Selective cross-linking of interacting proteins using self-labeling tags

Article abstract of DOI:10.1021/ja907818q

We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence scanning. S-CROSS requires the expression of the putative

Full text of DOI:10.1021/ja907818q

Published on Web 11/16/2009
Selective Cross-Linking of Interacting Proteins Using
Self-Labeling Tags
Arnaud Gautier,† Eiji Nakata,‡ Grazˇvydas Lukinavicˇius, Kui-Thong Tan, and
Kai Johnsson*
´
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fe´de´rale de Lausanne
(EPFL), CH-1015 Lausanne, Switzerland
Received September 15, 2009; E-mail: kai.johnsson@epfl.ch
Abstract: We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting
proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence
scanning. S-CROSS requires the expression of the putative interacting proteins as fusion to CLIP-tag or
SNAP-tag, two protein tags that can be specifically labeled with synthetic probes. Bifunctional molecules
that contain the substrates of the two tags connected via a fluorophore are used to selectively cross-link
interacting proteins in cell lysate. The amount of trapped complex can be then quantified after SDS gel
electrophoresis by in-gel fluorescence scanning. On the basis of a detailed kinetic analysis of the cross-
linking reaction, we showed that the cross-linking efficiency can be used as an indicator of interaction
between two proteins, allowing thereby the unambiguous identification of interacting protein pairs. We
validated our approach by confirming a number of interactions through selective cross-linking and showed
that it permits the quantitative and simultaneous analysis of multiple homotypic and heterotypic protein
complexes and the differentiation between strong and weak protein-protein interactions.
Introduction
(iii) immunoprecipitation of protein complexes from cell lysates
(coimmunoprecipitation or co-IP).8
Protein-protein interactions play key roles in all biological
processes from the regulation of signaling and metabolic
pathways to the formation of multiprotein enzymatic complexes
and cellular structures. Our understanding of these processes
therefore requires the identification and characterization of the
underlying protein-protein interactions. To map the so-called
protein interactome, genome-scale studies based on the yeast-
2-hybrid approach,1 protein arrays,2 or affinity purification
combined with mass spectrometry3 have been undertaken. These
studies have provided large sets of potentially novel protein-
protein interactions that require validation by independent
experiments and further characterization. To do so, a wide range
of techniques is currently available.4 The most popular are: (i)
cell-based assays employing autofluorescent proteins and Fo¨rster
resonance energy transfer (FRET) measurements;5 (ii) protein
complementation assays based on split protein sensors;6,7 and
FRET-based assays permit the study of dynamics and
localization of protein-protein interactions within cells. How-
ever, FRET measurements between two proteins tagged with
autofluorescent proteins are technically demanding and generally
have a low signal-to-noise ratio.9,10 In protein complementation
assays, the two interacting proteins are expressed as fusion
proteins to respectively two fragments of a reporter protein, a
so-called split protein sensor, which is reassembled upon
interaction of the two fusion proteins.6 Different reporter proteins
have been used to design split protein sensors that reveal
protein-protein interactions in living cells.11 Split protein
sensors are widely used now to study protein-protein interac-
tions but have the limitations that the effective formation of
active reporter depends on the geometry of the studied protein
complex and that, with few exceptions,12 the affinity of the
fragments makes the formation of active reporter irreversible.
The latter point prevents the dissociation of protein complexes
and makes the approach less suited for studying dynamic
interactions. Co-IP remains the most heavily used method to
verify protein-protein interactions because of its ability to detect
endogeneous complexes and its technical simplicity. Its main
limitation is that interactions, in particular weak and transient,
can be broken up during the immunoprecipitation procedure.
To address this problem, cross-linking strategies have been
† Present address: Medical Research Council Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 0QH, United Kingdom.
‡ Department of Life System, Institute of Technology and Science,
Graduate School, The University of Tokushima, Minamijosanjimacho-2,
Tokushima 770-8506, Japan.
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439, 168–174.
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Nat. ReV. Drug DiscoVery 2007, 6, 569–582.
