Catalytic activation of histone acetyltransferase
Rtt109 by a histone chaperone
Erin M. Kolonkoa, Brittany N. Albaugha, Scott E. Lindnera, Yuanyuan Chenb, Kenneth A. Satyshura, Kevin M. Arnolda,
Paul D. Kaufmanb, James L. Kecka, and John M. Denua,1
aDepartment of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, 1300 University Avenue, Madison, WI 53706; and
bPrograms in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, 364 Plantation Street #506, Worcester,
Edited by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, and approved September 28, 2010 (received for review July 7, 2010)
Most histone acetyltransferases (HATs) function as multisubunit
complexes in which accessory proteins regulate substrate specifi-
city and catalytic efficiency. Rtt109 is a particularly interesting
example of a HAT whose specificity and catalytic activity require
association with either of two histone chaperones, Vps75 or Asf1.
Here, we utilize biochemical, structural, and genetic analyses to
provide the detailed molecular mechanism for activation of a HAT
(Rtt109) by its activating subunit Vps75. The rate-determining step
of the activated complex is the transfer of the acetyl group from
(>250-fold), not by contributing a catalytic base, but by stabilizing
the catalytically active conformation of Rtt109. To provide structur-
al insight into the functional complex, we produced a molecular
model of Rtt109-Vps75 based on X-ray diffraction of crystals of the
complex. This model reveals distinct negative electrostatic surfaces
on an Rtt109 molecule that interface with complementary electro-
positive ends of a symmetrical Vps75 dimer. Rtt109 variants with
interface point substitutions lack the ability to be fully activated
by Vps75, and one such variant displayed impaired Vps75-depen-
dent histone acetylation functions in yeast, yet these variants
showed no adverse effect on Asf1-dependent Rtt109 activities in
vitro and in vivo. Finally, we provide evidence for a molecular mod-
el in which a 1∶2 complex of Rtt109-Vps75 acetylates a heterodimer
of H3-H4. The activation mechanism of Rtt109-Vps75 provides a
valuable framework for understanding the molecular regulation of
HATs within multisubunit complexes.
p300 ∣ K56 acetylation ∣ K9 acetylation ∣ NAP1
Histone acetyltransferases, or HATs, transfer the acetyl group
of acetyl CoA onto the ε-amino group of lysine residues. HATs
are almost exclusively found within largermultisubunit complexes
with accessory proteins that modulate enzymatic activity and di-
rect substrate specificity (2–4). For example, the yeast HATs Esa1
(KAT5) and Gcn5 (KAT2) alone are ineffective catalysts toward
nucleosomal histones; both enzymes require association with
other protein subunits for efficient acetyl transfer on nucleosomal
substrates (5–7). Despite the prevalent reports of multisubunit
HAT complexes, the molecular mechanisms by which accessory
proteins regulate the acetyltransferases are largely unknown.
HAT complexes formed by the acetyltransferase Rtt109
(KAT11) are remarkable. Distinct histone chaperones (Vps75
and Asf1) help direct Rtt109 substrate selection for different
biological processes, and each stimulates the acetyltransferase
activity of Rtt109 (8–11). In Candida albicans, Rtt109 is required
for pathogenesis and, thus, could provide a unique target for anti-
fungal therapies (12). Notably, Rtt109 lacks sequence homology
to previously characterized HATs (11, 13–15), although Rtt109’s
structure has revealed similarity to the mammalian HAT p300
Rtt109 is responsible for acetylating multiple lysine residues on
nonnucleosomal histone H3 substrates (11, 17). Rtt109 acetylates
n eukaryotes, histone acetylation regulates nucleosome assem-
bly, chromatin folding, transcription, and DNA repair (1).
lysine 56 on the histone H3 core domain (H3K56), a mark that
occurs globally on newly synthesized histones in Saccharomyces
cerevisiae and Schizosaccharomyces pombe and is required for
genome stability (13, 15, 18–21). H3K56ac was recently detected
in humans, where it has been shown to be prominent in multiple
cancers and is enriched at genes that are key regulators of stem
cell pluripotency (22–24). H3K56ac is absent in yeast cells lacking
Asf1 (asf1Δ mutants), indicating an essential role for Asf1 in the
Rtt109-dependent acetylation of H3K56 (11, 13, 25). The Nap1
family histone chaperone Vps75 copurifies with Rtt109, but yeast
cells lacking Vps75 (vps75Δ mutants) display normal levels of
H3K56ac. Instead, vps75Δ cells display a reduction in acetylation
of H3K9 and H3K23 (8, 9). Additionally, the Rtt109-Vps75
complex contributes to H3K27ac, an overlapping function with
the HAT Gcn5 (26). In vitro, both Asf1 and Vps75 stimulate the
histone acetyltransferase activity of Rtt109 (8, 10, 11, 27, 28).
