Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities

Article abstract of DOI:10.1002/pro.3359

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10 lowers the rate of chemical acetylation of Lys-9, Lys-14, and Lys-18 by methyl acetyl phosphate in that order consistent with a higher pK_a of these Lys residues induced by phosphorylation; basicities increase up to 3 pK_a units as a function of distance from Ser-10 phosphate. Enzymic acetylation of Lys residues with high pK_a values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser-10, the amines of Lys-9, Lys-14, and Lys-18 have lowered pK_a values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9–10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate–Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.

Full text of DOI:10.1002/pro.3359

Article
Protein Science
DOI 10.1002/pro.3359
Contributions to Nucleosome Dynamics in Chromatin from
Interactive Propagation of Phosphorylation/Acetylation
and Inducible Histone Lysine Basicities
Lois R. Manning and James M. Manning
Department of Biology
Northeastern University
Boston, MA 02115
Corresponding Author: J.M. Manning
Department of Biology
Mugar Life Sciences Building
Room 134
Northeastern University
3
60 Huntington Ave.
Boston, MA 02115
Phone: 617-373-5267
Email:
j.manning@northeastern.edu
Supported in parts by NIH grants HL-15157, HL-18819 and HL-58512
Running Title: Inductive Post-translational Modifications in Histone H3
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/pro.3359
©
2017 The Protein Society
Received: Nov 09, 2017; Revised: Dec 03, 2017; Accepted: Dec 06, 2017
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Page 2 of 22
Abstract
The effect of phosphorylation on the basicities of amines in histone H3 peptides and their
acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10
lowers the rate of chemical acetylation of Lys-9, Lys-14 and Lys-18 by methyl acetyl phosphate in
that order consistent with a higher pK of these Lys residues induced by phosphorylation; basicities
a
increase up to 3 pKa units as a function of distance from Ser-10 phosphate. Enzymic acetylation
of Lys residues with high pKa values in nucleosomes is also expected to be enhanced by
phosphorylation, consistent with the known mechanism involving binding of protonated amines to
N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site,
its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a
phosphate on Ser-10, the amines of Lys-9, Lys-14 and Lys-18 have lowered pK values. Chemical
a
acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but
the phosphate inductive effect in histone H3 is more potent since the linkage between
phosphorylation and acetylation is propagated with a range extending 9-10 amino acids in either
direction from the phosphorylation site enhancing protonation of amino groups. We conclude that
lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic
and immediate interaction between phosphorylation/acetylation that may contribute to inactive
heterochromatin by compaction through such Ser phosphate--Lys amine electrostatic interactions
and their relaxation by acetylation in euchromatin.
Abbreviations: MAP: methyl acetyl phosphate; Ac: acetyl; Hb: hemoglobin;
-2-
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Protein Science
Introduction
A variety of agents that induce cellular proliferation such as growth factors, for example,
stimulate protein kinases that phosphorylate Ser and Tyr residues on many proteins having
1,2
important biological roles including those in chromatin.
Post-translational modifications in
3
histones include not only acetylation of Lys residues first reported by Allfrey and phosphorylation
4,5
of Ser residues but also methylation of Lys and Arg residues as well as ubiquitination of Lys
residues. These multiple side chain modifications in histones, most of which are on the N-terminal
tails, either promote or repress DNA transcription by somehow altering the constraints within the
4,5
nucleosome structure.
Some current models view these modifications as general types of
6,7
markers that create binding sites for proteins that influence chromatin. Histone H3 N-terminal
tails have received much attention regarding phosphorylation/acetylation; lysine-14 of histone H3 is
an acetylation site and serine 10 is a phosphorylation site. These modifications may be related
8
since the presence of a phosphate on Ser-10 led to enhanced enzymic acetylation of Lys-14.
9
Studies on histone H4 indicate a pattern of diacetylation that may also indicate a linkage
relationship. Our results described here show a relationship between phosphorylation and
acetylation propagated over a significant distance and we describe a mechanism whereby this is
accomplished. The term epigenetic has been used to describe these histone modifications since
they represent another level of regulating transcription superimposed on the DNA sequence
10
itself. The results here describe an interactive and inducible aspect of epigenetics.
