Insight into Rtt109 has also emerged from recent crystallographic analysis with Vps75 [20
]. Three independent groups have reported a total of five Rtt109-Vps75 structures, with only two of the structures being from the same crystal form (). Notably, the data obtained by Kolonko et al.
is of poor resolution, limiting structural detail and preventing refinement [20
]. Collectively the remaining structures reveal three potential interfaces between Rtt109 and Vps75 (interface I, II, III) (). These interfaces account for the high affinity between Rtt109 and Vps75, with interface I and II together burying 3725 Å2
]. These interfaces are both hydrophobic and electrostatic in nature, and occur in context of the constitutive Vps75 homo-dimer [17
]. The Vps75 homo-dimer is formed through an anti-parallel arrangement of the Vps75 dimerization helix, located upstream of the so-called earmuff domain (). The Vps75 homo-dimer is either present in the asymmetric unit () [20
], or generated through a crystallographic 2-fold symmetry axis () [22
]. Vps75 dimerization is essential for Rtt109 binding [22
], with interface I and II involving different Vps75 chains (). The relevance of these interfaces in solution has been verified through enzymatic assays with Rtt109 mutants [22
Crystal Structures of Rtt109-Vps75
Interfaces observed in Vps75-Rtt109 at a 2:1 [Su et al.] (a) and 2:2 [Tang et al.] (b) stoichiometry
Conformational differences between Vps75-Rtt109 at a 2:1 and 2:2 stoichiometry
A notable feature of interface I is the involvement of Rtt109 residues 130-175. Limited proteolysis indicates that these residues undergo a disorder-to-order transition upon Vps75 binding [22
]. These residues were disordered or removed from the Rtt109 alone structures (). This is consistent with the instability of Rtt109 observed in a Vps75 deletion strain [6
]. Further comparison of free- and Vps75-bound Rtt109, however, fails to identify differences in the putative active or substrate-binding site (). Thus, while Vps75 is important for Rtt109 stability, it does not appear to induce an ‘active’ Rtt109 conformation.
The major difference between the structures is the stoichiometry of the Rtt109-Vps75 complex. Structures by Tang et al.
have one Rtt109 per Vps75 monomer () [22
], while those by Su et al.
have one Rtt109 per Vps75 dimer () [20
]. This difference arises as the Tang et al.
structures do not contain interface III such that the Rtt109 is tilted outward compared to the Su et al.
structures (Figures and ). Interface III is missing as Tang et al.
used truncated Vps75, and/or because it might not be physiologically relevant (see next paragraph). The Tang et al.
structures also contain a kink in the dimerization helix of Vps75 such that the region involved in interface I is tilted upwards (). This contrasts the Su et al.
structures, where this region arcs downward. The outward shift of Rtt109 and kink in Vps75 facilitate binding of a second Rtt109 in the Tang et al.
structures. Importantly, this different stoichiometry is not a result of mixing different Rtt109-Vps75 ratios as in all cases the proteins were co-expressed and co-purified (). It may however, be a result of different purification protocols. Interestingly, unlike the Tang et al.
structures, the Su et al.
structures don’t contain acetyl-coA (). Resolving the issue of stoichiometry is important as it influences the size of the cavity between Vps75 and Rtt109. Based on the orientation of Rtt109, sequence conservation and mutagenesis, this cavity is most likely the histone-binding site [20
]. The size of the cavity will determine if Rtt109-Vps75 binds H3-H4 dimer and/or tetramer and if acetylation occurs on a single or both H3 chains. In vitro Rtt109-Vps75 can acetylate a H3-H4 dimer [20
] but, like Vps75 alone, it does not split a H3-H4 tetramer [15
A possible explanation for the different stoichiometries is that both exist, depending on the relative concentrations of Rtt109 and Vps75. This is supported by native gels with Vps75 shifting at 2:1 (Vps75:Rtt109), and super-shifting at 2:2 [17
]; or native gels where free Vps75 is only detected in greater than 2:2 mixtures [20
]. A 2:2 complex has also been observed by analytical gel filtration [28
]. Such an explanation implies that the protocol employed by Su et al.
enriched for Vps75. This is feasible as the proteins were expressed from separate plasmids, possibly in different copy number, as well as because Vps75 is a constitutive homo-dimer and Rtt109 is notorious for aggregation and degradation [21
]. As such, compared to Vps75, Rtt109 could easily have been depleted during purification. A 2:2 complex is also incompatible with interface III. Interface III may be an artifact as it is small, involves few sequence-specific contacts and occurs near the Vps75 nuclear localization sequence that is bound by proteins which import Rtt109-Vps75 [14
]. It is difficult to visualize how Rtt109 and import proteins could simultaneously bind Vps75. The Tang et al.
structure clearly reveals that a 2:2 complex can form, albeit in the absence of the Vps75 N-terminal tail. While the Vps75 N-terminal tail is not involved in the Vps75 interaction with H3-H4, it is required for maximal Rtt109-Vps75 activity in vitro [17
]. A 2:2 complex with equal affinity sites however, conflicts with difficult to interpret analytical ultracentrifugation data [17
] and the near maximal Rtt109 activity observed at 2:1 [20
]. The question is; does Rtt109-Vps75 have maximal activity in the cell? To truly resolve the Rtt109-Vps75 stoichiometry conundrum, more experiments are required. In particular, the stoichiometry of the complex and the influence of the Vps75 N-terminal tail can easily be quantified using relatively straightforward in vitro fluorescence-based techniques.