However, a biochemical and structural understanding for how
these discrete histone chaperones stimulate catalysis and direct
distinct cellular functions is lacking.
Here we utilize biochemical, structural, and genetic analyses to
explore the functional Rtt109-Vps75 complex and the molecular
mechanism of catalytic activation. We demonstrate that catalytic
activation of Rtt109 by Vps75 is achieved via enhanced acetyl
transfer that occurs due to stabilization of the active enzyme con-
formation. Additionally, we provide a model of the Rtt109-Vps75
complex derived from X-ray crystallographic studies and define
critical interacting surfaces in vitro and in vivo that are required
for specific Vps75 activation of Rtt109. Finally, we present
evidence supporting a molecular model in which a 1∶2 complex
of Rtt109-Vps75 acetylates histone H3-H4 heterodimers. This
report describes a functional HAT-chaperone complex at the
biochemical, genetic, and structural levels. These results provide
important clues toward our general understanding for the roles of
accessory proteins in multisubunit HAT complexes.
Mechanism of Rtt109 Activation by Vps75. Rtt109 alone is an ineffi-
cient acetyltransferase (kcat¼ 2.3 ? 0.7 × 10−3s−1for histone
H3); however, when Rtt109 is purified in complex with Vps75,
its enzymatic activity increases ∼100-fold (8). Rtt109-Vps75
complex can be separated by strong hydrophobic interaction
chromatography or expressed individually and recombined to
produce fully activated complex, demonstrating the reversibility
of binding (29). We hypothesized that the rate enhancement of
Author contributions: E.M.K., B.N.A., P.D.K., J.L.K., and J.M.D. designed research; E.M.K.,
B.N.A., S.E.L., Y.C., and K.M.A. performed research; E.M.K., B.N.A., S.E.L., Y.C., K.A.S.,
K.M.A., P.D.K., J.L.K., and J.M.D. analyzed data; and E.M.K., B.N.A., S.E.L., Y.C., K.M.A.,
P.D.K., J.L.K., and J.M.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1009860107PNAS ∣ November 23, 2010 ∣ vol. 107 ∣ no. 47 ∣ 20275–20280
the Rtt109-Vps75 complex could be due either to an increase in
the rate of acetyl transfer (kcateffect) or to the ability of the
histone chaperone to affect substrate binding affinity (Kmeffect).
To determine the activation mechanism of Vps75 on Rtt109, we
performed steady-state kinetic analyses with increasing H3
concentrations and varied levels (0.2×–8×) of chaperone relative
to enzyme (Fig. 1A). Each resulting dataset was fitted to the
Michaelis–Menten equation. The series of curves revealed a
substantial increase in kcatand kcat∕Kmvalues as the ratio of
Vps75 to Rtt109 increased, whereas no general trend for Km
values was observed (Fig. 1A). Similar trends were observed when
H3-H4 was employed as substrate (Table S1). These results indi-
cated that the mechanism of Rtt109 activation by Vps75 involves
enhanced catalytic turnover (kcat) but not increased binding
affinity for histone. Next, we explored the mechanism by which
Vps75 enhances the rate of catalysis by Rtt109. To first ascertain
the step of catalysis affected by Vps75, the rate-limiting step for
Rtt109 alone was determined. Previously, we identified the che-
mical step of acetyl transfer as rate limiting for the Rtt109-Vps75
complex (29). A change in the rate-limiting step in the absence
of Vps75 would suggest that the chaperone activates Rtt109 by
enhancing the rate of substrate binding or product release. To
determine the rate-limiting step for Rtt109 alone, pre-steady-
state kinetic analysis was performed and compared to the results
obtained with the complex (Fig. S1A) (29). For Rtt109-Vps75, the
amount of product formed over time produced a linear curve
yielding a rate of 0.53 ? 0.02 s−1. For Rtt109 alone, the amount
of acetylated product measured over time (0–480 s) yielded a
linear curve, indicating that the rate-limiting step for both unac-
tivated Rtt109 and Rtt109-Vps75 is the chemical step of acetyl
transfer. As observed for the complex, a lag or a burst phase
was undetectable for Rtt109 alone. In addition, the linear curves
yielded rates (2.30 ? 0.03 × 10−3s−1) that are consistent with
steady-state kcatvalues (2.3 ? 0.7 × 10−3s−1);thus, the stimulated
activity reflects enhanced acetyl transfer and not a change in the
rate of substrate binding or product release.