The proximity of these modified sites to one another presented an opportunity to determine
in synthetic peptides corresponding to segments of the N-terminal tails of histone H3 if these
modifications are causally related in some circumstances, e.g. whether or not a particular enzyme
activity is present, and how such modifications might potentially affect nucleosome dynamics on a
larger scale. The uniform nature of each peptide in contrast to nucleosome mixtures with varying
degrees of modifications permits unequivocal conclusions about the effects of a modification at
one particular site on a different location subject to another modification. The unstructured histone
H3 tail is attached at Gly-44 to the folded core segment of the rest of the histone which terminates
at residue 135. Each of the 4 types of core histones has similar features with a long unstructured
flexible N-terminal tail attached to the rest of the folded histone. The nucleosome contains a pair of
4,5
each core histone referred to as the octamer around which DNA is wrapped.
In this report, we
show that the non-enzymic chemical acetylation rates of Lys-9, Lys-14 and Lys-18 are influenced
by the presence of a phosphate on Ser-10, thus mimicking enzymic acetylation/phosphorylation of
8
histone H3. We employ an acetylating agent, methyl acetyl phosphate (MAP), that we used
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11,12
previously to reveal those amino groups of hemoglobin that specifically bind chloride ion.
This
13,14
reagent, which was introduced by Kluger and co-workers,
is a mild and stable acetylating
agent. Under our experimental conditions, the acetylation is relatively slow so that accurate kinetic
studies can be performed. At neutral pH, one of the oxygens attached to its phosphate is
monoanionic so, as our results below indicate, it first binds to a basic peptide and the acetyl group
then attacks the unprotonated amine nucleophile.
Results and Discussion
Kinetics of Acetylation of Histone H3 Peptides - Amino acid analysis and mass
spectrometric data for the synthetic peptides as well as their sequences and locations
corresponding to segments of the N-terminal tails of histone H3 are given in Table 1. The ε-amine
of the Lys in bold face for the peptides is the only available MAP acetylation site. For example, in
the peptides where Lys-14 acetylation kinetics was studied, the ε-amine of Lys-9 was first
completely blocked during synthesis (AcK). Likewise, in the peptides where Lys-18 acetylation
was evaluated, both Lys-9 and Lys-14 ε-amines were first blocked during synthesis. In all cases
the N-terminal Ala was acetylated during synthesis. The kinetic profiles of acetylation by MAP
follow a concentration-dependent pattern (Fig. 1) consistent with a peptide • MAP complex formed
reversibly, followed by irreversible acetylation.
Peptide + MAP ⇄ [Peptide • MAP] → Acetylated Peptide
14
Such a mechanism is consistent with that suggested by Kluger and De Stefano involving a
complex between a protonated amine and the MAP anion followed by proton transfer to generate
the unprotonated amine. Since the unprotonated amine is the reactive species with MAP, the
higher its concentration the faster the acetylation rate, i.e. at pH 7.5 amines with low pK values
a
react faster than those with high pK values. The pK values of the ε-amine groups of Lys residues
a
a
in the N-terminal tails of histones have been assumed to be in the 9-10 range but, in fact, they may
15
be different due to electrostatic/inductive effects of neighboring positively-charged groups as
described more fully below.
Effects of Phosphorylation at Ser-10 on Acetylation Rates- The results in Figure 1 show
a family of straight lines typically found for each peptide studied although the reaction times and
MAP concentrations required (and hence the slopes) were different reflecting the efficiency of
acetylation of a particular peptide. Plots of the acetylation rate of histone H3 peptides containing
free Lys-9, Lys-14 or Lys-18 and with either free or phosphorylated Ser-10 vs MAP concentration
are shown in Figures 2(A), 3(A), and 4(A), respectively. For each peptide, the data fit a hyperbolic
saturation profile consistent with initial formation of a peptide • MAP complex described above.
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Protein Science
There is a highly significant difference in the acetylation of Lys-9, Lys-14 or Lys-18 of histone H-3
depending whether or not a phosphate is present on Ser-10. The effect of phosphate at Ser-10 is
more readily observed in the double reciprocal plots from which quantitative data can be calculated
as described below.