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Selective Cross-Linking of Interacting Proteins
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Figure 1. Specific cross-linking (S-CROSS) of interacting proteins. (a) Mechanism of S-CROSS: Cross-linking of CLIP-tag and/or SNAP-tag fusion proteins
with bifunctional molecules in which the substrates of the two tags are connected via a fluorophore (either Cy5 or Cy3). This permits the irreversible
trapping of homo- and heterotypic protein complexes. (b) Experimental protocol: Pairs of proteins are fused to SNAP-tag and CLIP-tag and coexpressed in
mammalian cells. Cells are lysed in the presence of fluorescent bifunctional molecules, and the resulting lysate is then analyzed after SDS-PAGE by in-gel
fluorescence imaging. It should be noted that the mobility of cross-linked, nonlinear proteins in SDS-PAGE can deviate from the mobility that would be
expected for a linear protein of the same molecular weight. (c) Structures of the bifunctional molecules SC-Cy5, SS-Cy5, and SS-Cy3 synthesized for the
cross-linking of protein complexes.
developed to capture complexes by covalent bonding.13 Bis-
maleimide-based cross-linkers are now commercially available
for the cross-linking of cysteine-containing proteins in living
cells (BMOE and BHM, Pierce). However, this approach
suffers from a very poor selectivity of the chemical reaction
as any cysteine-containing protein can react with the cross-
linker.13 Recently, alternative methods based on light-induced
cross-linking of interacting partners have been introduced,
using proteins that incorporate genetically encoded photo-
cross-linker-containing amino acids14,15 or proteins that are
fused to protein tags that can be labeled with photo-cross-
linkers.16 Label transfer technologies that utilize a genetic
tag fused to the protein of interest to transfer a chemical label
(e.g., biotin) onto interacting partners have also been
reported.17,18 However, so far few interactions have been
studied using these techniques, and further studies are needed
to evaluate their generality.
complexes. Here, we introduce a technically simple method that
fulfills these criteria and can be used in a variety of different
organisms. This method enables the detection of protein-protein
interactions in cell lysates through the selective cross-linking
(S-CROSS) of protein complexes. S-CROSS is based on the
coexpression of proteins fused to SNAP-tag19 or CLIP-tag,20
two self-labeling tags that can be specifically and covalently
labeled with synthetic probes. Protein-protein interactions are
detected by lysing cells in the presence of bifunctional molecules
in which the substrates of the two tags are connected via a
fluorophore (Figure 1). The efficiency of the resulting cross-
linking of the fusion proteins depends on their proximity and
can be quantified after SDS gel electrophoresis by in-gel
fluorescence scanning. The use of two self-labeling tags with
nonoverlapping substrate specificity ensures selectivity and
directionality of the cross-linking reaction between different
proteins, facilitating the identification of multiple protein
complexes. We have previously shown that the cross-linking
of two SNAP-tag fusion proteins can be used as an indicator of
protein proximity, but detection required Western blotting and
experiments were restricted to SNAP-tag fusion proteins, which
significantly limited the practicality of the approach.21
In summary, although there are numerous methods available
for studying protein-protein interactions, there is a need for
methods that permit the analysis of multiple interacting partners
in a single experiment and the study of the stability of protein
In this work, we demonstrate the following key features of
S-CROSS: (i) the possibility to simultaneously analyze multiple
interactions with high selectivity; (ii) the possibility to dif-
(13) Agou, F.; Ye, F.; Veron, M. Methods Mol. Biol. 2004, 261, 427–442.
(14) Farrell, I. S.; Toroney, R.; Hazen, J. L.; Mehl, R. A.; Chin, J. W. Nat.
Methods 2005, 2, 377–384.
(15) Hino, N.; Okazaki, Y.; Kobayashi, T.; Hayashi, A.; Sakamoto, K.;
Yokoyama, S. Nat. Methods 2005, 2, 201–206.
(16) Baruah, H.; Puthenveetil, S.; Choi, Y. A.; Shah, S.; Ting, A. Y. Angew.
Chem., Int. Ed. 2008, 47, 7018–7021.
(19) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.;
Johnsson, K. Nat. Biotechnol. 2003, 21, 86–89.
(17) Fernandez-Suarez, M.; Chen, T. S.; Ting, A. Y. J. Am. Chem. Soc.
2008, 130, 9251–9253.
(20) Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R.; Kindermann, M.;
Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128–136.
(21) Lemercier, G.; Gendreizig, S.; Kindermann, M.; Johnsson, K. Angew.
Chem., Int. Ed. 2007, 46, 4281–4284.
