Although the structure of the Rtt109–acetyl-CoA complex does not provide direct insight into Vps75 or histone H3 interaction, it does suggest how aspects of these interactions might occur. Specifically, a mapping of surface conservation within the Rtt109 family onto the structure reveals that in addition to surface conservation in the catalytic and acetyl-CoA binding sites, another separate patch of surface conservation maps to the α8- and α9-helices ( and and Supplementary Fig. 1
). This surface patch overlaps with a highly electronegative patch on the Rtt109 HAT domain ( and Supplementary Fig. 7a
online). This observation suggests that this region of Rtt109 might be used for interaction with an electropositive surface of an Rtt109 interacting protein such as Vps75 or histone H3. Correlating with a possible Vps75 interaction through this region of Rtt109, an alignment of Vps75 homologs revealed a highly conserved basic patch of residues that are not present within other histone chaperones (Supplementary Fig. 7b
). We hypothesize that this segment of Vps75, with predicted helical secondary structure, would interact with the α8-α9 region of Rtt109 via helix-helix charge interactions. Notably, both motifs (from Rtt109 and Vps75) are either missing or not conserved in fission yeast and a number of other fungi (Supplementary Fig. 7b
), which is consistent with previous reports that an Rtt109–Vps75 interaction is not essential for H3K56 acetylation in vivo
and that fission yeast Rtt109 is functional for Lys56 acetylation in vivo18
In light of (i) the fact that the 130–179 loop of Rtt109, deleted in our current structure, is not present in fission yeast and some other fungi, and (ii) the argument above that Rtt109 may not form an interaction with Vps75 in fission yeast, we addressed the role of this loop for Rtt109 association with budding yeast Vps75. To this end, we used pull-down studies to compare the ability of full-length Rtt109 and the (Δ130–179) deletion construct as glutathione S-transferase (GST) fusion proteins to associate with full-length Vps75 protein. We find that while full-length GST-Rtt109 interacts strongly with Vps75, the GST-Rtt109 (Δ130–179) deletion construct interacts very weakly under the same conditions (Supplementary Fig. 7c
), which demonstrates that the 130–179 loop of Rtt109 from budding yeast is used for Vps75 interaction. This data, taken together with the lack of conservation of the α8-α9 region of Rtt109 between fission and budding yeast noted above, suggests that Rtt109 uses some unique features for interaction with Vps75 in budding yeast that are not conserved in fission yeast.
To explore what role Vps75 might play in H3K56 acetylation by the Rtt109–Vps75 complex, we titrated an excess amount of free Vps75 along with yeast H3/H4 tetramers into an Rtt109–Vps75 complex. We found that H3K56 acetylation was substantially inhibited (~ten-fold) compared to the same assay without added Vps75 (Supplementary Figs. 7d and 5a
). We propose that this observation results from a competition between the Rtt109–Vps75 complex and free Vps75 for the H3/H4 tetramer, with slow exchange of the free and Rtt109-bound Vps75 due to a strong interaction between Rtt109 and Vps75 (data not shown). Based on this result, we propose that one role that Vps75 plays during H3K56 acetylation is to bind H3/H4 substrate for presentation to Rtt109 and acetylation.
The loop connecting α7 to β7 is highly conserved within the Rtt109 homologs, but surprisingly a R318A Q319A E320A mutation in this loop was found not to compromise catalytic activity13
. This loop is only about 9 Å away from the putative H3K56 substrate binding site, which suggests that it may play some role in histone H3 substrate binding. Residues 319–325 of this loop are poorly ordered in our structure (Supplementary Fig. 4
), which suggests that the loop is highly mobile. Notably, removal of this loop in the structure reveals a highly conserved hydrophobic core (Phe192, Pro245, Trp312 and Val329) that was formally occupied by Phe321 of the loop. Given that the histone H3K56 substrate of Rtt109 contains a phenylalanine (in budding yeast) or tyrosine (in most other species) two residues N-terminal to the Lys56 substrate, it is possible that an extended H3 peptide could bind to Rtt109 such that Tyr/Phe54 and Lys56 of H3 can occupy the position of Phe321 and the lysine binding tunnel of Rtt109, respectively. A docking exercise shows that a single side chain rotamer rotation (of Arg194) could accommodate such a binding mode (Supplementary Fig. 8
online). Based on this observation, we hypothesize that the Rtt109 region proximal to the α7-β7 loop participates in histone H3 substrate binding specificity, and that the α7-β7 loop could serve as a lid to mask the hydrophobic pocket from solvent exposure when the enzyme is not bound to histone H3 substrate.
Our structural studies suggest that yeast Rtt109 is the evolutionary precursor of metazoan p300/CBP. However, the structural similarity is not matched by an overlap in key catalytic residues. In addition, although it is tempting to suggest that p300/CBP might be a functional paralog of Rtt109, there is no strong evidence for H3K56 acetylation in human cells20
, and we have found that H3K56 is not a good substrate for p300/CBP in vitro
(data not shown). Taken together, this suggests that fungal Rtt109 and metazoan p300/CBP are not functional homologs, at least for the only H3K56 substrate that has been identified for Rtt109 so far. Nonetheless, this distinction between fold and mechanism underscores the point that the conserved fold or templating function of the HAT domain supercedes in importance the precise catalytic strategy, presumably because of the chemical simplicity of the reaction of a thioester with an amine. It should be noted that p300/CBP uses a series of protein-protein interaction domains within one long intact polypeptide for localization of the substrate. In contrast, this substrate-targeting function in Rtt109 is likely mediated by its intermolecular binding partners Asf1 and Vps75, thus exploiting modularity in chromatin regulation.