Studying the Conformation of a Receptor Tyrosine Kinase in Solution by Inhibitor-Based Spin Labeling

Article abstract of DOI:10.1002/anie.201703154

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof-of-concept that inhibitor-based spin labeling enables the convenient introduction of site-specific spin labels into kinases for which covalent or tight-binding small-molecule modulators are available.

Full text of DOI:10.1002/anie.201703154

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Communications
Chemie
International Edition: DOI: 10.1002/anie.201703154
German Edition: DOI: 10.1002/ange.201703154
EPR Spectroscopy
Studying the Conformation of a Receptor Tyrosine Kinase in Solution
by Inhibitor-Based Spin Labeling
Dongsheng M. Yin, Jeffrey S. Hannam, Anton Schmitz, Olav Schiemann, Gregor Hagelueken,*
and Michael Famulok*
Abstract: The synthesis of a spin label based on PD168393,
a covalent inhibitor of a major anticancer drug target, the
epidermal growth factor receptor (EGFR), is reported. The
label facilitates the analysis of the EGFR structure in solution
by pulsed electron paramagnetic resonance (EPR) spectros-
copy. For various EGFR constructs, including near-full-length
EGFR, we determined defined distance distributions between
the two spin labels bound to the ATP binding sites of the EGFR
dimer. The distances are in excellent agreement with an
asymmetric dimer of the EGFR. Based on crystal structures,
this dimer had previously been proposed to reflect the active
conformation of the receptor but structural data demonstrating
its existence in solution have been lacking. More generally, our
study provides proof-of-concept that inhibitor-based spin
labeling enables the convenient introduction of site-specific
spin labels into kinases for which covalent or tight-binding
small-molecule modulators are available.
defined cysteine residues, producing nitroxide side chains
coupled to the protein either through a disulfide or a thioether
linkage. Thus, SDSL commonly requires the introduction of
non-native cysteine residues at the desired sites while
removing endogenous cysteines by serine or alanine substi-
tutions. These requirements can be a drawback in cases where
the mutations alter the structure or activity of the target
protein. Therefore, spin-labeling strategies that are not based
on cysteine mutation are of high interest. The use of non-
natural amino acids as sites for labeling is such an approach.
However, currently this method is restricted to proteins that
can be expressed in E. coli or in an E. coli-derived cell-free
[2]
expression system. An attractive strategy not requiring
amino acid substitutions is the use of spin label-bearing
natural ligands, such as lipids or cofactors that target a distinct
[
3]
site in the protein of interest. An analogous approach would
be particularly promising for protein kinases, because a large
variety of synthetic inhibitors targeting their ATP binding site
with high specificity and affinity are available and can thus in
principle be applied to label the protein. Moreover, more than
200 kinases, about 40 percent of the kinome, contain a cysteine
residue in the ATP binding site, which can be used to
covalently attach the inhibitor and thus the spin label, while
avoiding the labeling of any other cysteine residues in the
O
btaining structural information on proteins in solution
remains challenging, particularly when information on
dynamic processes is desired. Although impressive progress
is currently being made using single-particle cryo-electron
microscopy, this technique is still limited to a few highly
specialized laboratories and to proteins larger than about
[
4]
1
00 kDa. While NMR-based methods are limited to proteins
same protein. Especially for receptor tyrosine kinases
(RTKs) such as the epidermal growth factor receptor
(EGFR), this approach appears a promising way to answer
long standing questions about the conformational states of
of small to medium size, pulsed electron paramagnetic
resonance (EPR) spectroscopy is not restricted by these
limitations. However, it requires the site-directed introduc-
tion of paramagnetic spin labels into the protein of interest,
a process that is known as site-directed spin labeling
[
5]
these important molecules in solution. Therefore, we chose
the EGFR to demonstrate the principal applicability of
mutagenesis-independent SDSL for RTKs. In earlier efforts
we had observed that replacing the cysteine residues in the
EGFR kinase domain resulted in almost complete loss of
activity (for an example, see the Supporting Information,
Figure S1), indicative of structural changes resulting from the
mutations, precluding traditional, mutagenesis-based SDSL
for the EGFR. However, a large number of ATP-competitive
small molecule inhibitors have been developed for the
EGFR, some of which covalently target a cysteine residue
[
1]
(
SDSL). In the vast majority of SDSL approaches, the
spin label is attached by modification of the thiol moiety of
[*] D. M. Yin, Dr. A. Schmitz, Prof. Dr. M. Famulok
Max Planck Fellow Chemical Biology
Center of Advanced European Studies and Research (caesar)
Ludwig-Erhard-Allee 2, 53175 Bonn (Germany)
E-mail: m.famulok@uni-bonn.de
D. M. Yin, Dr. J. S. Hannam, Dr. A. Schmitz, Prof. Dr. M. Famulok
LIMES Chemical Biology Unit
Rheinische Friedrich-Wilhelms-Universitꢀt Bonn
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
[
6]
in the ATP-binding site.