(18) Liu, B.; Archer, C. T.; Burdine, L.; Gillette, T. G.; Kodadek, T. J. Am.
Chem. Soc. 2007, 129, 12348–12349.
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Figure 2. Inter- (a) and intramolecular (b) cross-linking reactions. The rate constants of the different steps of the intermolecular (a) and intramolecular (b)
cross-linking reactions are reported. Rate constants are given with a 95% confidence interval. The effective molarity of the cross-linking reaction within the
SNAP-FKBP-rapamycin-CLIP-FRB ternary complex was calculated by dividing the rate constants of the intramolecular cross-linking step by that of the
intermolecular counterpart. We found an effective molarity of about 100 µM (ratio k′2/k2 or k′4/k4). Kinetic constants k1, k2, k3, and k4 as well as k′1, k′2, k′3,
and k′4 were determined from experimental data shown in Figure S1 (Supporting Information). It should be noted that under these conditions SNAP-tag and
CLIP-tag operate well below saturation with respect to substrate concentration, resulting in a linear relationship between the substrate effective concentration
and the reaction rate.
ferentiate between strong and weak interactions; and (iii) the
simplicity of the approach and the possibility to complement
S-CROSS with other SNAP-tag- and CLIP-tag-based methods
to characterize protein-protein interactions.
tag fusion protein (SNAP-FKBP), FRB was expressed as a
CLIP-tag fusion protein (CLIP-FRB), and the cross-linking of
the two purified proteins by SC-Cy5 was studied in the presence
and absence of rapamycin. The cross-linking reaction of SNAP-
FKBP and CLIP-FRB with SC-Cy5 is a two-step process with
two possible routes as outlined in Figure 2. The first route starts
with the reaction of SNAP-FKBP with SC-Cy5 to yield SNAP-
FKBP-Cy5-BC, which then reacts with CLIP-FRB. The second
route starts with the reaction of CLIP-FRB with SC-Cy5 to give
CLIP-FRB-Cy5-BG, which then reacts in a second step with
SNAP-FKBP. We determined the rate constants of the different
steps for the cross-linking in the presence and absence of
rapamycin (Figure 2a,b). The individual rate constants in
absence of rapamycin were determined for each step under
pseudo first-order conditions (Figure S1a,b). The individual rate
constants in the presence of rapamycin were determined from
the analysis of reactions of preformed complex between SNAP-
FKBP and CLIP-FRB complex at varying concentrations of SC-
Cy5 using a multistep kinetic model (Figure S1c). Two
important results emerged from these kinetic studies. First, the
reaction of noninteracting CLIP-FRB or SNAP-FKBP with
SNAP-FKBP-Cy5-BC or CLIP-FRB-Cy5-BG is roughly 20-
fold slower than the reaction of the proteins with free SC-Cy5
(compare k4 to k1 and k2 to k3). The reason is most likely that
the reactive groups BC and BG within SNAP-FKBP-Cy5-BC
and CLIP-FRB-Cy5-BG are shielded by the protein to which
they are attached. Consequently, when two noninteracting
proteins are incubated with an excess of SC-Cy5, each protein
reacts preferentially with SC-Cy5, and very little amount of
cross-linked proteins is formed. For example, when incubating
purified SNAP-FKBP and CLIP-FRB (each 0.2 µM) with 2 µM
of SC-Cy5, no cross-linked product could be detected (Figure
S2a,b). The second important result is that the cross-linking
reaction is significantly faster when the two proteins interact
because of high effective molarity of the tags within the protein
complex. We calculated an effective molarity (Meff) of about
Results and Discussion
Design of Molecules for the Selective Cross-Linking of
Interacting Proteins. The self-labeling protein tags SNAP-tag19
and CLIP-tag20 are two engineered mutants of human O6-
alkylguanine-DNA alkyltransferase (hAGT), which react specif-
ically and rapidly with benzylguanine (BG) and benzylcytosine
(BC) derivatives, respectively. We synthesized bifunctional
substrates for the heterocross-linking of SNAP- and CLIP-tagged
proteins (named SC-Cy5) and for the homo- and heterocross-
linking of two SNAP-tag fusions (named SS-Cy3 and SS-Cy5)
(Figure 1c). SC-Cy5 contains a BG and a BC subunit connected
by a Cy5 dye, while SS-Cy3 and SS-Cy5 are made of two BG
subunits tethered via a Cy3 and Cy5 dye, respectively. The
syntheses of these molecules are described in the Supporting
Information. The cyanine dyes allow for a sensitive detection
by in-gel fluorescence imaging: the detection limit for labeled
proteins in polyacrylamide gel is about 1-10 fmol (data not
shown).
