1. Introduction
Histones are structural scaffold proteins made up of multiple subunits
(four sets of the dimers H3–H4 and H2A–H2B) and form a hetero-octamer.
They facilitate the packing and ordering of DNA into nucleosomes by
allowing the double-helical molecules to wrap around their
hetero-octamer structure. These nucleosomes in turn assemble into
chromatin, which further condenses into a chromosome and regulates
transcriptional activity by allowing certain parts of the DNA to become
accessible to transcription factors. When chromatin is highly packed,
forming a structure known as heterochromatin, transcription is repressed
owing to the inaccessibility of the nucleic acid sequences to
transcription factors. By contrast, in the loosened form of chromatin
known as euchromatin, transcription is actively carried out.
Histone chaperone proteins assist in the formation of nucleosomes by
associating with the various histone subunits during the different steps
of hetero-octamer assembly. One such histone chaperone protein,
nucleosome assembly protein 1 (Nap1), is widely conserved among
eukaryotes. Aside from depositing the histone subunits in nucleosomes,
Nap1 has been implicated in the transportation of histones across the
nuclear membrane.1 Generally, the entry and exit of
cargo proteins across the nuclear pore are facilitated by karyopherins
known as importin and exportin, respectively. Proteins need to bind to
these karyopherins to pass through the nuclear pore. When a cargo
protein is to be transported from the nucleus to the cytosol, the
karyopherin acting as an exportin recognizes and binds to the nuclear
export signal (NES), a leucine-rich sequence on the cargo protein.
GTP-bound Ran, a low-molecular-weight GTPase, regulates the direction of
transport by binding to and activating an exportin in the nucleus. Once
the cargo protein has moved out of the nucleus into the cytosol, the
complex dissociates and Ran hydrolyzes its bound GTP into GDP.
A study of the crystal structure of yeast nucleosome assembly protein 1
(yNap1) has revealed that it is a homodimer, with the NES sequence being
part of the long α-helix.2 Furthermore, mutagenesis
experiments on the budding yeast have suggested that the NES is
necessary for the transport of yNap1 from the nucleus to the
cytosol.3 However, when yNap1 forms a homodimer, each
protein interface has a masking region, known as the accessory domain,
which masks the NES mutually.2 Although the accessory
domain is generally conserved among eukaryotes, the Nap family members
Yeast VPS75, human SET, and human TSPY lack this domain as they do not
have an NES sequence, suggesting an evolutionary link between
them.4-6 In terms of its biological function, the
accessory domain might allow exportin to access the NES to properly
transport the complex from the nucleus. However, the exact role of this
domain in nucleocytoplasmic transport has yet to be elucidated.
To shed some light on this subject, in this study, we focused on
phosphorylation in the accessory domain because several proteomic
studies have identified multiple phosphorylation sites on
yNap1.7-11 For example, in vitro experiments on
the budding yeast and on Drosophila melanogaster have confirmed
that multiple residues in the accessory domain are phosphorylated by
casein kinase 2 (CK2).8,12 One yeast study revealed
that non-phosphorylatable (alanine) and phosphomimetic (aspartic acid)
mutations in the accessory domain could lead to elongation of the S
phase (the cell cycle during which DNA is replicated and nucleosomes are
assembled),8 suggesting that proper yNap1 transport
requires nucleosome assembly. To investigate the phosphorylation effect
in the accessory domain, we performed all-atom molecular dynamics (MD)
simulations of the following systems: (1) non-phosphorylated yNap1
(Nap1-nonP) and (2) phosphorylated yNap1 (Nap1-P). In both systems, we
quantitatively measured the solvent-accessible surface area (SASA) of
the NES to address how it is exposed to the protein surface to increase
its accessibility. By determining a difference in the SASA distribution
between both systems, the phosphorylation effect on the NES recognition
by an exportin could be surmised.