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Vacuolar protein sorting-associated protein 75 (PDB ID: 3DM7), Vps75, located in the nucleus, is a histone chaperone not only responsible for the assembly and disassembly of genes but also for the acetylation of the histone H3-Lys-56 (1,2). Recent research has shown that when Vps75 forms a complex with Ty1 transposition protein 109, a histone acetyltransferase (HAT), the Vps75-Rtt109 complex acetylates histone H3-Lys-9 in the H3 tail (3). Vps75 improves the effectiveness of importing and retaining Rtt109 inside the nucleus, and this is an efficient mechanism increasing the direct interaction between the complex and genetic material (6). Vps75 is found in Saccharomyces cerevisiae, commonly known as Brewer’s yeast and is a member of the Nucleosome Assembly Protein (NAP) family of histone chaperones. The NAP family characteristically consists of three main regions: a conserved central NAP domain, non-conserved N-terminus, and a highly acidic C-terminus. This NAP histone chaperone family is thought to function in exchanging histones during nuclear processes, in transporting the histones into the nucleus, and in assembling nucleosomes (2).

Histone chaperones such as Vps75 are proteins involved in the interaction between histones and other proteins or DNA (2). A theory for overall nucleosome formation in the yeast S. cervisiae is that the coordination of histone chaperons in a hierarchical manner. Initially, Anti-silencing factor 1, Asf1, binds to the H3-H4 dimer that is newly synthesized and then presents the dimer for acetylation. At this stage, Vps75 binds to Rtt109, generating a unique complex that promotes acetylation of Histone-3 at the Lysine-56 position. The acetylation is significant because H3K56Ac increases the affinity for Chromatin assembly factor 1 (CAF-1) and Rtt106, other yeast histone chaperones, to bind to Histone-3. Lastly, CAF-1 and Rtt106 assembles the DNA into nucleosomes (11). Histone acetylation and nucleosome rearrangement not only contributes to genome stability but also regulates replication, transcription, and damage repair of DNA (4,3). The histone modification takes part in telomeric silencing and transcriptional activation (7). Vps75 has a unique function of preventing Rtt109 degradation, ensuring that Rtt109 will modify the histones. Unlike the other NAP members, Vps75 favors binding to histones H3-H4 over H2A-H2B. The binding to the H3-H4 complex can result in a structural change which proposes that the configuration of Vps75 can potentially change.

This 55-kDa (exactly 54853.6 Da) histone chaperone is characterized by an isoelectric point of 4.83 as deduced by the ExPASy ProtParam, which uses the primary sequence to derive the physico-chemical properties of the protein (8). As deduced from the sequence provided in the Protein Data Bank, Vps75 structure consists of 45% helices and 13% beta sheets (12) . The overall homodimeric protein has a relatively symmetrical form. As seen in homologous NAP family histone chaperones, Vps75 has two domains that adopt a “headphone” organization. Domain I consists of a 40-residue helix that pairs with a symmetrically related subunit in an antiparallel orientation (1,3). These opposing subunits are stabilized by the Hydrogen-bonds and van der Waals interactions between both of the dimer components. A Proline residue near the end of the domain I alpha helix creates a kink, leading to a “hockey stick-like” structure (3). Following this kink is domain II containing alpha/Beta globular regions, also known as the “earmuff segments” of the headphone which are positioned at both ends of domain I. The two globular domains contain a 4-stranded Beta sheet and 6 alpha helices, just below the Beta-sheets, that associates with a Beta-hairpin region. Together, the alpha helices and the Beta-sheets enclose a hydrophobic core region. Ultimately, a cleft is formed near the center of the homodimer such that it is surrounded by the domain I dimerization domain on top and by domain II on opposite sides (1).