Kinetic Analysis of a Model Cross-Linking Reaction. To test
if these cross-linkers can be used to detect protein-protein
interactions, we studied the kinetics of the cross-linking reaction
between two purified proteins in vitro. As a model system, we
chose the rapamycin-inducible interaction between FKBP (the
FK506 binding protein) and FRB (the FKBP-rapamycin binding
domain of mTOR).22 FKBP and FRB form a stable dimer in
the presence of rapamycin (KD of 12 nM23) but do not interact
in the absence of rapamycin. FKBP was expressed as a SNAP-
(22) Chen, J.; Zheng, X. F.; Brown, E. J.; Schreiber, S. L. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 4947–4951.
(23) Banaszynski, L. A.; Liu, C. W.; Wandless, T. J. J. Am. Chem. Soc.
2005, 127, 4715–4721.
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Figure 3. Detection of hetero- and homotypic interactions. (a-c) HEK 293 cells coexpressing SNAP-FKBP and CLIP-FRB were treated for 1 h with
different concentrations of rapamycin prior to lysis in the presence of 4 µM SC-Cy5. (a) Analysis of cross-linking by SDS-PAGE and in-gel fluorescence
scanning. For clarity, in this and the following figure the names of the cross-linked proteins are given without the name of the tags. (b) Coomassie staining
of gel (a). (c) Cross-linking efficiency (mean ( SD of three independent experiments) determined in (a) versus rapamycin concentration. (d-f) HEK 293
cells expressing SNAP-FKBP were treated for 1 h with different concentrations of AP1510 prior to lysis in the presence of 1 µM SS-Cy5. (d) Analysis of
cross-linking by SDS-PAGE and in-gel fluorescence scanning. (e) Coomassie staining of gel (d). (f) Cross-linking efficiency (mean ( SD of three independent
experiments) determined in (c) versus AP1510 concentration. (c and f) Data were fitted with sigmoidal dose-response equations.
100 µM for the cross-linking reaction of SNAP-FKBP and
CLIP-FRB within the complex by dividing the rate constant of
the cross-linking step within the protein complex by the rate
constant of the cross-linking step between two noninteracting
proteins (k′2/k2 or k′4/k4). The high effective molarity favors
cross-linking over the competing reaction with another molecule
of SC-Cy5. For example, when incubating purified SNAP-FKBP
and CLIP-FRB (each 0.2 µM) with 2 µM of SC-Cy5 in the
presence of rapamycin, 75% of cross-linked proteins and 25%
of labeled monomers are formed, and the reaction is complete
after 20 min (Figure S2c,d). This kinetic analysis explains why
the cross-linking efficiency can be used as an indicator of
interaction between two proteins. It should be noted that the
effective molarity of the tags within a protein complex will
depend on their proximity and relative orientation and will
therefore differ between different protein complexes (vide infra).
Despite the central importance of effective molarities in protein
complexes, few values have been experimentally determined
so far. However, the effective molarity measured here for the
complex between SNAP-FKBP and CLIP-FRB is comparable
to that measured for an inhibitor of human carbonic anhydrase
(HCA) attached to SNAP-tag in a SNAP-mCherry-HCA fusion
protein (Meff ) 80 µM).24
Characterization of Heterotypic Interactions in Cell Extracts.
We next analyzed the rapamycin-dependent interaction between
FKBP and FRB in mammalian cell extracts. SNAP-FKBP and
CLIP-FRB were transiently coexpressed in HEK 293 cells, and
the cells were incubated with varying concentrations of rapa-
mycin. Cells were lysed in the presence of 4 µM of SC-Cy5,
incubated for 1 h at room temperature, and directly analyzed
by SDS-PAGE and in-gel fluorescence scanning (Figure 3a,b).
