosome.

Reversible acetylation of core histone tails plays an important role in the regulation of eukaryotic transcription, in the formation of repressive chromatin complexes, and in the inactivation of whole chromosomes. The high-resolution X-ray structure of the nucleosome core particle, as well as earlier evidence, suggests that the histone tails are largely responsible for the assembly of nucleosomes into chromatin fibers and implies that the physiological effects of histone acetylation may be achieved by modulation of a dynamic inter-conversion between the fiber and a less condensed nucleofilament structure. In addition, the tails and adjacent regions serve as recognition sites for chromatin assembly and transcription remodeling machinery and the interactions that occur may also be responsive to histone acetylation.

Chromatin Fiber Folding: Requirement for the Histone H4 N-terminal Tail

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.

Baculovirus expression system for heterologous multiprotein complexes

The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant multiprotein production and multigene transfer applications.

DNA binding within the nucleosome core.

The high resolution structure of the nucleosome core particle of chromatin reveals the form of DNA that is predominant in living cells and offers a wealth of information on DNA binding and bending by the histone octamer. Recent studies imply that chromatin is highly dynamic. This propensity for unfolding and refolding stems from the structural design of the nucleosome core. The histone-fold motif, central to nucleosome structure, is also found in other proteins involved in transcriptional regulation.