We then investigated the possibility that Vps75 stimulates the
enzymatic activity of Rtt109 by contributing an ionizable catalytic
residue to the active site. We have previously shown that the kcat
pH profile of Rtt109-Vps75 exhibits a single critical ionization
(pKa¼ 8.5), which likely corresponds to the substrate lysine that
must be unprotonated for catalysis (29). To determine if Vps75
contributes a general base to the active site of Rtt109, we per-
formed a kcatpH profile analysis of Rtt109 alone (Fig. S1B).
The unactivated enzyme yielded a similar pKað8.1 ? 0.1Þ to that
determined for the activated complex (pKa¼ 8.5) (29), indicat-
ing that Vps75 does not enhance acetyl transfer by contributing
an active-site general base or by lowering the observed pKa.
We envisioned two scenarios for the activation of Rtt109 by
Vps75. First, Rtt109 could exist in an equilibrium between active
and inactive conformers, where only a small fraction of Rtt109 is
capable of catalyzing the acetyl transfer. In this case, Vps75 could
enhance the rate of acetyl transfer by stabilizing the active con-
formation of Rtt109. Second, formation of the Rtt109-Vps75
complex could enhance catalysis by lowering the activation en-
ergy of acetyl transfer; thus, the activated complex and Rtt109
alone represent two distinct transition states with different ener-
gies of activation. To distinguish between these possibilities, we
determined the effect of temperature on the kcatof Rtt109 and
Rtt109-Vps75 using S. cerevisiae H3-H4 tetramers as the sub-
strate. The kcatvalues were determined at various temperatures,
and the data were fitted using the natural logarithm of the
Arrhenius equation lnðkÞ ¼ ð−Ea∕RÞð1∕TÞ þ lnðAÞ (Fig. 1B).
The energy of activation, Ea, was calculated from the resulting
slope of the plot, which is equal to −Ea∕R, where R is the
ideal gas constant, 8.314472 JK−1mol−1. No significant differ-
ence was observed between the activation energy of Rtt109 alone
(Ea¼ 6.0 ? 0.4 × 104Jmol−1) and Rtt109-Vps75 (Ea¼ 6.1?
0.4 × 104Jmol−1). These data are consistent with the pH rate
analysis and argue against Vps75 contributing to transition state
stabilization. Thus, Rtt109 alone and the Rtt109-Vps75 complex
utilize the same transition state, supporting a model in which
Vps75 enhances catalytic turnover via stabilization of the active
conformation of Rtt109.
To provide further evidence for this model, we asked if the
activation of Rtt109 at various chaperone concentrations could
be attributed to the fraction of bound complex. We calculated
the apparent dissociation constant (appKd) of Rtt109-Vps75 using
the kinetic data of Fig. 1A. The concentration of Vps75 dimer
was plotted against the fraction of catalytically active complex
(Fig. S2A). The calculatedappKdvalue of 13 ? 3 nM is consistent
with the Kddetermined using an equilibrium binding method
(10 ? 2 nM) (29). Because the magnitude of activation correlates
with the fraction of bound complex, this result provides further
evidence that catalytic activation is attributed to Vps75 binding
and stabilization of the active Rtt109 conformation.
Structural Insights into the Rtt109-Vps75 Complex. To provide a
structural understanding of this unique HAT-chaperone complex,
turation curves were performed to determine the Kmand kcatfor Rtt109 with
varying amounts of chaperone. Data from two trials performed in duplicate
are shown. Kinetic constants obtained from the saturation curves are listed
below with standard error. (B) Arrhenius plots of Rtt109 and Rtt109-Vps75.
The natural logarithm of kcatvalues for Rtt109 alone (circles) and Rtt109-
Vps75 (triangles) are plotted against the reciprocal of absolute temperature.
