Nucleosome Core Particle at 2.8 Angstrom Resolution


by Philip Rosen

Introduction:
The nucleosome core particle is essential in the packaging of DNA in the chromosomes. It is able to condense a Eukaryotic genome into a cell’s nucleus. This is no small feat, considering the linear human genome is about 2 meters long, while the cell nucleus is only 5 micrometers in diameter. Many cellular functions, such as transcription and replication are effected by the structure of the chromatin, and so understanding the structure of the nucleosome is key to understanding the activation and repression of these functions. The nucleosome core particle consists of fourteen turns of B-form DNA around an octamer of histone proteins. The octamer contains two copies each of four different proteins: H2A, H2B, H3, and H4.

One can think of this arrangement as the dimerization of tetramers. These four proteins are arranged with C2 Symmetry .
In vivo, the nucleosome core particle contains a fifth protein, H1. H1 helps to bind the DNA to the core particle such that the number of base pairs bound to the nucleosome increases from 146 to 166 with the addition of H1. Unfortunately, H1 was not part of the crystal structure because it is released from the nucleosome core particle upon digestion of the chromosomes. An 8-Angtrom resolution structure was solved in 1984,


and it wasn’t until 1997 that the 2.8-Angstrom structure was solved. This is because a suitable, pre-defined DNA sequence had to be found that would cause high-resolution diffraction. Also, the core particle had to be synthesized from recombinant proteins.
The core particle contains many basic proteins (here, the DNA is in orange, the basic proteins are in cyan, the neutral proteins are in yellow, and the acidic proteins are in red), with many arginines and lysines.


Histone-Histone Interactions:

Because the nucleosome core particle expresses C2 symmetry, one can talk about the interactions concerning half the molecule, and each quality on that half can be referenced to the other half, as well.
Each Histone has a basic Helix-Turn-Helix-Turn-Helix motif, where three alpha helices are separated from one another by a loop in the protein. The helices are designated, from their N-Terminus to their C-Terminus, as alpha-1, L1, alpha-2, L2, alpha-3. The H3 and H4 histones use this motif to interact because the L1 of one histone is in close proximity to the L2 of the other. Hydrogen bonds between the two histones are then able to form.
H3-H4 Helix-Loop-Helix-Loop-Helix
L1/L2 interactions


The H2A and H2B proteins also use this same motif to interact.
H2A-H2B Helix-Loop-Helix-Loop-Helix
L1/L2 interactions

(Note that only the interactions between the L1 of H3 and the L2 of H4 form more than one hydrogen bond - 3 such bonds are formed.)
Zoom Out
The H2B and H4 chains interact through hydrogen bonds formed by amino acids at the C-Terminus of the alpha-2 helix of H4 and the N-Terminus of the alpha-3 helix of H2B. Hydrophobic forces between tyrosines of both alpha-2 helices and the alpha 3 helix of H4 also hold the two chains together.


The interactions between the two H3 chains is similar. Hydrogen bonds form between histidines at the C-Terminus of alpha-2 and aspartic acids at the N-Terminus of alpha-3. Furthermore, it is speculated that cysteins on the alpha-2 helices form a disulfide bridge. While the measured distance between the two sulfides is too long for such a bond, experimental evidence suggests its existance.

Among other factors, the presence of hydrophobic forces between H4 and H2B (which is located near the surface of the core particle) and the absence of hydrophobic forces between the two H3 histones (which is located in the middle of the core particle), account for the fact that, at low salt concentrations, the tetramer is more stable than the octamer.
Zoom Out
There are also interactions between the two H2A and H2B chains, which contribute to the dimerization of the tetramers. On the L1 of the H2A histone, an asparagine (ASN 38) makes a hydrogen bond to both a histidine (His 79) at the C-Terminus of the alpha-2 helix of the H2B on the other tetramer and a glutamic acid (Glu 41) on the L1 loop of the H2A on the other tetramer.

Zoom Out


Histone-DNA Interactions:
The binding of the DNA to the nucleosome core particle is not sequence-specific. This is extremely important because the nucleosome core particle needs to bind DNA of all different sequences. The interactions between the DNA and the histone fall into one of five categories:
  1. Helix dipoles - A net positive charge at the N-terminus at the alpha-2 helices of all four histones as well as the N-terminus of the alpha-1 helices of H2B, H3, and H4. These dipoles lock specific phosphate groups of the DNA backbone in place. The N-terminus of the Alpha-2 helix of each histone is hi-lighted in red when that histone is zoomed-in upon. The N-termini of the alpha-1 helices of H2B, H3, and H4 are hi-lighted in dark blue when these histones are zommed-in upon
    2. .
  2. Main chain nitrogens on L1 or L2 make hydrogen bonds to the DNA backbone.
  3. Nonpolar contacts with deoxyribose groups.
  4. Hydrogen bonds or salt links between the phosphate oxygen atoms and the side chains of basic proteins.