In this and the following experiments, we used cross-linker
concentrations between 1 and 4 µM so that they exceed the
concentrations of the SNAP and CLIP fusion proteins in the
extract (typically between 5 and 500 nM, see Table S1),
minimizing thereby nonspecific cross-linking (vide supra).
Cross-linking efficiency increased 200-fold with increasing
rapamycin concentration, from 0.3% in the absence of rapa-
mycin to 60% at rapamycin concentrations above 30 nM. The
EC50 of rapamycin was determined to be 6 nM (Figure 3c),
which is in agreement with previously determined values.23
Characterization of Homotypic Interactions in Cell Extracts.
We next showed that S-CROSS can reveal homotypic protein
complexes by cross-linking of SNAP-tag fusion proteins using
SS-Cy5. HEK 293 cells expressing SNAP-FKBP were incubated
with varying concentrations of AP1510, a synthetic molecule
promoting the homodimerization of FKBP,25 and lysed in the
presence of SS-Cy5. The cross-linking efficiency increased from
2.5% in the absence of AP1510 to 60% at AP1510 concentra-
tions above 300 nM (Figure 3d-f).
Simultaneous Detection of Multiple Interactions. The ap-
proach furthermore permits the simultaneous detection of
multiple protein-protein interactions in a single experiment.
To demonstrate this, we incubated HEK 293 cells coexpressing
SNAP-FKBP and CLIP-FRB with a fixed concentration of
AP1510 and varying concentrations of rapamycin. In the
presence of AP1510 and absence of rapamycin, SNAP-FKBP
should be mainly as a homodimer, whereas addition of rapa-
mycin should shift the equilibrium toward the more stable
SNAP-FKBP-CLIP-FRB heterodimer. Cells were lysed in the
presence of both SC-Cy5 and SS-Cy3 to simultaneously trap
the SNAP-FKBP-CLIP-FRB heterodimer and the homodimer
of SNAP-FKBP; in-gel fluorescence scanning then permitted
one to unambiguously distinguish the Cy3-cross-linked FKBP
homodimer from the Cy5-cross-linked FKBP-FRB heterodimer
(Figure 4a,b). The data demonstrate how addition of rapamycin
leads to the formation of the FKBP-FRB heterodimer at the
(25) Amara, J. F.; Clackson, T.; Rivera, V. M.; Guo, T.; Keenan, T.;
Natesan, S.; Pollock, R.; Yang, W.; Courage, N. L.; Holt, D. A.;
Gilman, M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10618–10623.
(24) Brun, M. A.; Tan, K. T.; Nakata, E.; Hinner, M. J.; Johnsson, K. J. Am.
Chem. Soc. 2009, 131, 5873–5884.
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Figure 4. Simultaneous detection of several protein-protein interactions. (a,b) HEK 293 cells coexpressing SNAP-FKBP and CLIP-FRB were treated for
1 h with 1 µM AP1510 and different concentrations of rapamycin prior to lysis in the presence of 2 µM SC-Cy5 and 1 µM SS-Cy3. (a) Analysis of
cross-linking by SDS-PAGE and in-gel fluorescence scanning; overlay of Cy3 (green) and Cy5 (red) channels. (b) Cross-linking efficiencies (mean ( SD
of three independent experiments) determined in (a) versus rapamycin concentration. (c,d) HEK 293 cells coexpressing SNAP-FKBP, CLIP-FRB, and
CLIP-ALK5 were treated for 1 h with different concentrations of rapamycin prior to lysis in the presence of 4 µM SC-Cy5. (c) Analysis of cross-linking by
SDS-PAGE and in-gel fluorescence scanning. (d) Cross-linking efficiency (mean ( SD of two independent experiments) determined in (c) versus rapamycin
concentration. (b and d) Data were fitted with sigmoidal dose-response equations.
expense of the homodimer of FKBP. This experiment illustrates
moreover how the combined use of SC-Cy5 and SS-Cy3 enables
the detection of different cross-linked protein complexes with
the same mobility in SDS-PAGE.