Data were fitted to the natural logarithm of the Arrhenius equation, and
activation energy values with standard error were calculated using the slope
of the lines.
Enzymatic characterization of Rtt109 activation by Vps75. (A) H3 sa-
www.pnas.org/cgi/doi/10.1073/pnas.1009860107Kolonko et al.
the complex of Rtt109 and Vps75 was crystallized for diffraction
studies. Crystals of a His-tagged fragment of Rtt109 (residues
1–405) bound to a fragment of Vps75 (residues 9–223) were
obtained after coexpression in Escherichia coli and purification
through three chromatographic steps. The resulting crystallized
complex diffracted to a 4.25-Å resolution (Table S2). Molecular
replacement was performed using the previously determined
Rtt109 (16) and Vps75 (8) structures as search models. The asym-
metric unit contained two Rtt109 monomers and four Vps75
monomers, which were arranged into apparent trimeric arrange-
ments with two Vps75 monomers capping a single Rtt109 mono-
mer (Fig. 2A). Interestingly, the Vps75 monomers pack together
by interaction of the long helices that mediate similar homodi-
meric Vps75 interactions previously observed (8, 10), although
this arrangement was not a constraint used in the molecular re-
placement. The limited resolution of the diffraction data did not
permit stable refinement of the Rtt109-Vps75 model; however,
this structural model provided an excellent platform for directed
biochemical experiments that probe the interfaces between sub-
units. Using native PAGE, Rtt109 was titrated with increasing
concentrations of Vps75 and complex formation was assessed
(Fig. S3). Consistent with the formation of a trimeric complex,
2 M equivalents of Vps75 to Rtt109 produced maximal levels
of complex. These data are also in agreement with the kinetic
data (Fig. 1 and Fig. S2A) as 2M equivalents of Vps75 to
Rtt109 resulted in near maximal activation.
To investigate the importance of the two electrostatic inter-
faces observed between subunits within the Rtt109-Vps75 com-
plex (Fig. 2B), two sets of amino acid substitutions in Rtt109
were generated and functionally assessed for their ability to affect
Vps75-dependent histone acetylation. Within the two α5 helices
of the Vps75 dimer, residues R173 and K177 are predicted to
interact with Rtt109 through E374 and E378 on helix α9 and
E299, E300, and D301 on helix α6 (Fig. 3). Thus, we made three
sets of substitutions within Rtt109 at these residues [E374A/
E378A (A2), E374K/E378K (K2), and E299K/E300K/D301K
(K3)] (Fig. 3). To verify that the point substitutions did not affect
the overall structural integrity of Rtt109, steady-state kinetics of
the unactivated enzyme variants were performed (Fig. 3). All
variants showed similar activity toward H3 in comparison to wild-
type enzyme, suggesting that these substitutions do not grossly
affect the structure of Rtt109 in the absence of Vps75. We then
assessed the ability of Vps75 to activate the Rtt109 variants by
determining the initial rates of acetylation at increasing chaper-
one concentrations (1–25× relative to Rtt109) under saturating
substrate conditions (Fig. 3). The data show a >10-fold reduction
in activation for the charge reversal variants at both interfaces,
whereas the neutralization variant had a less dramatic effect.
If the loss in activation results from a decrease in binding affinity
between Rtt109 and Vps75, then the kcatfor wild type and the
variants should be equivalent but require more Vps75 to reach
the kcatfor K2 and K3; however, the kcatfor these variants is
>10-fold lower than that of wild-type Rtt109. Equilibrium bind-
ing measurements yielded Kdvalues for K2 and K3 of 12 ? 1 and
16 ? 3 nM, respectively (Fig. S2); thus, both variants are able
to bind to Vps75 with similar affinity as wild-type Rtt109
[Kd¼ 10 ? 2 nM (29)]. We next asked if the point substitutions
in Rtt109 caused a change in the Kmvalue for H3. Steady-state
kinetic analyses of Rtt109-Vps75 variants K2 and K3 revealed no
significant change in H3 Km(7.5 ? 1 and 4.6 ? 1 μM, respec-
tively, Fig. S4), relative to wild-type Rtt109 (6.5 ? 2 μM, Fig. 1A).