    H2A - DNA
    H2A - DNA Interactions - Magenta
    H2A - DNA Interactions - CPK
    H2B - DNA
    H2B - DNA Interactions - Magenta
    H2B - DNA Interactions - CPK
    Zoom Out
    H4 - DNA
    H4 - DNA Interactions - Magenta
    H4 - DNA Interactions - CPK
    H3 - DNA
    H3 - DNA Interactions - Magenta
    H3 - DNA Interactions - CPK
    Zoom In The image has zoomed into this spot because in this small area of the H3 histone, are examples of DNA-protein interactions #2, #3, and #4.
    • Interaction #2 (main chain nitrogen on L1/L2 to DNA backbone) is exemplified by the hydrogen bond between Ser 86 and the oxygen of the phosphate group of a nucleic acid (in this case, a Cytosine).
    • Interaction #3 (non-polar contacts between hydrophobic groups) is exemplified by the interaction between Pro 66 and a deoxyribose group.
    • Interaction #4 (salt links or hydrogen bonds between the side chain of a basic amino acid and the DNA backbone) is exemplified here through Arg 72 hydrogen bonding to an oxygen of the phosphate group of a nucleic acid (in this case, an Adenine).

    Zoom Out

  5. The fifth type of contact made between the DNA and histone proteins are the minor groove insertions. There are fourteen minor grooves which face the nucleosome core particle. There is an arginine side chain inserted into ten of those minor grooves. The N-terminus tail of the H2B chain is inserted into two minor grooves, and the N-terminus tail of the H3 in inserted into the remaining two. For eight of the ten arginines in the minor groove, there is an adjacent threonine which forms a bond with the arginine hydroxyl group. This apparently restrains the arginine side chain so that it does not form a bond with the bases of the DNA.
Zoom Out


Histone Tails:
Each histone polypeptide has two tails:
  1. The C-Terminus tails. These tails are globular.
  2. The N-terminus tails. These tails are randomly coiled. They fit through the gyres of the DNA. We have already discussed how the N-terminal tails of the H2B and H3 histones penetrate the minor groove. the H3 N-terminal tails are solely responsible for holding the last 13 base pairs at each end of the DNA helix (in the absence of H1).
The structures of the N-terminus tails and some of the C-terminus tails were not fully determined because of weak electron density.

The histone tails are thought to play a role in connecting adjacent nucleosomes. Evidence for this is the presence of acidic amino acids on the H2A and H2B histones which are able to bind the N-terminal tail of H4.


DNA Distortions:
The DNA of the nucleosome is overwound. It makes 14 helical turns, with an average of 0.3 base pairs per turn less than regular B-DNA. The actual number of base pairs per turn varies from turn to turn (with the arginines/N-terminal tails that penetrate the minor groove as the divisional points). They range from 9.4 bp per turn to 10.9 bp per turn. Despite the fact that the DNA itself is overwound, the linking number of DNA decreases overall with the nucleosome core particle. This is because the DNA makes a left-handed torroidal around the nucleosome. When the two halves of the torroid are super-imposed, it can be noted that the DNA does not line up.



Summary:
The determination of the structure of the nucleosome core particle was a milestone in biochemistry because knowing how the DNA interacts with the proteins can help to determine the organization of DNA in the chromosome. Once all the details are known, we will have a much better understanding of the regulation of such cellular functions as replication and transcription. The Basic helix-turn-helix-turn-helix motif is the basis for the histone-histone and histone-DNA interactions. The basic amino acids arginine and lysine are responsible for many of the histone-DNA interactions, making the protein basic. The structures of the N-terminal tails were not fully determined, but they are believed to be involved with interactions between adjacent nucleosome molecules.


Bibliography:
  1. Luger, K. et al. Crystal Structure of the Nucleosome Core Particle at 2.8 Angstrom Resolution. Nature. 389. 251-260 (1997).
  2. Rhodes, D. The Nucleosome Core all Wrapped Up. Nature. 389. 231, 233 (1997).
  3. Voet, D., Voet, J. Biochemistry - Second Edition. John Wiley and Sons, Inc. New York. 1125-1130. 1995.