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.