These results suggest that the K2 and K3 substitutions do not
significantly alter substrate binding or the overall affinity between
Rtt109 and Vps75. Native PAGE was performed to provide
additional evidence of the variants to form complexes with Vps75
(Fig. S5). Although the Rtt109 K2 mutant generated Rtt109-
Vps75 complexes that were indistinguishable from that of wild
type, interestingly, Rtt109 K3-Vps75 generates a slightly faster-
running complex, suggesting that the hydrodynamic properties
the Rtt109-Vps75 complex. Rtt109 is shown in green and the Vps75 homodi-
mer is shown in blue. (B) Electrostatic maps of Vps75 dimer (Top) and Rtt109
(Bottom) illustrate the charge–charge interaction surfaces seen in the crystal
structure. The active-site channel is positioned directly in the center of the
substrate cavity formed by the complex. Structural figures and electrostatic
maps were made using MacPyMOL.
Structure of the Rtt109-Vps75 complex. (A) 2Fo-Fcdifference map of
Point substitutions were made at acidic residues of Rtt109 (green) proposed
to interact with basic patches of Vps75 (blue overlaid with a vacuum electro-
statics surface generated in MacPyMOL). E374 and E378 were replaced with
either Lys or Ala, whereas E299, E300, and D301 were changed to Lys. Initial
rates of acetylation by Rtt109 variants alone or with increasing [Vps75] were
measured to determine kcatvalues for each Rtt109 variant. Kinetic constants
obtained from three trials of samples performed in duplicate are listed with
error being one standard deviation for unactivated values and the standard
error for Rtt109-Vps75 complex values.
Variants of Rtt109 at the proposed Rtt109-Vps75 complex interface.
Kolonko et al.PNAS
November 23, 2010
are different from that observed with wild-type Rtt109 and the
K2 variant (Fig. S5B). However, quantitative titrations revealed
that the K3-Vps75 complex forms in a 1∶2 M ratio (Fig. S5C), just
as that observed with the wild-type complex (Fig. S3 A and B).
Collectively, these observations indicate that perturbation of
the electrostatic interfaces yields Rtt109-Vps75 complexes that
fail to attain full catalytic activation, but are otherwise similar.
To demonstrate that the impairments in catalytic activation are
specific to Rtt109-Vps75 contact surfaces (Fig. 3), we investigated
the ability of A2, K2, and K3 variants to be activated by Asf1
using H3-H4 as substrate (Fig. S6A). The results indicate that the
Rtt109 point substitutions (A2, K2, and K3) caused no significant
change in Asf1-dependent acetylation compared to wild-type
To provide in vivo support for the functional importance and
chaperone specificity of the electrostatic interaction sites re-
vealed in our biochemical and structural studies, we investigated
the Asf1- and Vps75-dependent cellular functions of these
Rtt109 variants. Yeast strains carrying the mutant rtt109-K2 and
-K3 alleles were created and grown on either synthetic media
[synthetic complete media lacking leucine (SC-Leu)] as a positive
control for cell growth or on rich media [yeast extract/peptone/
dextrose (YPD)] containing camptothecin (CPT, 8 μM) to
measure genotoxic stress resistance (Fig. S6B and Fig. 4A). CPT
covalently traps topoisomerase I leading to double-stranded
DNA breaks (30), making Rtt109-Asf1 (and not Rtt109-Vps75)
stimulated histone deposition essential for survival (31, 32). Both
Rtt109 variants as well as wild-type Rtt109 were able to restore
CPTresistance to the rtt109Δ strain, suggesting that the acetyla-
tion of H3K56 by Rtt109-Asf1 is unaffected by the K2 or K3
To assess the in vivo effect of Rtt109 variants on Vps75-depen-
dent H3 tail acetylation, we examined the acetylation of H3 by
immunoblotting. We previously demonstrated that Rtt109-Vps75
readily acetylates residues K9, K14, and K23 in vitro, and K56 on
free H3 alone and residues K9 and K23 in cells (8). Here, we
examined the primary sites of Rtt109-Vps75-catalyzed acetylation
of reconstituted H3-H4 tetramers. Using quantitative MS/MS
analysis, the acetylation sites were determined after 30 min of
reaction, which represents non-steady-state conditions (Fig. 4B).