Herein we report the development and application of an
EPR spin label derived from the covalent type I EGFR
Prof. Dr. O. Schiemann, Dr. G. Hagelueken
Institute of Physical and Theoretical Chemistry
Rheinische Friedrich-Wilhelms-Universitꢀt Bonn
Wegelerstrasse 12, 53115 Bonn (Germany)
E-mail: hagelueken@pc.uni-bonn.de
[7]
inhibitor PD168393 (Supporting Information, Figure S2).
We developed a variant of the synthesis described previously
[
8]
for the fluorophore-labeled version of PD168393 (Support-
ing Information, Scheme S1). The synthesis follows standard
procedures to 7-fluoroquinazoline 4 (Supporting Information,
Scheme S1). Subsequently, Boc-ethanolamine was installed
with a crown ether catalyzed ipso-substitution to give 5. The
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201703154.
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acrylamide 7 was elaborated in a two-step procedure, firstly
reacting aniline 6 with 3-chloropropionic acid chloride,
followed by b-elimination with triethylamine. Finally, 1 (Fig-
ure 1A) was obtained after Boc deprotection of 7 and
reaction with commercially available spin label 8. The short
ethanolamine linker was chosen to reduce the steric flexibility
of the spin label relative to the inhibitor moiety, which should
result in sharper distance distributions.
(MBP) as carrier (for details on the constructs see the
Supporting Information, Figure S3). Owing to the presence of
the juxtamembrane segment of the EGFR, which is required
for efficient receptor dimerization and activation, the fusion
protein is active in a cell-free autophosphorylation assay.
Both, PD168393 and 1 reduced the autophosphorylation of
the construct to the same extent (Figure 1B).
[
9]
MBP-EGFR-ICD998 labeled with 1 was then used for
pulsed electron–electron double resonance (PELDOR)
measurements. Crystal structures for the full-length ICD are
currently not available. Instead, an EGFR construct compris-
[
10]
ing amino acids 645–998 was the first to be crystallized and
extensively characterized by crystallography, including
[8]
a structure covalently linked to PD168393 (PDB: 2J5F).
In this structure, the EGFR was observed in an asymmetric
[11]
dimer, also known as the active conformation. The EGFR
kinase domain also crystallizes in a symmetric dimer that has
[
11]
been proposed to reflect the inactive conformation. In the
inactive and active EGFR dimer structures, the binding sites
of 1 are about 30 ꢀ or about 65 ꢀ apart, respectively
(Figure 2A). Such a large difference in distances is well-
Figure 1. An inhibitor-derived EGFR EPR probe. A) To generate a cova-
lently attached EPR probe, the irreversible EGFR inhibitor PD168393
was modified with the spin label 1-oxyl-2,2,5,5-tetramethylpyrroline-3-
carboxylic acid via an ethanolamine spacer to yield 1. B) The inhibitory
activity of 1 was verified in an EGFR autophosphorylation assay in
direct comparison with PD168393. Phosphorylated MBP-EGFR-ICD998
suited to distinguish the active and inactive conformation of
the EGFR by PELDOR spectroscopy. To verify that the
protein was efficiently labeled with 1, we conducted cw-X-
band EPR measurements (Figure 2C) and determined an
average spin-labeling degree of at least 80%, in accordance
with the inhibition of autophosphorylation (Figure 1B).
The Q-band PELDOR time trace for 1-labeled MBP-
EGFR-ICD998 revealed a slow initial decay and a pro-
nounced modulation of the echo intensity with an average
(
pY992) was visualized by anti-EGFR(pY992) antibody and total
amount of MBP-EGFR-ICD998 (MBP) by anti-MBP antibody. PD:
PD168393. rel. phos.: phosphorylation levels at 1 and 3 min relative to
the value at 0 min (set as 1 for each sample).