Double reciprocal plots of the hyperbolic profiles in Figures 2(A), 3(A), and 4(A) permit an
accurate assessment of kinetic constants for the effect of Ser-10 phosphorylation on Lys-9, Lys-14
and Lys-18 acetylation, respectively, as shown in the B panels of these figures. Analogous to a
Lineweaver-Burk double reciprocal plot for an enzyme-catalyzed reaction, the intercept on the
negative x-axis gives a value for MAP complex with each peptide which is referred to a K . The
b
higher the K value, the slower the acetylation rate with MAP meaning that there is less of the
b
unprotonated reactive form of the amine and more of the protonated form. The y-intercept provides
the maximum acetylation rate at infinite MAP concentration. The slope of each line (K /Vmax)
b
represents the efficiency of the acetylation of Lys-9, Lys-14 and Lys-18 when Ser-10 is
phosphorylated or not. The acetylation kinetics, i.e., the distance between the pairs of straight lines
in the B panels converge with each other in the order Lys-9, Lys-14, and Lys-18 due to decreasing
slopes for phosphorylated peptides and increasing rates for unphosphorylated peptides.
The kinetic constants of acetylation of the phosphorylated and the unphosphorylated
peptides corresponding to residues 7-13, 7-15 and 7-19 containing free ε-amino groups at Lys-9,
Lys-14 and Lys-18, respectively, are shown in Table 2. The maximal rates of acetylation of Lys-9,
Lys-14 and Lys-18 at infinite MAP concentration, as determined from the y- intercepts, are very
similar whether or not Ser-10 is phosphorylated. The major effect of phosphorylation of Ser-10 is
an increased Kb value in the order Lys-9 > Lys-14 > Lys-18. The acetylation rates for all peptides
either with or without a phosphate at Ser-10 are not proportional to MAP concentration as shown
by the hyperbolic nature of the profiles in Figures 2(A), 3(A) and 4(A), indicative of formation of a
complex. Thus, the possibility that the phosphorylated peptide repelled the MAP reagent appears
unlikely since, if that were so, low MAP concentrations would have been proportionally less
effective than high concentrations, which is opposite to the observed behavior.
Chemical vs Enzymic Acetylation - Since the reactive species in the chemical acetylation
by MAP at pH 7.5 is the unprotonated amine, higher Kb values reflect more of the protonated form
(
high pK ) for any given Lys acetylation site. For the three unphosphorylated peptides, the Kb
a
values (Table 2) increase moderately in the order Lys-9, Lys-14 and Lys-18, i.e., more MAP is
required for acetylation suggesting intrinsically lower pK values for those closer to the N-terminus.
a
However, when Ser-10 is phosphorylated, the results indicate the opposite, i.e., higher K values
b
from the phosphorylation site (Ser-10) indicating that acetylation is diminished due to pKa
increases in the order Lys-9 > Lys-14 > Lys-18. Hence, this inductive/electrostatic effect is present
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when the phosphate group is either immediately adjacent to the amine (Lys-9) or at a distance
Lys-14 and Lys-18), and it acts both in the N-terminal and C-terminal directions.
(
16
The mechanism of enzymic acetylation involves a protonated amine binding to the N-
acetyltransferase active site followed by catalytic proton removal to generate the reactive
unprotonated species. Therefore, a higher pK for Lys 9 (or any other amine) would be expected
a
to favor enzymic acetylation (depending also on other parts of the sequence required for binding)
leading to increased acetylation. However, chemical acetylation by MAP would be decreased,
consistent with less of the unprotonated amine.
Electrostatic Effects in Amino Acids and Their Neutralization - The above conclusions
on the effects of Ser-10 phosphorylation are completely consistent with the profiles of the chemical
acetylation of glycine and glycinamide by MAP [Fig. 5(A), 5(B)], where electrostatic effects
15
affecting pK values are well known.
The shapes and amplitudes of the hyperbolic saturation
a
profiles as well as the double reciprocal plots for acetylation of glycine and glycinamide are
qualitatively similar to those observed for the Lys-9, Lys-14 and Lys-18 in phosphorylated and
unphosphorylated histone H3 peptides although the absolute values vary (note difference in
intercept values). The well known effect of the negatively-charged α-carboxylate of glycine on its
positively-charged α-amino group (pK = 9.7) is abrogated when the α-carboxylate charge is
a
17
neutralized by amidation in glycinamide (pK = 7.7). Thus, the experimental design to use MAP to
a
measure the inductive effect of phosphorylation is validated.
pKa Effects on Acetylation and Tetramer Weakening in Hemoglobins- Fetal
hemoglobin F is an example of the importance of electrostatic interactions in protein networks (also
18
referred to as “salt bridges”) and its weakening by acetylation. Thus, natural human fetal
hemoglobin (Hb F ) has an especially strong electrostatic network involving the positively-charged
0
19,20
N-terminal Gly of its γ-subunit, which has a pKa of 8.1.
