A small molecule receptor that selectively recognizes trimethyl lysine in a histone peptide with native protein-like affinity

Article abstract of DOI:10.1039/c000255k

A small molecule receptor that mimics the HP1 chromodomain's affinity for trimethyl lysine has been identified from a dynamic combinatorial library. Discrimination for trimethyl lysine over the lower methylation states parallels that of the native protein receptor. These studies demonstrate the feasibility of small molecule receptors as potential sensors for protein posttranslational modifications. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c000255k

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A small molecule receptor that selectively recognizes trimethyl lysine
in a histone peptide with native protein-like affinityw
Lindsey A. Ingerman,a Matthew E. Cuellarb and Marcey L. Waters*a
Received (in Austin, TX, USA) 7th January 2010, Accepted 27th January 2010
First published as an Advance Article on the web 12th February 2010
DOI: 10.1039/c000255k
A small molecule receptor that mimics the HP1 chromodomain’s
affinity for trimethyl lysine has been identified from a dynamic
combinatorial library. Discrimination for trimethyl lysine over
the lower methylation states parallels that of the native protein
receptor. These studies demonstrate the feasibility of small
molecule receptors as potential sensors for protein post-
translational modifications.
groove that forms additional sequence specific interactions
with the adjacent residues of the histone tail.4
Typically antibodies are used to identify PTMs, but these
have some inherent limitations; for example, antibodies
cannot be used to identify unknown PTMs due to their
sequence specificity.5 Recently, DNA aptamers have been used
to develop protein affinity reagents for acylated lysine PTMs,
emphasizing the need for new molecular tools for the recognition
of PTMs.6 The development of small molecules which mimic
Understanding the role of post-translationally modified
histone proteins in gene expression is crucial to the study of both
development and disease, and cancer in particular.1 Many
post-translational modifications (PTMs) have been shown to
function by recruiting non-histone proteins to chromatin,
dictating the higher-order chromatin structure in which
DNA is packaged, and resulting in gene expression or gene
silencing depending on the type and location of the PTM.2
One such covalent modification is the site-specific methylation
of Lys, which can be mono-, di-, or tri-methylated. Depending
on the position of the Lys residue and the degree of methyl-
ation, different proteins are recruited, resulting in variable
transcriptional outcomes.3 A significant protein–protein inter-
action induced by Lys methylation is the binding of histone 3
(H3) K9Me3 to the HP1 chromodomain (Fig. 1a), which
results in gene silencing. This binding event is the result of
the recognition of the trimethylammonium in an aromatic
cage via cation–p interactions, as well as an extended surface
chromodomains is
a field which has remained largely
unexplored.7 Synthetic receptors have some significant
potential advantages, including better reproducibility, lower
cost, lower molecular weight, and the possibility of being used
within cells. We report here the use of dynamic combinatorial
chemistry (DCC) to identify synthetic receptors for trimethyl
lysine (KMe3) that bind with comparable affinities and
selectivities as the native HP1 chromodomain.4
DCC allows the formation of a library of hosts under
equilibrium conditions.8 In the presence of a molecular target
such as methylated lysine, favorable host–guest binding inter-
actions drive the synthesis and amplification of the best
receptor(s) at the expense of other oligomers. By screening
the same library against all Lys methylation states, this
method provides a rapid approach to screen for selectivity as
well. A range of building blocks (Fig. 2) were investigated.
