Engineering Methyllysine Writers and Readers for Allele-Specific Regulation of Protein-Protein Interactions

Article abstract of DOI:10.1021/jacs.9b05725

Protein-protein interactions mediated by methyllysine are ubiquitous in biological systems. Specific perturbation of such interactions has remained a challenging endeavor. Herein, we describe an allele-specific strategy toward an engineered protein-protei

Full text of DOI:10.1021/jacs.9b05725

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Communication
Engineering Methyllysine Writers and Readers for Allele-
Specific Regulation of Protein-Protein Interactions
Simran Arora, W. Seth Horne, and Kabirul Islam
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b05725 • Publication Date (Web): 13 Sep 2019
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Engineering Methyllysine Writers and Readers for Allele-Specific
Regulation of Protein-Protein Interactions
Simran Arora, W. Seth Horne, and Kabirul Islam*
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Department of Chemistry, University of Pittsburgh, Pittsburgh PA 15260 USA
Supporting Information Placeholder
ABSTRACT: Protein-protein interactions mediated by
methyllysine are ubiquitous in biological systems. Specific
perturbation of such interactions has remained a challenging
endeavor. Herein, we describe an allele-specific strategy
towards an engineered protein-protein interface orthogonal to
the human proteome. We develop a methyltransferase (writer)
variant that installs aryllysine moiety on histones that can only
be recognized by an engineered chromodomain (reader). We
establish biochemical integrity of the engineered interface,
provide structural evidence for orthogonality and validate its
applicability to identify transcriptional regulators. Our
approach provides an unprecedented strategy for specific
manipulation of the methyllysine interactome.
Figure 1. A strategy for allele-specific protein-protein interactions.
Methyllysine writer (KMT) is first engineered to introduce
‘bumped’ lysine and disrupt interaction with wild-type readers.
Binding is restored using ‘hole-modified’ reader, allowing
conditional manipulation of methyllysine pathways.
Lysine methylation in histones is
a posttranslational
modification that regulates gene expression.1 This process is
catalyzed by lysine methyltransferases (KMTs), classified as
‘writers’, using S-adenosylmethionine (SAM) (Figure 1).2-3
Biological functions of methyllysine are manifested via specific
recognition by conserved protein modules termed ‘readers’
(Figure 1).4-6 In humans, an array of ~60 KMTs and >200
proteins with reader domains are involved in establishing and
recognizing >5000 methyllysine sites.7 Context-dependent
function of the methyllysine network is largely unexplored,
mainly due to a lack of tools capable of interrogating various
components of the ‘interactome’ simultaneously.8-10 Probing
how a specific writer-histone-reader axis contributes to
chromatin-dependent processes requires precise and sequential
perturbation of the methyllysine writers and readers.
We chose trimethylation on H3 at lysine 9 (H3K9me3) as a
host system to test the above approach. H3K9 methylation is a
marker of transcriptional repression.1 In humans, a large set of
KMTs methylate H3K9, and the modification is ‘read’ by
chromodomain and PHD finger containing proteins.5,18 Herein,
we first develop ‘hole-modified’ H3K9me3 methyltransferases,
G9a and Suv39H2, to install lysine modifications that are not
recognized by cognate wild-type readers CBX1, 3, and 5; we go
on to engineer CBX proteins that bind ‘bumped’ histone and
restore the lost interaction. Finally, we provide structural
evidence for orthogonality and demonstrate suitability of the
engineered system to control
signaling pathway.
a methyllysine-dependent
Allele-specific chemical genetics (often called the ‘bump-
and-hole’ tactic) has emerged as a powerful tool to examine
member-specific protein function.11-16 We envisioned
developing allele-specific writer-histone-reader pairs, fully
orthogonal to the human proteome, for precise manipulation of
methyllysine interaction (Figure 1). We reasoned a ‘hole-
modified’ KMT could accept SAM analogues to modify histone
with bulky groups that would perturb the binding of a wild type
reader through steric repulsion. Interaction with the ‘bumped’
histone could be restored with a ‘hole-modified’ reader variant,
thus providing control over methyllysine-dependent
interactions. Although engineered KMTs are known,17 evolving
a reader to recognize non-native protein modifications is
unexplored. We hypothesized that the hydrophobic ‘aromatic
cage’ in reader domain that recognizes methyllysine5 could be
expanded to accommodate alkyllysine in histone and create an
orthogonal interface.