This network contributes to the 70-fold
increased tetramer stability of Hb F compared to adult Hb A, which has Val at that position with a
19,20
pKa of 7.1.
This N-terminal Val in Hb A has significantly reduced net charge and is not
acetylated but the corresponding positively charged Gly found in fetal hemoglobin can be
acetylated, which very significantly destabilizes this electrostatic network. Consequently, acetylated
20
Hb F stability is nearly the same as that for Hb A. These conclusions correlating pKa values,
1
acetylation, and weakening of subunit interactions in Hb F were confirmed in recombinant
hemoglobins where Ala was substituted for Val at either or both N-terminal sites of Hb A subunits
21
thereby conferring increased positive charge and the ability to be acetylated. Described below is
a model for ε Lys pKa changes and acetylation susceptibility in histones and nucleosome
condensation/decondensation based on these same principles.
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Protein Science
Estimates of Increased Lysine Basicity Induced by Phosphorylation- The K value for
b
each amine group represents the efficiency with which it is acetylated by MAP and is a function of
17
its pK value. From the known pK values for glycine and glycinamide (9.7 and 7.7, respectively )
a
a
and their K values as calculated from Figure 5(B) and listed in Table 2, one can approximate pKa
b
values for the H3 peptide Lys groups since their Kb values are known (Table 2). These estimates
shown in Table 2 for their unphosphorylated and phosphorylated forms, respectively, are 7.7 and
1
0.8 for Lys-9, 7.8 and 9.1 for the Lys-14 peptides, and 8.0 and 8.2 for the Lys-18 peptides. These
results indicate that the effect of the phosphate at Ser-10 is to raise the pK of adjacent Lys amines
a
[
analogous to the effect of the carboxyl group of glycine in increasing its pKa (Fig. 5, Table 2)].
Thus, their chemical acetylation by MAP would be hindered and enzymic acetylation would be
expected to be enhanced due to increased basicity of Lys in the phosophorylated peptides making
8
them better substrates for the N-acetyltransferase. This conclusion is supported by a report
comparing the relative efficiencies of enzymic acetylation at Lys-14 for phosphorylated and
unphosphorylated peptides with H3 sequences; in that study the phosphate group binds to the
enzyme but our results are without enzyme. The 3-order magnitude pKa increase found for the
Lys-9 peptide is an especially large enhancement and represents the potency of Ser-10
phosphorylation by itself. The 1.3 pKa increase for Lys-14 when Ser-10 is phosphorylated attests
to the relatively large range of the electrostatic effect.
The pKa values of the lysine amines in unphosphorylated histone tails are sometimes
assumed to be in the 9-10 range based on known values for free lysine although this may not be
an accurate analogy. Indeed, our pKa estimates of the unmodified Lys amines (Table 2) are
significantly lower due likely to the inductive effect of neighboring Arg side chains. Their pKa range
of 7.7-8.0 means they would carry a significantly reduced net positive charge and be less favorable
substrates for N-acetyltransferases.
A Role for pKa Changes in Hyper- and Hypoacetylation- The shifts towards higher pKa
noted above in H3 peptides occur immediately upon phosphorylation and provide a possible
explanation for the rapid increased acetylation of histone H3 in nucleosomes upon phosphorylation
22
of Ser-10 in histone H3. The terms hyperacetylated and hypoacetylated are loosely defined
states of transcriptionally active euchromatin and transcriptionally inactive heterochromatin,
4,5
respectively, with no known clear demarcation between them. Since our results show a strong
correlation between pKa values and the degree of acetylation for susceptible sites, perhaps the
low pKa estimated values in Table 2 for the Lys sites in unphosphorylated histones and therefore
expected low levels of enzymic acetylation would be equivalent to hypoacetylated nucleosomes.