tivation domain, promoting its ubiquitinylation and degradation
by the proteasome. The p53-Mdm2 interaction can be disrupted
by the Mdm2 antagonist nutlin-3a, a cis-imidazoline analogue
that binds to the p53-binding site of Mdm2.27 We were able to
detect the interaction of SNAP-Mdm2 and CLIP-p53 through
S-CROSS in cell lysates and to demonstrate that prior incubation
of the cells with nutlin-3a at concentrations above 1 µM
disrupted the p53-Mdm2 interaction (Figure 5a,b). S-CROSS
furthermore enabled us to analyze the oligomeric state of p53
and Mdm2 as well as the interaction between the two proteins
simultaneously. p53 forms a tetramer (which is best described
as a dimer of dimers28), whereas Mdm2 forms dimers through
a RING domain interaction.29 By coexpressing p53 and Mdm2
as SNAP-tag fusions and cross-linking with SS-Cy5, we detected
three different protein complexes in a single experiment: the
two homotypic (SNAP-p53)2 and (SNAP-Mdm2)2 complexes
and the heterotypic SNAP-Mdm2-SNAP-p53 complex; of these
only the SNAP-Mdm2-SNAP-p53 complex was sensitive to
nutlin-3a (Figure 5c). Deleting the tetramerization domain of
SNAP-p53, yielding SNAP-p53∆, prevented its efficient cross-
linking with SS-Cy5 (Figure 5d) but did not affect its interaction
with CLIP-Mdm2 (Figure 5e). These experiments illustrate the
versatility of S-CROSS for the facile and simultaneous analysis
of multiple protein-protein interactions, and the complemen-
tarity of SS-Cy5 and SC-Cy5 to discriminate hetero- and
homocross-linked complexes.
The ability to simultaneously detect and quantify two
interactions in one experiment is an important feature of
S-CROSS. We therefore attempted to demonstrate more ex-
amples of such applications and in particular to extend S-CROSS
to the simultaneous analysis of interactions between more than
two proteins. A natural binding partner of FKBP is the
transforming growth factor ꢀ type 1 receptor ALK5.26 This
interaction is disrupted by the rapamycin-dependent complex
formation between FKBP and FRB.22 HEK 293 cells coex-
pressing SNAP-FKBP, CLIP-FRB, and the cytosolic domain
of ALK5 fused to CLIP-tag were incubated with varying
concentrations of rapamycin and lysed in the presence of SC-
Cy5. The results illustrate how rapamycin disrupts the SNAP-
FKBP-CLIP-ALK5 interaction and induces the formation of the
SNAP-FKBP-CLIP-FRB complex (Figure 4c,d). It should be
noted that it is possible to detect the exchange of SNAP-FKBP
binding partners only because there is no interaction between
the two tags prior to addition of the cross-linker. This is an
important difference between S-CROSS and most split protein
sensors.
We next applied S-CROSS to the tumor suppressor p53, a
transcription factor involved in apoptosis, DNA repair, cell cycle
arrest, and senescence. The activity of p53 in normal cells is
regulated through its interaction with the E3 ubiquitin ligase
Mdm2. Mdm2 controls p53 by binding its N-terminal transac-
(27) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.;
Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi,
N.; Liu, E. A. Science 2004, 303, 844–848.
(28) Joerger, A. C.; Fersht, A. R. Annu. ReV. Biochem. 2008, 77, 557–82.
(29) Poyurovsky, M. V.; Priest, C.; Kentsis, A.; Borden, K. L. B.; Pan,
Z. Q.; Pavletich, N.; Prives, C. EMBO J. 2007, 26, 90–101.
(26) Wang, T. W.; Donahoe, P. K.; Zervos, A. S. Science 1994, 265, 674–
676.
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Figure 5. (a,b) HEK 293 cells coexpressing SNAP-Mdm2 and CLIP-p53 were treated for 1 h with different concentrations of nutlin-3a prior to lysis in the
presence of 4 µM SC-Cy5. For clarity, the names of the cross-linked proteins are given without the name of the tags. (a) Analysis of cross-linking by
SDS-PAGE and in-gel fluorescence scanning. (b) Cross-linking efficiency (mean ( SD of three independent experiments) versus nutlin-3a concentration.
Data were fitted with a sigmoidal dose-response equation. (c) HEK 293 cells coexpressing SNAP-Mdm2 and SNAP-p53 were incubated with or without
10 µM nutlin-3a for 1 h prior to lysis in the presence of 1 µM SS-Cy5 followed by analysis of cross-linking by SDS-PAGE and in-gel fluorescence scanning.