To access the ‘bumped’ histone peptides, we synthesized a
series of SAM analogues carrying bulky sulfonium alkenyl and
aryl groups (Figure 2A, Scheme S1).17,19 Each of the SAM
derivatives was incubated with previously developed Y1154A
mutant of G9a17 and tetramethylrhodamine (TAMRA) labeled
H3K9 peptide. The products were purified and confirmed by
MALDI-MS (Figure S1). The mutant showed complete
alkenylation of the substrate (Figure S1); however, it was not
efficient at incorporating benzyl modification. We envisioned
an aryl group as a lysine modifier would improve binding with
engineered CBX by maintaining - interaction in the
remodeled aromatic cage. We surmised that expanding the
pocket of G9a further could facilitate its use of benzyl-SAM.
Indeed, Y1154G mutation led to complete substrate benzylation
(Figure 2B), demonstrating that G9a can be engineered to
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accept aryl SAM analogues for expanding the repertoire of
‘bumped’ histone variants. Under our assay conditions, we did
not observe any dibenzylation of the substrate by G9a mutant
(Figure S2).
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binding (Figure S10-12); however, a Y26F/F50G variant with
intact aromatic sidechain at F26 maintained robust affinity
towards benzyl peptide (Kd = 5.50.9 M; Figure 2C, S10).
Importantly, interaction with H3K9me3 peptide was reduced to
a Kd of ~80 M. We further confirmed the binding constants by
isothermal titration calorimetry using H3K9me3 and benzylated
(H3K9bn) peptides. Both native and engineered readers showed
comparable affinity to their cognate peptides (Figure 3A, B,
S13, Table S1). Furthermore, when tested against the H3K9me2
peptide, a major enzymatic product of G9a, wildtype and
engineered CBX1 bound with Kd of 18.3 M and >300 M,
respectively (Figure S14). Taken together, our mutational study
led to an orthogonal chromodomain capable of recognizing
‘bumped’ histone peptide—a new approach to modulate
histone-reader interactions.
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Figure 2. Engineering of G9a and CBX1. (A) G9a-Y1154A mutant
transfers alkenyl groups from the corresponding SAM analogues to
TAMRA-labeled H3K9 peptide. (B) MALDI-MS showing
benzylation of the substrate peptide by G9a-Y1154G mutant using
benzyl-SAM. (C) Heat-map diagram showing dissociation
constants (Kd) for the binding of modified H3K9 peptides by wild-
type CBX1 and ‘hole-modified’ mutants.
As a model reader domain for allele-specific engineering, we
selected the CBX1 chromodomain, which recognizes
H3K9me3 through its aromatic cage.20 First, using
a
fluorescence polarization assay, we determined the dissociation
constants (Kd) of wild-type CBX1 chromodomain from
TAMRA-labeled H3K9me3 and various ‘bumped’ variants
(Figure 2C, S3). The reader protein bound H3K9me3 tightly,
with a Kd (2.00.1 M) close to the reported value.21 CBX1
showed 53-fold weaker binding affinity (Kd = 10620 M)
towards allyl-modified peptide. Importantly, it failed to bind
peptides with modifications bulkier than allyl (Kd > 500 M,
Figure S3), presumably due to a steric clash inside the aromatic
cage.
Figure 3. Characterization of the histone-reader pairs. (A, B)
Isothermal titration calorimetric (ITC) measurements for binding
between wild-type CBX1 and H3K9me3 (A), and Y26F/F50G-
CBX1 and H3K9bn (B). (C, D) View of the aromatic cage from
crystal structures of wild type CBX1 chromodomain bound to
H3K9me3 peptide (C) and ‘hole-modified’ CBX1 (Y26F/F50G)
bound to H3K9bn (D).
To gain further insight into the orthogonality of the bump-
hole pair, we grew crystals of the complex of native CBX1 with
H3K9me3 as well as the complex of Y26F/F50G-CBX1 with
H3K9bn by hanging drop vapor diffusion. We collected
diffraction data on each complex, solved the native structure
(PDB 6D07) by molecular replacement with a model derived
from a structure of CBX5 bound to H3K9me3,21 and solved the
mutant structure (PDB 6D08) using refined model of the native
CBX1 complex. Both structures were refined to 2.1 Å
resolution (Figure 3C, D, Table S2). The overall fold and
binding interactions observed for native CBX1 in complex with
H3K9me3 are identical to those noted in closely related
members CBX3 and CBX5 (Figure S15).21 The backbone
coordinates for the bump-hole mutant complex were virtually
identical to native CBX1 (0.6 Å Cα rmsd, Figure S15).
Inspection of the aromatic binding pocket shows that the benzyl
‘bump’ effectively fills the hole created by the F50G mutation
(Figure 3D) and that removal of the side chain in CBX1 has no
measurable effect on local backbone conformation.