Enhanced and rapid increases of acetylation upon phosphorylation due to the inductive effect
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resulting in higher pKa values reported here could form the basis for hyperacetylated nucleosomes
in transcriptionally active euchromatin.
Propagation of Interactive Histone Modifications and Implications for Chromatin
Condensation / Decondensation - The peptides studied here represent segments of the N-
terminal tail of histone H3. In the nucleosome, the N-terminal tails of the histones extend into
23
adjacent nucleosomes but their structures are not well defined. Our results demonstrate that the
effects of one type of histone modification can directly influence other modification sites at a
considerable distance, i.e., the effect is propagated. Thus, uncharged Ser-OH assumes a negative
charge upon phosphorylation and, as a result, an increased positive charge is induced in nearby
Lys residues. For histone H3, there are two phosphorylation sites (Ser-10 and Ser-28) which are
6
1
8 amino acids apart on its N-terminal tails. Four of the five acetylation sites on these N-terminal
tails (Lys-14, Lys-18, Lys-23 and Lys-27) are between the Ser-10 and Ser-28 phosphorylation sites
and the fifth site, Lys-9, which can also be methylated, is immediately adjacent to Ser-10. When
the K values in Table 2 are plotted vs distance from the Ser-10 phosphorylation site, a straight line
b
is found (Fig. 6). The K value for the Lys-9, Lys-14 and Lys-18 peptides containing Ser-10-
b
phosphate all fall on this line. The plot shows that the potency extends 9-10 residues from the
phosphorylation site. Since the findings for acetylation of Lys-9 and of Lys-14 indicate that the
propagation acts in both directions, the full effective range is estimated to be about 18-20 amino
acid residues, which is close to the distance between the two Ser-10 and Ser-28 phosphorylation
sites in histone H3. Perhaps the divalent anionic nature of the phosphate groups on Ser-10 and
Ser-28 with the negatively-charged oxygens opposite one another promoting enhanced
protonation of adjacent Lys residues is responsible for this bidirectional propagation effect (see
Fig. 6).
SerOP---Lys Electrostatic Pairings in Transcription vs Mitosis / Meiosis-- Nucleosome
clusters undergo a dynamic and reversible process of condensation as in transcriptionally inactive
heterochromatin and decondensation as in transcriptionally active euchromatin. The core histone
N-terminal tails intercalate between adjacent nucleosomes and are involved in the condensation /
23-25
decondensation process
although the mechanism is unknown. The large range of the
propagation effect described here for histone H3 peptides and the enhanced Lys protonation
favoring it as an N-acetyltransferase substrate suggests a possible role in transcription. Upon Lys
acetylation in gene expression the charge network would be disrupted, as described above for
Hb F , and decondensation as in transcriptionally active euchromatin could occur; this model is
1
consistent with the N-acetyltransferase activity of some transcription factors. On the other hand,
the magnitude and bi-directionality of this inductive effect operating through the Ser-10
---
phosphorylation may contribute to nucleosome condensation in heterochromatin when SerOP Lys
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Protein Science
sites are fully charged and the acetylating systems are absent as shown at the bottom of Figure 6
resulting in a hinge-like property to bring different parts of the tails and their attached nucleosomes
together representing a mechanism for nucleosome condensation as in mitosis / meiosis; the
importance of phosphorylation of histones in this and other nuclear events besides gene
26
transcription is well known. Multiple phosphorylations would be expected to enhance such
compaction. Methylation of Lys-9 of histone H3 (Fig. 6, bottom) correlates with gene
silencing/condensation consistent with a stabilization of the electrostatic interaction of methylated
Lys-9 with Ser-10-phosphate and the preclusion of acetylation at Lys-9 once methylated.
All core histone N-terminal tails as well as the H1 and H5 linker histones have Ser---Lys
separated pairings similar to those in histone H3 subject to phosphorylation and acetylation.
Hence, multiple phosphorylated Ser hinge points would be amplified when the core histones act in
concert brought about by electrostatic charges as a basis for nucleosome compaction in the
absence of acetylation and relaxation upon acetylation resembling an accordion-like action.
Similary, outward-facing DNA phosphates of the nucleosome could also partner with some histone
lysines with induced high pKa values to create a similar mechanism that would be abrogated by
acetylation.