(d) HEK 293 cells expressing SNAP-p53 or SNAP-p53∆ were lysed in the presence of 1 µM of SS-Cy5 and subsequently analyzed by SDS-PAGE and
in-gel fluorescence scanning. (e) HEK 293 cells coexpressing SNAP-p53∆ and CLIP-Mdm2 were treated with 0 or 10 µM of nutlin-3a for 1 h prior to lysis
with 4 µM of SC-Cy5 and subsequently analyzed by SDS-PAGE and in-gel fluorescence scanning.
Figure 6. Detection of weak interactions. (a) HEK 293 cells expressing SNAP-FKBP-F36M were incubated with or without 1 µM of different ligands
(FK506, rapamycin) for 1 h and then lysed in the presence of 1 µM SS-Cy5 and subsequently analyzed by SDS-PAGE and in-gel fluorescence scanning. The
concentration of SNAP-FKBP-F36M in the extract was 250 nM. The ligands FK506 and rapamycin disrupt the homodimer and serve as control. (b) Efficiency
of the cross-linking of SNAPFKBP-F36M and SNAP-p53 tetramer with 1 µM SS-Cy5 at different concentrations of fusion protein in lysates. Gels are shown
on the right of the panel.
Detection of Weak Protein-Protein Interactions. Weak
protein-protein interactions can be difficult to detect by
conventional approaches such as affinity purification.30 In
contrast, S-CROSS permits the detection of weak interactions
as the cross-linking reaction can trap a complex that is in rapid
equilibrium with its monomers. As an example for a weak
interaction, we characterized the homodimer formed by the
FKBP mutant FKBP-F36M. The mutation F36M induces the
dimerization of FKBP with an associated KD of 30 µM.31
Incubating lysates of HEK 293 cells expressing SNAP-FKBP-
F36M with SS-Cy5 revealed that at SNAP-FKBP-F36M con-
centration of 250 nM the cross-linking efficiency reached 49%
(Figure 6a), although, at that concentration, only 2% of the
complex should be present at the equilibrium if KD ) 30 µM.
This means that the protein complex is enriched during the cross-
linking process. Modeling the influence of protein concentration,
KD, and effective molarity on the cross-linking efficiency
revealed that the observed cross-linking efficiency for SNAP-
FKBP-F36M can be rationalized by assuming an effective
molarity for the cross-linking reaction within the homodimer
of about 500 µM (Figure S3). This value is comparable to the
effective molarity experimentally determined for the cross-
linking reaction within the SNAP-FKBP-CLIP-FRB complex
(vide supra). The higher effective molarity value predicted in
the case of SNAP-FKBP-F36M is consistent with the shorter
distance between the N-termini in the homodimer of FKBP-
F36M (38 Å) as compared to the FKBP-FRB heterodimer (49
Å) (Figure S3d). Our experiments and the modeling suggest
that weak interactions with KD of at least 30 µM can be detected
by S-CROSS if the effective molarity for the cross-linking
reaction within the protein complex is greater than 100 µM and
(30) Berggard, T.; Linse, S.; James, P. Proteomics 2007, 7, 2833–42.
(31) Rollins, C. T.; Rivera, V. M.; Woolfson, D. N.; Keenan, T.; Hatada,
M.; Adams, S. E.; Andrade, L. J.; Yaeger, D.; van Schravendijk, M. R.;
Holt, D. A.; Gilman, M.; Clackson, T. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 7096–7101.
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Figure 7. Characterization of the MEK1-ERK2 interaction by studying protein colocalization and selective cross-linking. (a) CHO-K1 cells expressing
ERK2-CLIP were labeled for 30 min with BC-505 (10 µM). Cells expressing SNAP-MEK1 were labeled for 30 min with TMR-star (2 µM). Cells coexpressing
ERK2-CLIP and SNAP-MEK1 were labeled for 30 min with BC-505 (10 µM) and TMR-star (2 µM). Cells were fixed with formaldehyde after labeling and
imaged by confocal microscopy. Green channel: detection of ERK2-CLIP labeled with BC-505. Red channel: detection of SNAP-MEK1 labeled with TMR-
star. Structures of TMR-star and BC-505 are shown in Figure S4. (b) HEK 293 cells coexpressing SNAP-MEK1/ERK2-CLIP were lysed in the presence of
4 µM SC-Cy5 or in the presence of BG-647 and BC-647 (both 5 µM) and subsequently analyzed by SDS-PAGE and in-gel fluorescence scanning; Cy5
channel shown. Control experiments were performed coexpressing the two proteins with FKBP fusions that should not interact with the two proteins:
SNAP-MEK1 with CLIP-FKBP (lane 2), ERK2-CLIP with SNAP-FKBP (lane 3), and SNAP-MEK1, ERK2-CLIP, and SNAP-FKBP (lane 4).