Acetylation of H3 on K9 (100%), K23 (82%), K27 (31%), and
K56 (100%) was clearly observed. Under steady-state conditions,
we compared the site specificity of the wild type and the
K3-Vps75 complex toward free histone H3. Consistent with pre-
vious observations (8, 27), the Rtt109-Vps75 complex displays a
marked preference for H3K9. Although the rate was >10 times
slower (Fig. 3), the K3-Vps75 complex displays a similar prefer-
ence for H3K9 (Fig. S6C), suggesting that the K3 variant does not
alter the enzyme specificity. Once we verified the sites of acetyla-
tion on the H3-H4 substrate, we used antibodies against H3K9ac,
K27ac, and K56ac to investigate the Vps75-dependent activity of
Rtt109 variants in vivo. Whole-cell extracts of rtt109Δ and rtt109Δ
gcn5Δ strains carrying the plasmid-borne RTT109 alleles were
probed for acetylation at K56, K9, and K27 of histone H3. The
latter strain was used to investigate acetylation at H3K9 and
H3K27, as both Rtt109 and Gcn5 acetylate these sites (8, 26, 27).
In agreement with the genome stability data (Fig. 4A), both K2
and K3 variants supported normal levels of H3K56 acetylation
(Fig. 4C and Fig. S6 D and E). Interestingly, although normal
levels of H3K9 and H3K27 acetylation were observed in cells ex-
pressing wild-type Rtt109 and the variant Rtt109-K2, the variant
Rtt109-K3 generated reduced levels of K9 and K27 acetylation in
the rtt109Δgcn5Δ strain. These findings were supported by immu-
noblotting titrations of each variant using the double knockout
strain (Fig. S6 D and E). Thus, we have developed variants of
Rtt109 that selectively perturb the Vps75-dependent acetylation
of H3 in vitro and in vivo. Additionally, the results demonstrate
that the Rtt109-Vps75 interfaces revealed with our crystal model
are unique to the functional interaction between Rtt109 and
Vps75, and that activation of Rtt109 by Asf1 is mediated through
a distinct mechanism.
A Model for H3-H4 Dimer Acetylation by Rtt109-Vps75. With a struc-
tural and biochemical model of the Rtt109-Vps75 complex in
hand, we next investigated the relevant histone substrates that
would effectively fit into the cavity formed between Rtt109-
Vps75. From our structural model, the available space for
substrate binding is approximately 27 × 32 Å, which would allow
for the binding of an H3-H4 dimer but not a tetramer. We there-
fore modeled the histone H3-H4 dimer (33) into the Rtt109-
Vps75 structure by positioning Lys56 from an H3-H4 dimer into
the Rtt109 active site and minimizing structural clashes between
the models (Fig. 5). This position is meant to illustrate a possible
arrangement ofthe Rtt109-Vps75-H3-H4 complexand todemon-
strate the capacity of the Rtt109-Vps75 complex to accommodate
the histone dimer. However, this model does not exclude the
potential for structural conformations that could take place upon
substrate binding, which would allow for alternative arrange-
ments of histones.
Histone H3-H4 complexes exist in an equilibrium between
heterotetrameric and heterodimeric forms (34, 35); thus from
previous kinetic analyses, it was unclear if the H3-H4 dimer is
an efficient substrate for Rtt109-Vps75. The ability of Rtt109-
Vps75 to acetylate a dimeric form of H3-H4 was assessed kine-
tically. We compared the steady-state acetylation rates of recon-
stituted histone tetramers versus a variant histone construct,
H3(A110E)-H4, which has been demonstrated to exist in only
dimeric form in solution (34). Substrate saturation curves were
fitted to the Michaelis–Menten equation, revealing kcatvalues
type Rtt109 and the Rtt109 variants are able to rescue genome stability of the
rtt109Δ strain. Yeast strains carrying the indicated RTT109 alleles were plated
on rich media (YPD) containing 8 μM camptothecin (CPT) to indicate resis-
tance to genotoxic stress. Plates were grown at 30 °C for 3 d prior to photo-
graphy. (B) Histone H3 K9, K23, K27, and K56 are the primary sites of
acetylation on H3-H4 tetramers for Rtt109-Vps75, as determined by quanti-
tative MS/MS analysis. (C) H3K56ac is not affected by the point substitutions
in Rtt109; however, H3K9ac and H3K27ac are reduced in the rtt109Δgcn5Δ
strain with the mutant Rtt109 allele for K3 substitutions. Whole-cell alkaline
lysis extracts of the indicated strains were analyzed by immunoblotting,
with the indicated antiacetyl histone antibodies and reprobed with anti-H3
antibody as the loading control.