These monomers combine aromatic groups to facilitate
cation–p interactions, carboxylates for water solubility, and
thiols which can undergo disulfide exchange to establish an
equilibrium. Disulfide exchange is compatible with most
protein functional groups in aqueous solution at close to
neutral pH.9 A and B have been shown previously to form
receptors that interact favorably with alkylated ammonium
groups.10z
Dynamic combinatorial libraries (DCLs) were prepared by
mixing equimolar amounts of 3–4 building blocks in water at
pH 8.5. Upon reaching equilibrium, the resulting DCLs
were analyzed by LC–MS. The introduction of the dipeptide
Ac-KMe3-G-NH2 as a template resulted in significant changes
Fig. 1 (a) Structure of the HP1 chromodomain bound to the H3
K9Me3 peptide (green) with the aromatic pocket shown in blue
(pdb: 1KNE). (b) Structure of rac-A2B and rac-A2C.
a Department of Chemistry, CB 3290, University of North Carolina,
Chapel Hill, NC 27599, USA. E-mail: mlwaters@unc.edu;
Fax: +1 919 962 2388; Tel: +1 919 843 6522
b Department of Medicinal Chemistry, University of Minnesota,
308 Harvard St. SE, Minneapolis, MN 55455, USA
w Electronic supplementary information (ESI) available: Experimental
procedures, LC, MS, fluorescence anisotropy, and ITC data. See DOI:
10.1039/c000255k
Fig. 2 Monomers used in DCLs.
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25–30 mM, which is equivalent to its binding affinity to the
HP1 chromodomain (Table 1).48 Moreover, H3 K9Me3 binds
to both isomers of A2B with greater than 2-fold selectivity over
its dimethylated counterpart, exhibiting slightly better selectivity
than the native protein.4 The mono- and unmethylated peptides
exhibited considerably weaker affinity for rac-A2B, with no
appreciable binding to the unmethylated histone tail. Thus, the
trend in both affinity and selectivity of A2B for the histone tail
peptide parallels that of the native protein.4
Fig. 3 Extent of amplification of rac-A2B and meso-A2B with
H3 K9Men: Fam-QTAR-KMen-STG-NH2, n = 0–3
Ac-KMen-G-NH2 guests relative to the untemplated library.
A mutant H3 tail with Gly in place of Arg8 exhibited
comparable affinity to that of the native sequence (Table 1),
indicating that the adjacent basic Arg does not contribute
significantly to binding. Additionally, Lys9 was mutated to
Gly, which resulted in total loss of binding to rac-A2B. NMR
studies of the KMe3 dipeptide in the presence of rac-A2B
exhibit upfield shifting of the lysine methyl groups by 0.83 ppm
at 25 1C, indicating that the binding event is indeed occurring
at the site of modification.** These findings confirm that the
key binding interaction is between the trimethylammonium
group and the aromatic pocket, as in the histone–chromodomain
protein–protein interaction.
in the composition of several libraries. For example,
amplification of receptors rac-A2B and meso-A2B from one
library,y as well as A2C and AC3 from another, was observed
as identified by ESI-MS (Fig. 1b). Interestingly, amplification
of only a single isomer of the isomeric receptor A2C was
observed. When a library of A and B was screened in the
presence of an 8-residue histone tail sequence containing KMe3,
a similar degree of amplification of rac- and meso-A2B was
observed, indicating that other nearby sidechains do not
significantly influence binding.
Using biased libraries in which building blocks A and B as
well as A and C were each mixed in a 2 : 1 ratio (7.5 mM
total), the selectivity of the receptors for different methylation
states of lysine was investigated, using Ac-KMen-G-NH2
(n = 0–3) as the guests. The amplification of both A2B
diastereomers was dependent on the extent of methylation,
with approximately 10-fold amplification with KMe3 and less
than 2-fold amplification with Lys (Fig. 3), suggesting significant
selectivity. In contrast, no selectivity was observed for
receptors A2C and AC3, as similar amplification was observed
for all methylation states as well as unmethylated Lys
(see ESIw). This lack of selectivity may be the result of
additional electrostatic and/or hydrogen bonding interactions
with each guest due to the position of the carboxylic acid in
C as compared to B. Because of this lack of selectivity,
A2C and AC3 were not investigated further.
There are likely several factors that result in the observed
selectivity of A2B. First, the binding pocket may be too large
for Lys and KMe. In addition, Lys, KMe, and KMe2 can all
form hydrogen bonds with water, and binding to the pocket
may require some degree of desolvation, which would be
unfavorable. Lastly, model systems have shown that cation–p
interactions are enhanced with increasing methylation of
the ammonium group.11 Thus, KMe3 is expected to have
stronger cation–p interactions with A2B than with the lower
methylation states.
