Distance restraints.
5. Discussion
5.1. Asf1 and Vps75 activate Rtt109 via substrate presentation mechanisms.
The structure of the RVAH complex presented in this work rationalizes the in vivo activity data, showing that both Asf1 and Vps75 are important for H3 K9 acetylation, but only Asf1 is essential for H3 K56 acetylation (Fig. 1.3.2, 1.4.2). The proposed mechanism of how the chaperones regulate Rtt109-dependent acetylation of H3 is summarized in Fig. 5.1.1.
Figure 5.1.1. Regulation of Rtt109 activity by Asf1 and Vps75. Asf1 presents the H3:H4 dimer to Rtt109 in a correct orientation, where the H3 N-terminal tail faces towards the active center and H3 K56 is placed in the vicinity of the catalytic pocket. Vps75 facilitates the formation of the complex and attracts the disordered H3 N-terminal tail to the Rtt109 active center via both its folded and unstructured domains.
Mutations of H3 E94, which affect the formation of the RVAH complex, as shown by NMR data in Fig. 4.5.3, decrease both H3 K56 and K9 acetylation in vivo (Zhang et al, 2018). Mutations that affect local interactions of Rtt109 and the Asf1-bound histones, such as deletion of the H4 C-terminus (95-102) or the Rtt109 C-terminal tail, decrease the acetylation of H3 K56 but don’t affect, or affect less significantly acetylation of H3 K9 by the RVAH complex in vitro (Fig. 4.5.4). On the other hand, deletion of the Vps75 CTAD decreases the acetylation of K9, but not K56 (Fig. 4.6.4). These results, taken together with the structure of the RVAH complex, are consistent with a model in which H3 K56 acetylation is promoted by Asf1, acting to position the H3 core with respect to Rtt109 in such a way that K56 faces the Rtt109 active site; Vop75 aids the formation of a stable complex, but does not exert an active function in catalysis. On the other hand, acetylation of H3 K9 is critically dependent on Vps75: the H3 N-terminal tail is first released from Asf1 by both Rtt109 and Vps75, and then is confined near the Rtt109 active center by the concerted action of both the acidic cavity
and the CTAD of Vps75. This is shown by the CSP data on the H3 N-terminal tail and the Vps75 CTAD as well as by the low resolution SANS envelopes (Fig. 4.5.7 and section 4.6).
The 206 EE 207 acidic patch located in the Vps75 cavity further aids H3 N-terminal tail
acetylation by capturing R52 and R53 of H3 and mildly inhibiting H3 K56 acetylation, as demonstrated by molecular dynamics simulations and in vitro activity assays (Fig. 4.6.3). The H3 52 RR 53 is the only double arginine patch in the H3 N-terminal tail, despite the fact that
all of the H3 lysine residues that can be acetylated have an arginine residue in the vicinity. However, H3 52 RR 53 might be engaged by the Vps75 cavity more efficiently due to both its
ability to make a higher number of hydrogen bonds and its proximity the 206 EE 207 . It is
important to note that although Vps75 206 EE 207 has a mild inhibitory effect on H3 K56ac, the
overall acetylation of H3 K56 is not inhibited but rather promoted by the presence of Vps75
in vitro (Fig. 4.3.4). This is most likely due to the role of Vps75 in the formation of the tight complex between Rtt109 and Asf1–H3:H4.
In vitro , CSP experiments and SEC did not detect a tight complex between Rtt109 and Asf1–H3:H4 in the absence of Vps75 (Fig. 4.2.6). However, Rtt109–Asf1–H3:H4 can acetylate H3 K56 both in vitro and in vivo . One explanation of this is that a transient short-lived interaction between Rtt109 and Asf1-bound histones is sufficient for the acetylation of H3 K56; on the other hand, efficient acetylation of the disordered N-terminal tail, which is not actively guided towards the active center in the absence of Vps75, requires a longer lifetime of the complex. In addition to this, the Rtt109–Asf1–H3:H4 stability and activity in vivo might be influenced by such factors as PTMs of H3 and the long CTAD of Asf1.
Some Rtt109 homologs lack the ability to bind to their corresponding Vps75 (Chen et al , 2019b; Zhang et al , 2018) . The conservation of the residues involved in the interaction between the Asf1–H3:H4 and Rtt109 in A. fumigatus and S. cerevisiae together with the role of characterized Rtt109 homologs in the DNA damage response, suggest that the mechanism of H3 K56 acetylation by Rtt109 is conserved throughout fungi. The degree of the H3 N-terminal tail acetylation by Rtt109 homologs as well as the role of Vps75 homologs appears to be more variable.
While the structural model of the RVAH complex explains the preference of Rtt109 for the acetylation of H3, and how Vps75 promotes acetylation of the H3 N-terminal tail, it does not explain the selectivity for specific lysine residues in the H3 tail. It appears that the selectivity
of Rtt109 towards H3 K9, H3 K14 , K23, K27 and K56, but not K18, K36 or K37 is 9 determined by the structure of the active center of the acetyltransferase rather than by histone chaperones. All lysine residues targeted by Rtt109 have a similar surrounding amino-acid sequences (Fig. 5.1.2A).
Figure 5.1.2. Residues following H3 lysines determine the acetylation by Rtt109. A. Lysines
which are acetylated by Rtt109 are followed by small residues (similar residues are highlighted by a green background). B,C . Particular views of the A. fumigatus Rtt109–Asf1–H3:H4 structure (PDB entry 5zba). Rtt109 is shown in beige, Asf1 in pink, H3 in green and H4 in grey.
As an example of how the neighbouring amino acids influence lysine acetylation, the H3 S57Q mutation abolishes K56 in vivo without affecting K9, K23 or K27 (Zhang et al , 2018). In the X-ray structure of the A. fumigatus Rtt109–Asf1–H3:H4 from Zhang et al , H3 S57 is engaged in a hydrogen bond with D260 (corresponding to D287 in S. cerevisiae ), which could explain its importance (Fig. 5.1.2B). However, H3 K23 and K14, which can be acetylated by Rtt109, are followed by alanines, which are similar to serines in size but cannot form hydrogen bonds with their side chain. This, together with the effect of the mutation of a small S to a large Q, suggests that the size of the neighbouring amino acids is the defining factor. From the crystal structure it is unclear why a large residue could not be accommodated in this position.
In addition to the placement of H3 K56 in the catalytic pocket, the Zhang et al structure shows that a part of the H3 tail (residues ~45-51) is wrapped around the H3:H4 dimer via interactions with H4 (residues 44-47) mediated mostly by the backbone atoms (Fig. 5.1.2C). Interestingly, our CSP data indicates that this H4 surface is affected by the presence of the Vps75 CTAD (Fig. 4.5.7). While I hypothesize that these CSPs arise from a direct interaction between the Vps75 CTAD and H3:H4, they may also be caused by the modulation of the interaction of the H3 tail with H3:H4.
9 Rtt109 is able to acetylate H3 K14 in vitro , however in vivo Gcn5 is the major HAT responsible for
It would be interesting to obtain a more detailed molecular view of the H3 N-terminal tail and its interactions within the complex, including the residues surrounding the catalytic pocket of Rtt109, the Vps75 CTAD and the complex cavity.