Materials and Methods
Materials
16
Methyl acetyl phosphate (MAP) was synthesized according to procedures of Kluger and
13,14
colleagues.
O
৷
O
৷
CH – C – O –P – OCH
3
3
|
O-
11
The identity of the product was established by NMR and elemental analysis. MAP is stored dry
at -30°; fresh solutions are made immediately prior to use. MAP is completely stable during the
reaction time described here (30 min), consistent with the linearity of the kinetics. Synthetic
peptides corresponding to partial histone H3 sequences were prepared at Rockefeller University
Protein/DNA Technology Center. Each peptide, which gave a single peak by HPLC and had the
expected mass and amino acid composition (see Table 1), was first dissolved in H O. To this
2
solution, 1 mM Hepes was added and the final pH was adjusted to ~ 7 with NaOH. Peptide
concentrations were determined by amino acid analysis on a Beckmann 6300 instrument and also
27
by the Fluram fluorescamine reagent (Roche or Sigma), as described below. There was good
agreement between these measurements.
-10-
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Methods
Acetylation of peptides (0.2-0.3 mM concentration determined by amino acid analysis or
Fluram) with varying MAP (10-100 mM) was performed at 25° in 10 mM Hepes buffer, pH 7.5 (final
pH of the reaction mixture, which did not change significantly during the 30 min reaction). This pH
was chosen so that the concentration of protonated amine would be much higher than that of the
unprotonated species thus permitting formation of its complex with anionic MAP (see below). The
total concentration of free amino groups in each peptide was determined with Fluram against a
standard of L-alanine and used as the 100% value at time zero. In this assay a sample of the
reaction mixture containing 0-100 nmoles of amino groups (50 ꢀl) was added to 0.5 ml of 0.5M
sodium borate pH 8.0 and mixed with a 0.5 ml solution of freshly-prepared 0.025% Fluram in HPLC
grade acetone using a vortex mixer. After 10 minutes at room temperature, 1.5 ml H O was then
2
added while mixing thoroughly. Fluorescence due to the amine-fluorescamine adduct was
recorded on a Shimadzu RF-5301 spectrofluorimeter with the excitation wavelength set at 390 nm
and the emission wavelength at 480 nm.
To start the acetylation reaction, a freshly prepared MAP solution was added to the peptide.
Aliquots were removed at selected time intervals and the concentration of free amino groups
remaining was measured with the Fluram reagent. For each peptide, a family of straight lines was
obtained on a semi-logarithmic plot of percent amine remaining as a function of time using
varying MAP concentrations (10 mM to 100 mM) such as shown in Figure 1. For secondary plots,
the amount of free amine remaining was subtracted from the initial amount to determine the
amount acetylated; the rate of acetylation was then calculated and plotted vs the MAP
concentrations used. For all peptides, a hyperbolic profile was found (see A panels of Fig. 2-4). A
reciprocal plot of these data generated straight lines (see B panels of Fig. 2-4) from whose x and y
intercepts kinetic constants were calculated.
Acknowledgements – We thank W.T. Jenkins and R. Kluger for their suggestions.
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Ashiuchi M, Yagami T, Willey RJ, Padovan JC, Chait BT, Popowicz A, Manning LR,
Manning JM (2005) N-terminal acetylation and protonation of individual hemoglobin
subunits: Position-dependent effects on tetramer strength and cooperativity. Protein Sci
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4:1458-1471.
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Clayton AL, Hazzalin CA, Mahadevan LC (2006) Enhanced histone acetylation and
transcription: A dynamic perspective. Mol Cell 23:289-296.
Davey C, Sargent DF, Luger K, Maeder A, Richmond TJ (2002) Solvent mediated
interactions in the structure of the nucleosome core particle at 1.9A resolution. J Mol Biol
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W, Kurumizaka H (YEAR) Contribution of histone N-terminal tails to the structure and
stability of nucleosomes. FEBS Open Bio 3:363-369.
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7.
Rossetto D, Avvakumov N, Cote J (2012) Histone phosphorylation. A chromatin modification
involved in diverse nuclear events. Epigenetics 7:1098-1108.
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amines in the picomole range. Science 178:181-872.