In conclusion, we report the identification of a synthetic
receptor for KMe3 that exhibits both comparable affinity and
selectivity to the native HP1 chromodomain. The mass of A2B
is less than 900 Da, as compared to approximately 6300 Da for
the HP1 chromodomain and about 150 kDa for a typical
antibody. The comparable binding affinity to the native
protein is impressive, particularly given that the synthetic
receptor appears to bind only to the KMe3 sidechain, whereas
the chromodomain binds to the surrounding sequence as well.
The observed selectivity for higher methylation states with
A2B can be attributed to differences in the magnitude of
the cation–p interactions, as well as differences in size and
desolvation penalty. The difference in selectivity observed
Both A2B isomers were isolated for further studies using
semi-preparative HPLC. Fluorescence anisotropy was used to
measure the dissociation constant of rac- and meso-A2B to a
peptide consisting of residues 5–11 of the histone 3 protein
(H3 K9Men), with each of the methylation states at Lys9,
appended with an N-terminal carboxyfluorescein (Fam) for
fluorescence detection.z The H3 K9Me3 peptide was found to
bind both rac- and meso-A2B with binding affinities of about
Table 1 Dissociation constants for H3 histone tail peptides with varying methylation states at K9 as determined by fluorescence anisotropya
b
Peptide
rac-A2B Kd b/mM
meso-A2B Kd /mM
HP1 chromodomain Kd c/mM
H3 K9Me3
H3 K9Me2
H3 K9Me
H3 K9
25 ꢁ 3
28 ꢁ 4
73 ꢁ 9
N.A.d
N.A.d
N.A.d
10e (21 ꢁ 2 at 25 1C)f
58 ꢁ 10
166 ꢁ 50
>1200
34 ꢁ 8
15e (39 ꢁ 7 at 25 1C)f
96e
>1000e
—
H3 K9Me3 R8G
a
b
c
Conditions: 27 1C, 10 mM phosphate buffer, pH 8.5. Errors are from the fit. Values for rac- and meso-A2B binding to H3 K9Me3 with no
d
fluorophore are 20 mM and 13 mM, respectively, as measured by ITC (see ESIw). Not available due to limited material (meso-A2B), although
e
binding is expected to be comparable to that of rac-A2B. Conditions: 15 1C in pH 7.5 phosphate buffer. Values are taken from ref. 4c.
f
Conditions: 25 1C in 50 mM phosphate buffer, pH 8.0, 25 mM NaCl (Eisert and Waters, unpublished results).
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when B is replaced with C is surprising, and demonstrates that
subtle structural changes can have a significant effect on
binding. Such sequence-independent receptors have the
potential to be widely used in the investigation of unknown
protein Lys methylation sites,12 unlike antibodies which are
sequence selective. This work suggests that small molecule
receptors identified via DCC have a promising future as
affinity reagents for PTMs, as they can easily be synthetically
modified with a fluorophore, cell-penetrating peptide, or other
tag. We are now investigating the application of these
receptors to sensing of trimethyl lysine in proteins.
2 (a) T. Jenuwein and C. D. Allis, Science, 2001, 293, 1074–1080;
(b) T. Kouzarides, Cell, 2007, 128, 693–705.
3 C. Martin and Y. Zhang, Nat. Rev. Mol. Cell Biol., 2005, 6,
838–849.