Variants of Rtt109 do not affect Rtt109-Asf1 activity in vivo. (A) Wild-
www.pnas.org/cgi/doi/10.1073/pnas.1009860107Kolonko et al.
of 0.41 ? 0.02 s−1
1.4 ? 0.4 μM and 2.4 ? 0.7 μM for H3-H4 and H3(A110E)-H4,
respectively (Fig. 5). Thus, both substrates display similar effi-
ciency within the error of the experiment. The results indicate
that the H3-H4 mutant dimer is an efficient substrate and that
Rtt109-Vps75 does not require an obligate H3-H4 tetramer for
acetylation. Collectively, the biochemical evidence along with our
structural model provides compelling evidence that the activated,
high-affinity Rtt109-Vps75 trimer represents a functionally rele-
vant complex that can acetylate an H3-H4 dimer.
and 0.39 ? 0.04 s−1
Rtt109-Vps75 and Rtt109-Asf1 complexes are associated with
discrete biological functions in vivo and catalyze different histone
acetylation reactions. However, the mechanisms by which Vps75
and Asf1 activate Rtt109 and direct lysine specificity are
unknown. Here we investigated the mechanism of activation of
Rtt109 by Vps75 using biochemical, structural, and genetic
approaches. We demonstrated that Rtt109 can be activated
>250-fold by Vps75 via an increase in the rate of acetyl transfer,
which occurs through the stabilization of the catalytically active
form of Rtt109. To gain insight into the specific interactions of
Rtt109 and Vps75, we have produced a molecular replacement
solution of the Rtt109-Vps75 complex. The subunit arrangement
was comprised of a 1∶2 complex of Rtt109-Vps75 that suggested
and chaperone. We validated the two distinct interfaces bio-
chemically and genetically using point substitutions. These struc-
ture-based variants disrupted Vps75-dependent activity and
function but did not adversely affect the Asf1-dependent func-
tions of Rtt109 in vitro and in vivo. Additionally, the structure
revealed a charged cavity capable of acetylating a heterodimer
of H3-H4. By demonstrating that the H3-H4 dimer is an efficient
substrate for Rtt109-Vps75, we provided kinetic evidence for a
model involving a functional Rtt109-Vps75-H3-H4 complex
Several lines of biochemical evidence suggest that interaction
surfaces other than those available in our structural model con-
tribute to the formation of the Rtt109-Vps75 complex. However,
the limited resolution of the structure impedes our ability to mod-
el potential interfaces that are not present in the previously de-
termined Rtt109 and Vps75 structures, which were used as search
models. We demonstrated that although the two electrostatic
contact sites illustrated in the structural model are important
for catalytic activation, they contribute very little to overall bind-
ing affinity (Fig. 3 and Fig. S2B). Though not observed in any
solved structures to date, previous studies have implicated impor-
tant binding regions within residues 130–179 of Rtt109 (16, 36)
and the α8 helix (residues 209–222) of Vps75 (28). Consistent
with this idea, the Rtt109-Vps75 complex is stable to high salt
and requires the use of strong hydrophobic chromatography to
disrupt the native complex (29). Based on these data, we propose
that the high-affinity interaction between Rtt109 and Vps75 is
mediated primarily through these hydrophobic interfaces, likely
involving the unstructured loop of Rtt109 (residues 130–179).
The electrostatic contact surfaces, which we investigated in the
present study, are critical to permit transition to the fully active
conformer of Rtt109 and allow for the optimal positioning of
acetyl CoA and the attacking lysine from histone substrates.
This study represents the biochemical, structural, and genetic
analysis of a HAT-regulatory subunit complex. The activation
mechanism of Rtt109-Vps75 provides a valuable framework for
understanding the molecular regulation of other HATs within
multisubunit complexes. For example, it appears likely that the
Esa1 and Tip60 HATs experience a similar regulatory mechanism
(5, 37). Interaction of Esa1 with two subunits (Epl1 and Yng2),
stimulates the kcatof Esa1 by ∼100-fold, without alteration of
the Kmfor histone substrates (5). Stabilization of the active HAT
conformation might be one critical method of minimizing
spurious activity, allowing the cell to coordinate the timing and
specificity of acetylation by linking transferase activity to the
amount or type of accessory proteins.