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Protein Science
Table 1
Peptide Characterization
1
2
3
Peptide
Histone H3 Sequence
(by synthesis)
Amino Acids
Mass
Mass
Designation
(by amino acid analysis) (expected) (found)
Lys-9-NH /Ser-10-OP
K
R
T
.8
.9
.8
S
G
A
.5
1.8
[1.0]
797.3
718.4
797.1
717.2
2
AcARKSTGG (7-13)
|
P
Lys-9-NH /Ser-10-OH
K
R
T
.8
.9
.8
S
G
A
.7
1.8
[1.0]
2
AcARKSTGG (7-13)
Ac
|
Lys-14-NH /Ser-10-OP
K
R
T
1.6
.7
.8
S
G
A
.5
1.8
[2.0]
1038.5
959.5
1037.7
2
AcARKSTGGKA (7-15)
|
P
Ac
|
Lys-14-NH /Ser-10-OH
K
R
T
1.7
.9
.8
S
G
A
.7
1.8
[2.0]
957.9
2
AcARKSTGGKA (7-15)
Ac
Ac
|
|
Lys-18-NH2/Ser-10-OP
Lys-18-NH2/Ser-10-OH
K [3.0]
S
E
P
.7 1589.8
1590.7
AcARKSTGGKAPRKQ (7-19)
R
T
G
1.3
.9
2.4
.3
1.4
2.6
|
P
A
Ac
|
Ac
|
AcARKSTGGKAPRKQ (7-19)
K [3.0]
S
E
P
.8 1510.9
1514.2
4
R
T
G
1.8
.8
2.2
1.2
1.3
2.5
A
Glycine
Glycinamide
75.1
74.1
76.3
75.3
1
.
.
Each peptide was amidated on its COOH-terminus and its N-terminus was completely acetylated.
Lys residues in bold contain free – NH2 groups and are the site of acetylation by MAP. Bold P indicates
phosphate. The sequence numbers are given in parenthesis.
The values in brackets indicate the amount of that amino acid found after acid hydrolysis, which was set
to the theoretical value; all other values are expressed relative to it.
Glutamic acid (E) arises from glutamine (Q) upon acid hydrolysis.
2
3
4
.
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Table 2
Kinetic Constants
a
.b
c
Compound
Kb
mM)
Vmax
K /V
NH2
b
max
-1
-1
(
(min )
(mM min ) pKa
(estimate)
Lys-9-NH /Ser-10-OP
243.9
27.2
30.4
27.8
22.8
25.9
8.02
0.98
5.29
1.25
10.8
2
Lys-9-NH /Ser-10-OH
7.7
9.1
7.8
2
Lys-14-NH /Ser-10-OP
120.5
32.4
2
Lys-14-NH /Ser-10-OH
2
Lys-18-NH /Ser-10-OP
65.8
43.1
22.7
26.5
2.90
1.63
8.2
8.0
2
Lys-18-NH /Ser-10-OH
2
pKa
9.7
d
Glycine
163.9
26.8
57.8
70.4
2.84
0.38
e
Glycinamide
7.7
a
.
.
.
Reciprocal of x-axis intercept of double reciprocal plots.
Reciprocal of y-axis intercept of double reciprocal plots.
b
c
Slope of line in double reciprocal plots.
d
17
.
.
pK value of amine is 9.7.
a
e
15
pK value of amine is estimated to be 7.7 by analogy with the value for the methyl ester.
a
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Protein Science
Figure Legends
Figure 1. Relationships of Acetylation Rate to MAP Concentration. The experimental conditions
are given in the text. Each peptide studied as well as glycine and glycinamide gave a similar
family of straight lines although different slopes were found and different MAP concentrations were
used depending on the reactivity of a given amine. The log plots for the acetylation of Lys-14 in
the unphosphorylated peptide (Fig. 3) are shown here.
Figure 2. The Effect of Phosphorylation of Ser-10 on Acetylation of Lys-9 in a Histone H-3
Peptide. The experimental conditions are given in the text. Panel A: the acetylation rate was
calculated as the amount of amine reacted at varying MAP concentrations. Panel B: the
reciprocals of the points in Panel A were plotted. The values for the x- and y-intercepts and the
slopes are given in Table 2.