Vps75 is homologous to the SET/Taf-1Beta/NHAT protein (PDB ID: 2E50) found in humans. As seen in Dali searches, a Z-score of 14.9 and root mean squared deviation of 2.7 (9) indicates a similarity in tertiary structure to SET/Taf-1Beta/NHAT. A BLAST search was performed and an E-value of 3E-47 (10) shows there is a similarity in primary structure to SET/Taf-1Beta/NHAT. Furthermore, Vps75 and Nap1 (PDB ID: 2Z2R) share analogous structural features. However, the Z-score is 13.1 (9), indicating that compared to the tertiary structure of SET/Taf-1Beta/NHAT, the tertiary structure of Nap1 is not as similar to that of Vps75. The dimerization helices of domain I in all three of these proteins are comparable in length (~4.5 nm) (1). Also, the alpha helices and beta sheets constructing the hydrophobic core in Vps75 show some extent of structural alignment with that of Nap1 and of SET/Taf-1Beta/NHAT protein. Specifically, the averaged distance between the C-alpha atoms of residues 56 to 223 of a Vps75 monomer and the corresponding atoms of Nap1 and SET/Taf-1Beta/NHAT protein are around 0.19 nm.

Despite previously mentioned similarities between the three proteins, essential functions of Vps75 are attributable to the distinctive structural elements. The cleft in Vps75 is not as restricted in size compared to that of Nap1 and Human SET/Taf-1Beta/NHAT homologs, both of which have helices and beta sheets enclosing the cleft. Another evident divergence between the three is the surface electrostatic potential of the cleft. Vps75 has an electronegative ring of charge concentrated immediately under the dimerization helices of domain I and electropositive patches at the bottom of domain II. Contrastingly, Nap1 and SET/Taf-1Beta/NHAT protein has an electronegative surface throughout the entire cleft region. The histone substrate specificities of the Vps75 can be attributed to this difference in size and electrostatic potential (1).

The interface between the dimerization and earmuff domains is highly conserved throughout the Vps75 homologues, specifically, residues from domain I such as Phe-15, Leu-18, and Ile-5, interact with residues from the top surface of domain II. This specific interaction defines the size of the central cleft, ultimately signifying that the relative position of these domains affect the function of Vps75. Predominantly, both the cleft region of the dimer and the bottom of the earmuff domains has the greatest degree of conservation among the homologues, suggesting that these areas are functionally significant (1).

In support of this structure-functional theory, the Rtt109 histone acetyltransferase is thought to interact with Vps75 in a 2:1 stoichiometric ratio where two molecules of Vps75 associates with one molecule of Rtt109(1). If the Rtt109 concentration is increased, there is also a possibility that two molecules of Rtt109 will interact with Vps75 (1,4). Alpha helix-5 and the C-terminal end of alpha helix-8 of the earmuff domains is critical for binding with Rtt109. Once the Rtt109 molecule binds to the Vps75 homodimer, a cylindrically-shaped enclosure, containing the active site interiorly, is formed. Even though the Vps75 structural homologs have similar histone chaperone functions, Vps75 is the only protein that stimulates Rtt109 enzymatic HAT activity (5). The negatively charged dimer cleft region complements well with the electropositive H3-H4 complex in histone binding. The Vps75 cleft is capable of not only interacting with a H3-H4 dimer but also a symmetrical (H3-H4)2 tetramer (1).

Vps75 functions are commonly introduced in relation to Rtt109 activity. However recent research shows that Vps75 regulates transcription of genes different from those regulated by Rtt109 (6). So, Vps75 might have functions independent of Rtt109. These findings about Rtt109 independent activity are still being confirmed and consolidated. In the cases where Rtt109 is absent or deleted, Vps75 is responsible for promoting other HATs to modify the H3 tail. Alternatively, Vps75 might play a role in histone exchange and chromatin assembly instead of promoting other histone modifications. Vps75 may help in the exchange of histones with various modifications and of canonical histones with variant histones (6). By “refreshing” histones with new histones, the chromatin structure in the yeast becomes increasingly responsive to any changes in the environment.