(n = 3) modulation depth of 26% (green in Figures 2B,C; see
To verify that 1 retained the inhibitory function of
PD168393, the ability of 1 to inhibit the EGFR kinase activity
was determined. For this purpose, a truncated version of the
intracellular domain (ICD) of the EGFR (EGFR-ICD998)
was purified as a fusion protein with maltose-binding protein
the Supporting Information, Figure S4 for the uncorrected
PELDOR time traces). Analysis of the time trace with
DeerAnalysis2016 resulted in a distance distribution with
a well-defined peak at 6.3 nm (Figure 2D). To accurately
compare this distance to the available structures of EGFR,
[
12]
[
15]
Figure 2. PELDOR on 1-labeled EGFR constructs. A) The previously proposed asymmetric (active) and symmetric (inactive) EGFR dimers are
shown as cartoon models. The two individual monomers of each dimer are colored blue and green and the position of the membrane is indicated
as gray shading. The two EGF ligands on the extracellular domain of the asymmetric dimer are shown as magenta and yellow cartoons. Models of
two 1 spin labels (SL) in each dimer are shown as blue spheres and the distance vectors between them as red lines. B) Background-corrected
PELDOR time traces of the EGFR constructs indicated on the top (see the Supporting Information, Figure S4 for raw data). The gray line marks
constructs that only contain the ICD. The modulation depth of each time trace is marked on the y axis (as V =1Àl). C) Modulation depth l in
l
percent (lower y-axis) and spin labeling efficiency (upper y-axis) for EGFR-ICD constructs. Note that IQ+VR is a mixture of the two individual
constructs and thus no labeling efficiency is given. The coloring is the same as in (B). D) Distance distributions calculated from (B) using
DeerAnalysis2016. The samples are colored according to (B) and the mtsslWizard predictions for the symmetric and asymmetric states are
represented by gray shading. VR: MBP-EGFR-ICD998(V924R), IQ: MBP-EGFR-ICD998(I682Q), 998: MBP-EGFR-ICD998, wt: MBP-EGFR-ICD.
2
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while accounting for the flexibility of the attached nitroxide
group (Figure 1A), 1 was implemented into the in silico spin
ure S6). Mixing MBP-EGFR-ICD998(I682Q) and MBP-
EGFR-ICD998(V924R) at equimolar concentrations should
[
13]
[11]
labeling software mtsslWizard.
Using this program, the
regain the ability to form an asymmetric dimer. Indeed, we
spin–spin distance was predicted for both the active- and
inactive conformations of the EGFR. Figure 2D shows that
the experimental distance distribution fits almost perfectly to
the distance that was predicted for the asymmetric active
dimer (Figure 2D, green). Interestingly, no significant amount
of 1-labeled MBP-EGFR-ICD998 in the symmetric inactive
conformation was detectable (Figure 2D). The cw-X-band
spectra of 1-labeled EGFR indicated a rather rigid spin label.
For this reason, we investigated, whether the PELDOR signal
was orientation selective. Time traces with different fre-
quency offsets between pump and probe pulses were recorded
and the Pake patterns calculated. This analysis did not reveal
evidence for orientation selectivity (Supporting Information,
Figure S5). A possible reason for this combination of broad
cw-X-band spectra and absence of orientation selectivity
would be multiple distinct orientations of the spin label,
where each orientation is individually stabilized by interac-
tions with the protein surface.
observed a significantly increased average modulation depth
of 24% (vs. 26% for MBP-EGFR-ICD998) and the same
active dimer distance as for the wild-type sample. These
findings support the view that the measured 6.3 nm interspin
distance indeed derives from the asymmetric dimer as found
in crystal structures. Our results confirm that the interface
mutations severely limit the ability of EGFR to form the
active dimer. However, they do not shift the equilibrium to
the symmetric dimer but rather to a monomeric form of
EGFR. These findings are in accordance with cellular studies
showing that full-length EGFR containing either the I682Q or
V924R mutation had a drastically reduced ability to form
dimers even when dimerization was stimulated by treatment
[
14]
of the cells with EGF. In contrast however, the isolated
EGFR(V924R) kinase domain crystallizes in the form of the
[
9a]
symmetric dimer.
Therefore, to investigate the effect of the mutants on
dimerization by an EPR-independent method, we analyzed
the PELDOR samples by analytical gel filtration. MBP-
EGFR-ICD998 produced a relatively broad peak (Fig-
ure 3A). The maximum of the peak eluted at the position
of the 150 kDa calibration standard fitting the theoretical
mass of the dimer of 160 kDa. In contrast, the two mutants
I682Q and V924R produced sharper peaks at higher elution
The region distal to the kinase domain, the so-called C-
terminal tail, has been described as an autoinhibitory region
[
14]
in the EGFR.
The lack of most of this region in our
construct might shift the equilibrium of inactive and active
states to the latter. Therefore, we repeated the measurement
with MBP-EGFR-ICD, that is, the full-length ICD containing
the complete autoinhibitory region (Figure 2B, red). Also for
this construct, we found exclusively the asymmetric, active-
like dimer (Figure 2D). Interestingly, this construct reprodu-
cibly led to PELDOR time traces with a lower modulation
depth compared to the C-terminally truncated EGFR-
ICD998 construct (Figures 2B,C). Since the labeling efficien-
cies (Figure 2C, upper y-axis), as well as the inhibition of the
autophosphorylation (Supporting Information, Figure S6)
were comparable between the two samples, this result may
be explained by an increased amount of monomeric EGFR-
ICD compared to EGFR-ICD998. Further experiments will
be needed to clarify whether this observation is of any
functional relevance.