Figure 3. The Effects of Phosphorylation of Ser-10 on Acetylation of Lys-14 in a Histone H3
Peptide. The experimental details were like those in the legend to Figure 2 except that a different
peptide was used (see text and Table 1).
Figure 4. The Effects of Phosphorylation of Ser-10 on Acetylation of Lys-18 in a Histone H3
Peptide. The experimental details are like those in the legend to Figure 2 except that a different
peptide was used (see text and Table 1).
Figure 5. The Acetylation of Glycine and Glycinamide by Methyl Acetyl Phosphate. In this
experiment the effect of the carboxyl group of glycine mimicks that of the phosphate group on Ser-
1
0 described in Figures 2, 3 and 4.
Figure 6. Propagation of the Phosphorylation Effect. (Top) The binding constants (K ) in Table 2
b
were plotted against their distance from the Ser-10 phosphorylation site. The K value for the Lys-
b
9
peptide was assumed to be the same on either side of the Ser-10 phosphorylation site and this
value (Table 2) was set at 1 on the x axis (after subtraction of the average basal K values for the
b
unphosphorylated Lys-9, Lys-14 and Lys-18 peptides in Table 2). The K values for the Lys-14
b
and Lys-18 were assigned as 4 and 8 residues, respectively. (Bottom) The locations of the 5 Lys
residues that are capable of being acetylated in histone H3 are shown. Lys-9 is also known to be
a methylation site. The solid arrows represent experimentally determined values, the dashed lines
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Page 16 of 22
were not measured. The formal negative and positive charges are shown above the oxygen atoms
and the nitrogen atoms, respectively
-18-
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Protein Science
Figure 1. Relationships of Acetylation Rate to MAP Concentration. The experimental conditions are given in
the text. Each peptide studied as well as glycine and glycinamide gave a similar family of straight lines
although different slopes were found and different MAP concentrations were used depending on the
reactivity of a given amine. The log plots for the acetylation of Lys-14 in the unphosphorylated peptide (Fig.
3) are shown here.
114x93mm (300 x 300 DPI)
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Figure 2. The Effect of Phosphorylation of Ser-10 on Acetylation of Lys-9 in a Histone H-3 Peptide. The
experimental conditions are given in the text. Panel A: the acetylation rate was calculated as the amount of
amine reacted at varying MAP concentrations. Panel B: the reciprocals of the points in Panel A were
plotted. The values for the x- and y-intercepts and the slopes are given in Table 2.
266x662mm (300 x 300 DPI)
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Protein Science
Figure 3. The Effects of Phosphorylation of Ser-10 on Acetylation of Lys-14 in a Histone H3 Peptide. The
experimental details were like those in the legend to Figure 2 except that a different peptide was used (see
text and Table 1).
266x658mm (300 x 300 DPI)
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Figure 4. The Effects of Phosphorylation of Ser-10 on Acetylation of Lys-18 in a Histone H3 Peptide. The
experimental details are like those in the legend to Figure 2 except that a different peptide was used (see
text and Table 1).
267x646mm (300 x 300 DPI)
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Protein Science
Figure 5. The Acetylation of Glycine and Glycinamide by Methyl Acetyl Phosphate. In this experiment the
effect of the carboxyl group of glycine mimicks that of the phosphate group on Ser-10 described in Figures
2, 3 and 4.
265x682mm (300 x 300 DPI)
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Figure 6. Propagation of the Phosphorylation Effect. (Top) The binding constants (Kb) in Table 2 were
plotted against their distance from the Ser-10 phosphorylation site. The Kb value for the Lys-9 peptide was
assumed to be the same on either side of the Ser-10 phosphorylation site and this value (Table 2) was set
at 1 on the x axis (after subtraction of the average basal Kb values for the unphosphorylated Lys-9, Lys-14
and Lys-18 peptides in Table 2). The Kb values for the Lys-14 and Lys-18 were assigned as 4 and 8
residues, respectively. (Bottom) The locations of the 5 Lys residues that are capable of being acetylated in
histone H3 are shown. Lys-9 is also known to be a methylation site. The solid arrows represent
experimentally determined values, the dashed lines were not measured. The formal negative and positive
charges are shown above the oxygen atoms and the nitrogen atoms, respectively.
183x230mm (300 x 300 DPI)
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