Reagents. Reagents were purchased from Sigma-Aldrich, Fisher Scientific, or
RPI unless otherwise noted. [3H]-acetyl CoA (2–25 Ci∕mmol) was obtained
from Morevek, acrylamide/bisacrylamide from Bio-Rad, site-directed muta-
genesis kits from Stratagene, anti-H3K9ac and K27ac from Upstate, and
anti-H3 from Abcam.
Expression and Purification of Proteins. Recombinant untagged Rtt109,
His6-tagged Rtt109, His6-tagged Vps75, His6-tagged Asf1, and Xenopus laevis
and S. cerevisiae histones were expressed in either Rosetta 2 (DE3) pLysS
(Novagen) or BL21-CodonPlus (DE3)-RIPL (Stratagene) competent cells and
purified as described previously (8, 29, 33). Rtt109 and Vps75 concentrations
were determined as previously described (29). Histone concentrations were
determined using extinction coefficients of ε ¼ 4;040 for X. laevis H3 and
ε ¼ 2;560 and 5;120 M−1cm−1for S. cerevisiae H3 and H4, respectively,
which were derived from the method described by Gill and von Hippel
(38). S. cerevisiae H3-H4 oligomers and H3(A110E)-H4 dimers were reconsti-
tuted as previously described (39). Unincorporated H3 and H4 monomers
were removed by purification over a HighLoad 16/60 Superdex 200 prep
grade column (GE Healthcare). Protein concentrations were determined
using an extinction coefficient of ε ¼ 7;680 M−1cm−1.
HATAssays. Reactions were analyzed by filter binding and scintillation count-
ing of products (40). Unless otherwise noted, reaction buffers were com-
prised of 50 mM Tris (pH 7.5 at 25°C) and 1 mM DTT and reaction
contained 40 μM [3H]-acetyl CoA (0.2–0.4 Ci∕mmol). The details of specific
experiments are described in SI Text.
Structure Determination of 6xHisRtt109(1-405)-Vps75(9-223). The details of ex-
pression, purification, crystallization, X-ray diffraction data collection, and
structure determination are described in SI Methods. Briefly, Rtt109(1-405)
and Vps75(9-223) were coexpressed in E. coli using a pET-derived vector con-
taining a 6xHis epitope tag and a pET3a vector, respectively. Recombinant
protein was purified using three chromatographic steps, and crystals were
Vps75-H3-H4 complex. K56 from the histone dimer was manually positioned
in the Rtt109 active site, and structural clashes were minimized between the
models. Kinetic analysis with yH3-H4 tetramer or yH3(A110E)-H4 dimer.
Kinetic constants obtained from substrate saturation curves of three trials
of samples performed in duplicate are listed with standard error.
H3-H4 dimer is a substrate for Rtt109-Vps75. Model of the Rtt109-
Kolonko et al.PNAS
November 23, 2010
generated in a hanging drop vapor diffusion crystallization experiment. Download full-text
Diffraction data were indexed and scaled using HKL2000 (41). Molecular
replacement was carried out using Phaser (42) with the previously deter-
mined Rtt109 structure (16) and a monomer Vps75 structure (8) as search
Mass Spectrometry Quantitation Assay. Enzymatic acetylation sites were
determined using chemical acetylation combined with MS/MS peptide se-
quencing as described previously (43). The details of specific experiments
are described in SI Text.
Yeast Assays. The details of yeast strains are described in SI Text. For immu-
noblotting experiments, alkaline whole-cell extracts were prepared from
2 mL of cells grown to an OD600of 0.8 in YPD or Leu media (44). The final
volume of the extracts was 30 μL, from which 2–10 μL was loaded onto 17%
SDS-PAGE for Western blot analysis.
ACKNOWLEDGMENTS. We thank K. Luger (Colorado State University, Fort
Collins, CO)for generously supplying histone plasmids. We thank all members
in the research group of John Denu for helpful discussions. We also acknowl-
edge the University of Wisconsin–Madison Human Proteomics Program,
funded by the Wisconsin Partnership Fund. This work was supported by
National Institutes of Health Grants GM059785 (to J.M.D.) and GM055712
(to P.D.K.) and a postdoctoral fellowship (to E.M.K.) from the American Heart
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