Superimposition of the two complexes revealed that F50 of wild
In order to develop an orthogonal reader, we analyzed a
published structure of the CBX1 chromodomain and identified
residues Y26, W47, F50, D54 and T56 that recognize
H3K9me3.22 We generated the corresponding alanine mutants
and measured their binding affinity towards all the modified
peptides (Figure 2C, S4-8). Compared to wild type CBX1, the
mutants showed decreased binding towards the trimethylated
peptide, indicating that an intact hydrophobic pocket is essential
for optimum recognition of H3K9me3.21 Among the CBX1
mutants examined, F50A showed significant gain in binding
affinity towards the pentyl and benzyl peptides (Kd of 14.12.8
and 18.54.5 M, respectively), while affinity for H3K9me3
decreased by almost 32-fold (Kd of 63.56.2 M) compared to
wild-type reader (Figure 2C, S6). Encouraged by this result, we
prepared F50G mutant with a larger cavity in an effort to further
improve the binding efficiency. F50G indeed showed a 3-fold
improvement in affinity towards H3K9bn (Figure 2C, S9).
To improve orthogonality of the engineered readers, we
generated several double mutants by combining F50G with
mutations at Y26. Most mutations led to complete loss of
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type CBX1 undergoes steric interference with benzyl group of
histone. Next, we expressed full-length CBX1 variant carrying
the engineered chromodomain.26 Nuclear extracts were
incubated with biotinylated histone peptides, each carrying
either unmodified, methylated or benzylated lysine, pulled
down with avidin beads and analyzed using antibodies for
CBX1, 3 or 5 (Figure 5B, S20). While the trimethylated peptide
enriched all three wildtype CBX proteins, the benzylated bait
was specific for hole-modified CBX1 (Figure 5B, S21).
Furthermore, biotinylated peptides carrying methyllysine at a
site different from H3K9 failed to interact with the mutant
(Figure 5C, S21), confirming competency of the engineered
system to mediate allele-specific histone-reader interactions.
Although, we only examined CBX1, 3 and 5, detailed
proteomic analysis of enriched proteins will be required to
evaluate specificity of H3K9bn across all the methyllysine
readers.
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H3K9bn (Figure S15), explaining the observed orthogonality.
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Sequence analysis of the major KMTs revealed that Y1154
in G9a is highly conserved among writers (Figure 4A) and an
aromatic residue, equivalent to F50 in CBX1, is present in all
of the readers analyzed (Figure 4B).23 These bulky amino acids
are acting as gatekeepers that preclude binding of SAM and
methyllysine analogues to writers and readers, respectively. We
focused on another H3K9me3 methyltransferase, Suv39H2, and
the cognate reader CBX3.24-25 The hole-modified Y372G
mutant of Suv39H2, equivalent to G9a-Y1154G, efficiently
utilized benzyl-SAM to modify substrate peptide as well as full-
length H3 (Figure 4C, S16, 17).
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To demonstrate generality among reader domains, we
prepared F44G mutant of CBX3, equivalent to Y26F/F50G-
CBX1. Conveniently, CBX3 already carries a phenylalanine at
the position equivalent to Y26 in CBX1. Wild type CBX3
recognized H3K9me3 peptide effectively as judged by
fluorescence anisotropy, but no binding was observed towards
the benzylated homologue (Figure 4D). In contrast, the ‘hole-
modified’ CBX3 exhibited enhanced affinity for H3K9bn with
significant loss of binding towards the trimethyl mark (Figure
4E). These results establish that multiple histone writers and
readers can be engineered at gatekeeper sites to access
orthogonal protein-protein interfaces.
Finally, we examined interactions between the CBX1
variant and transcriptional complexes (Figure 5A). Binding of
full-length CBX proteins to H3K9me3 creates a docking site for
transcriptional regulators such as TIF1.26,27 We incubated
biotinylated H3K9bn peptide with nuclear extract of HEK293T
cells expressing the ‘hole-modified’ reader, and enriched the
‘interactome’ using avidin and analyzed by western blotting
with TIF1 antibody (Figure 5D, S21). We observed selective
pull-down of TIF1 by benzylated peptide, but not the
unmodified peptide, only when cells expressed the ‘hole-
modified’ CBX1 mutant. The ‘bumped’ peptide failed to
interact with transcriptional regulators without the engineered
reader. As a positive control, trimethylated peptide was shown
to enrich TIF1 using endogenous CBX proteins. This set of
results demonstrated that the engineered system maintains
native-like interactions between a specific methyllysine reader
and transcriptional regulators.