4 (a) S. A. Jacobs and S. Khorasanizadeh, Science, 2002, 295,
2080–2083; (b) P. R. Nielsen, D. Nietlispach, H. R. Mott,
J. Callaghan, A. Bannister, T. Kouzarides, A. G. Murzin,
N. V. Murzina and E. D. Laue, Nature, 2002, 416, 103–107;
(c) R. M. Hughes, K. R. Wiggins, S. Khorasanizadeh and
M. L. Waters, Proc. Natl. Acad. Sci. U. S. A., 2007, 104,
11184–11188; (d) For a review of cation–p interactions, see:
J. C. Ma and D. A. Dougherty, Chem. Rev., 1997, 97, 1303–1327.
5 (a) N. Blow, Nature, 2007, 447, 741–744; (b) C. C. Kazanecki,
A. J. Kowalski, T. Ding, S. R. Rittling and D. T. Denhardt,
J. Cell. Biochem., 2007, 102, 925–935.
We thank the Army Research Office and Defense Threat
Reduction Agency (DTRA) for support (W911NF06-1-0169).
6 B. A. R. Williams, L. Lin, S. M. Lindsay and J. C. Chaput, J. Am.
Chem. Soc., 2009, 106, 6330–6331.
7 C. S. Beshara, C. E. Jones, K. D. Daze, B. J. Lilgert and F. Hof,
ChemBioChem, 2009, 11, 63–66.
We also thank Prof. Michel R. Gagne and James W.
´
Jorgenson (UNC) for helpful discussions. We also gratefully
8 For reviews, see: (a) P. T. Corbett, J. Leclaire, L. Vial, K. R. West,
J.-L. Wietor, J. K. M. Sanders and S. Otto, Chem. Rev., 2006, 106,
3652–3711; (b) J.-M. Lehn, Chem.–Eur. J., 1999, 5, 2455–2463;
(c) S. Ladame, Org. Biomol. Chem., 2008, 6, 219–226;
(d) R. F. Ludlow and S. Otto, Chem. Soc. Rev., 2008, 37,
101–108; (e) S. Otto, Curr. Opin. Drug Discovery Dev., 2003, 6,
509–520.
acknowledge Sijbren Otto for donation of monomer E.
Notes and references
z Monomers A and B are based on a host originally reported by
Dougherty. See ref. 4d.
y Rac- and meso-A2B have been identified as receptors for other
cationic guests. See ref. 10.
9 (a) S. Otto, R. L. E. Furlan and J. K. M. Sanders, J. Am. Chem.
Soc., 2000, 122, 12063–12064; (b) O. Ramstrom and J.-M. Lehn,
ChemBioChem, 2000, 1, 41–48.
¨
z Binding affinities for H3 K9Me3 lacking the fluorophore were
confirmed by ITC (see ESIw).
8 Interestingly, rac-A2B binds to H3 K9Me3 approximately 6-fold
more tightly than to other trimethylated ammonium ions or NMe4
reported previously. See ref. 10.
** No NMR studies were performed with meso-A2B because of
limited availability of this compound due to significant co-elution
with rac-A2B.
10 (a) S. Otto, R. L. E. Furlan and J. K. M. Sanders, Science, 2002,
297, 590–593; (b) P. T. Corbett, J. K. M. Sanders and S. Otto,
Chem.–Eur. J., 2008, 14, 2153–2166.
11 (a) R. M. Hughes, M. L. Benshoff and M. L. Waters, Chem.–Eur. J.,
2007, 13, 5753–5764; (b) L. M. Salonen, C. Bucher, D. W. Banner,
W. Haap, J.-L. Mary, J. Benz, O. Kuster, P. l. Seiler,
W. B. Schweizer and F. Diederich, Angew. Chem., Int. Ed., 2009,
48, 811–814; (c) A. L. Whiting, N. M. Neufeld and F. Hof,
Tetrahedron Lett., 2009, 50, 7035–7037.
+
1 (a) S. R. Bhaumik, E. Smith and A. Shilatifard, Nat. Struct. Mol.
Biol., 2007, 14, 1008–1016; (b) G. G. Wang, C. D. Allis and P. Chi,
Trends Mol. Med., 2007, 13, 363–372.
12 J. Huang and S. L. Berger, Curr. Opin. Genet. Dev., 2008, 18,
152–158.
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