The spin–spin distance of 6.3 nm for the 1-labeled EGFR-
ICD dimer fits perfectly the distance deduced from models of
the asymmetric dimer. Nevertheless, it could result from
a dimer in another conformation having accidentally the same
interspin distance. Therefore, we analyzed MBP-EGFR-
ICD998(I682Q) and MBP-EGFR-ICD998(V924R), two
EGFR mutants having a reduced ability to form the
asymmetric dimer, due to point mutations in the interface
[11]
area. The PELDOR time traces of both mutants showed
reduced modulation depths of only 6% for V924R and 16%
for I682Q (Figures 2B,C, orange and purple, respectively).
The reduced modulation depths are not due to inefficient
labeling as shown by cw-X-band EPR measurements (Fig-
ure 2C) and inhibition studies (Supporting Information,
Figure S6). Analyzing the remaining dipolar signal with
DeerAnalysis2016 revealed the same distance as found for
the “wild-type” constructs (Figure 2D). Correspondingly,
some residual activity of the mutants was found in the
autophosphorylation assay (Supporting Information, Fig-
Figure 3. Analytical gel filtration of the PELDOR samples shown in
Figure 2. A) MBP-EGFR-ICD998. B) MBP-EGFR-ICD998(I682Q).
C) MBP-EGFR-ICD998(V924R). D) 1:1 mixture of I682Q and V924R
mutants. For each panel, the absorption at 280 nm was normalized to
values between 0 and 1 to allow easier comparison of the traces.
Vertical lines indicate the peak elution volumes of the calibration
standards.
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volumes (14.1 mL for both mutants), fitting the elution
volume of the EGFR kinase monomer (Figure 3B,C). The
I682Q + V924R mixture showed a slightly broadened peak
important receptors. We envision that in the future, a combi-
nation of SDSL and spin-label bearing small molecules will
greatly facilitate studies on protein dynamics in solution and
in cells by EPR methods.
(
1
compared to I682Q or V924R alone) at an elution volume of
3.8 mL (Figure 3D). The broadening can be explained by
a weak interaction between the monomers, leading to an
[16]
overall slightly faster passage through the column. This Acknowledgements
explanation is supported by the PELDOR result of the
I682Q + V924R mixture, where an increased modulation
depth, as compared to the individual proteins, but a slightly
reduced modulation depth, as compared to wild-type EGFR,
was observed (Figure 2C). Thus, the PELDOR as well as the
gel filtration measurements show that 1-labeled MBP-EGFR-
ICD998(V924R) exists mainly as monomer in solution.
Next, we measured the interspin distance distribution of
The Max-Planck-Society and Bonn University supported this
work through a Max-Planck-Fellowship to M.F. We thank Y.
Aschenbach-Paul, V. Fieberg, and F. Duthie for technical
assistance. We thank E. Schubert for collecting some of the
cw-X-band spectra.
EGFRDC, a construct containing the complete extracellular Conflict of interest
and transmembrane domains (Supporting Information, Fig-
ure S3). Using negative staining electron microscopy, stabili-
zation of the asymmetric dimer by PD168393 has been
The authors declare no conflict of interest.
[17]
reported for a very similar construct. EGFRDC, purified in
Triton-X 100 micelles, showed EGF-dependent autophos-
phorylation, which was inhibited by 1 (Supporting Informa-
tion, Figure S7). The PELDOR time trace of 1-labeled
EGFRDC is shown in Figure 2B (black) and indeed looks
very similar to the time traces of the ICD constructs. Note that
the modulation depth of this construct cannot be compared to
those of the other constructs because for technical reasons the
labeling was done differently (see the Supporting Informa-
tion, Methods). This does, however, not affect the determi-
nation of the interspin distances. Using DeerAnalysis2016 an
interspin distance fitting the asymmetric dimer was calculated
Keywords: covalent inhibitor spin probe ·
epidermal growth factor receptor · EPR spectroscopy · PELDOR ·
spin labels
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Version of record online: && &&, &&&&
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Communications
EPR Spectroscopy
Synthesizing and employing a spin-la-
beled covalent inhibitor led to a versatile
and convenient method to introduce
paramagnetic residues at a defined site
into a protein for EPR studies. The probe
allows the determination of the confor-
mation in solution of the near-full-length
epidermal growth factor receptor (EGFR)
bound to an inhibitor. This study is thus
a significant step forward in EPR analysis
of complex proteins.
D. M. Yin, J. S. Hannam, A. Schmitz,
O. Schiemann, G. Hagelueken,*
M. Famulok*
&&&& — &&&&
Studying the Conformation of a Receptor
Tyrosine Kinase in Solution by Inhibitor-
Based Spin Labeling
6
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