A
1
Allele-specific interaction
2
Reader-specific ‘interactome’
NH2
NH2
NH2
+
+
>200 Endogenous
methyllysine readers
Transcriptional
regulators
Mammalian
cell
'Bumped’
bait
Figure 4. Generality in methyllysine writer-reader engineering. (A,
B) Sequence alignments of KMTs (A) and methyllysine readers (B)
showing Y1154 of G9a and F50 in CBX1 are highly conserved. (C)
Substrate benzylation by Suv39H2-Y372G mutant on full-length
histone H3. (D) Binding of wild-type CBX3 towards H3K9me3 (Kd
= 12.20.2 M) and H3K9bn (Kd > 1mM). (E) Binding of CBX3-
F44G mutant towards H3K9me3 (Kd > 1mM) and H3K9bn ((Kd =
492.4 M).
Avidin
bead
Capture
Elution
Detection
Biotin
B
C
Anti HA
+ Y26F/
F50G
Anti-CBX1
Anti-CBX3
Anti H3
- Y26F/F50G
+ Y26F/F50G
D
Anti-CBX5
Anti HA
To determine if the engineered writer and reader could act in
tandem, we first benzylated full-length H3 and purified
mononucleosomes using Suv39H2 mutant followed by
enrichment with the hole-modified CBX1. We observed signal
for H3 only in the presence of benzyl-SAM as evident from
western blot data (Figure S18), demonstrating successful
reading and writing of H3K9bn on full-length substrates.
Anti-TIF1β
Anti H3
Anti H3
Y26F/F50G:
-
+
+
-
Figure 5. Allele-specific interactions demonstrated in cell extracts.
(A) Interaction between ‘hole-modified’ CBX1 and ‘bumped’
histone in presence of endogenous methyllysine readers (step 1).
The engineered complex binds to transcriptional regulators (step
2). (B) Western blot (indicated antibody) of nuclear extracts pulled-
down using biotin-attached H3K9me3 or H3K9bn peptide. CBX1
mutant carries HA tag. (C) Western blot (anti-HA antibody) of
nuclear extracts pulled-down using biotin-attached H3K4me3,
H3K27me3, H3K36me3 or H3K9bn peptide (D) Western blot
(anti-TIF1 antibody) of nuclear extracts pulled-down using biotin-
attached H3K9me3 or H3K9bn or unmodified peptide.
We further investigated whether the orthogonal pairs are
functional in cellular milieu (Figure 5A, S19). HEK293T cells
were cultured to express full-length hole-modified Suv39H2
variant and the isolated nuclei were incubated with benzyl-
SAM in a pulldown assay. Only the benzylated H3 was found
to be specifically enriched with the CBX1 mutant (Figure S19),
indicating that engineered pair could act on chromosomal
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In summary, we offer a strategy to modulate protein-protein
interactions particularly those governed by lysine methylation.
Development of orthogonal pairs for protein-protein
interactions is rarely attempted.28-30 Using a set of H3K9me3
writers and readers as paradigm, we showed that the remodeled
interface is orthogonal to the wild type. We established
biochemical integrity of the engineered interface, provided
structural rationale for orthogonality, demonstrated generality
of the approach, and validated functional compatibility of the
synthetic interface in recognizing transcriptional regulators. We
anticipate developing an in-cellulo assay to synthesize benzyl-
SAM by promiscuous SAM synthetase mutant using benzyl
methionine precursor.31 The engineered benzyllysine apparatus
would allow installing the unique modification on
chromosomal histone to interrogate a specific methyllysine
pathway within cell for orthogonal manipulation of mammalian
gene expression.
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ASSOCIATED CONTENT
Supporting Information. Methods for protein expression
and crystallization, biochemical assays, supplementary
figures and tables. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
kai27@pitt.edu
Notes
The authors declare no competing financial interest
(20) Lachner, M.; O'Carroll, D.; Rea, S.; Mechtler, K.; Jenuwein, T.,
Methylation of histone H3 lysine 9 creates a binding site for HP1
proteins. Nature 2001, 410, 116-20.
ACKNOWLEDGMENT
We thank the University of Pittsburgh, the National Institutes
of Health (R01GM123234) and the National Science
Foundation (MCB-1817692) for financial support; Dr. D.
Chakraborty and members of our laboratory for critical reading
and editing of the manuscript. Support for MALDI-TOF MS
instrumentation was provided by a grant from the National
Science Foundation (CHE-1625002).
(21) Kaustov, L.; Ouyang, H.; Amaya, M.; Lemak, A.; Nady, N.;
Duan, S.; Wasney, G. A.; Li, Z.; Vedadi, M.; Schapira, M.; Min, J.;
Arrowsmith, C. H., Recognition and specificity determinants of the
human cbx chromodomains. B. Biol. Chem. 2011, 286, 521-9.
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6
7
8
9
Protein-ligand
1
Protein-protein
2
N
‘bump-hole’
NH2
N
‘bump-hole’
N
N
CO2
H
H2N
WT
Reader
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
S
SAM
H3
C
HO
OH
NH3
Methyllysine
WT Writer
WT interface
NH2
N
N
H2
N
CO2
S
H
H2
N
N
N
MT
Reader
O
Benzyl
SAM
HO
OH
Benzyllysine
Engineered interface
MT Writer
ACS Paragon Plus Environment