the protein concentration in the sample is in the high nanomolar
range (Figure S3c). Calculations showed that the cross-linking
efficiency of a weak interaction should be strongly concentra-
tion-dependent at concentrations below the KD. Indeed, dilution
of SNAP-FKBP-F36M in the lysate led to a drastic drop of the
cross-linking efficiency (Figure 6b). In contrast, the cross-linking
efficiency in the case of the strong tetrameric SNAP-p5332 did
not show any significant drop upon dilution to concentrations
of about 1 nM (Figure 6b). The possibility to measure the cross-
linking efficiencies of interacting proteins as a function of their
concentrations is a convenient way to distinguish weak interac-
tions from strong ones.
Combining S-CROSS with Other SNAP-tag and CLIP-tag-
Based Methods. As SNAP- and CLIP-tag fusions can be labeled
with various membrane permeable fluorescent probes in living
cells, it is possible to complement S-CROSS experiments in
lysates with colocalization studies20 or FRET measurements33
within cells. To illustrate this, we studied the interaction of the
two kinases ERK2 and MEK134 by characterizing their colo-
calization through fluorescence microscopy and their physical
interaction by S-CROSS (Figure 7). The formation of the stable
complex between ERK2 and MEK1 results in a change of ERK2
localization, which is predominantly nuclear when not bound
to MEK1 and predominantly cytosolic when bound to MEK1.34
The fluorescence microscopy images shown in Figure 7a confirm
how expression of SNAP-MEK1 influences the localization of
ERK2-CLIP: ERK2-CLIP, which is predominantly localized in
the nucleus when expressed alone, is localized in the cytosol
when coexpressed with SNAP-MEK1. To validate that the
colocalization of the two proteins is due to a molecular
interaction, we performed S-CROSS experiments on cells
coexpressing SNAP-MEK1 and ERK2-CLIP. In these experi-
ments, a cross-linking efficiency of 80% between the two
proteins was observed (Figure 7b, lane 1). We furthermore
carried out control experiments by coexpressing SNAP-MEK1
and ERK2-CLIP together with SNAP-FKBP as an internal
control: we observed cross-linking between ERK2-CLIP and
SNAP-MEK1 but not between ERK2-CLIP and SNAP-FKBP,
demonstrating thereby the specific interaction between ERK2
and MEK1 (Figure 7b). The possibility to perform selective
controls in the same cells and in parallel to the actual experiment
is another important feature of S-CROSS that sets it apart from
other methods to study protein-protein interactions.
Conclusion
We have introduced a new method named S-CROSS for the
detection and characterization of protein-protein interactions
in cell extracts by specific covalent cross-linking. This approach
relies on the use of the self-labeling proteins SNAP-tag and
CLIP-tag to specifically and directionally cross-link interacting
proteins. The use of cross-linkers that incorporate fluorophores
allows the sensitive detection of protein complexes by in-gel
fluorescence imaging. This method should be applicable in all
cells or organisms amenable to genetic manipulation. As
compared to affinity purification methods, S-CROSS requires
the expression of both partners as fusion proteins, but it is
technically simpler and requires lower amount of material.
Moreover, S-CROSS can be used to detect interactions of
proteins expressed at level as low as 1 pmol/mg of total protein
(see Table S1 for examples), which is in the range of expression
(32) Natan, E.; Hirschberg, D.; Morgner, N.; Robinson, C. V.; Fersht, A. R.
Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 14327–14332.
(33) Maurel, D.; Comps-Agrar, L.; Brock, C.; Rives, M. L.; Bourrier, E.;
Ayoub, M. A.; Bazin, H.; Tinel, N.; Durroux, T.; Prezeau, L.; Trinquet,
E.; Pin, J. P. Nat. Methods 2008, 5, 561–567.
(34) Burack, W. R.; Shaw, A. S. J. Biol. Chem. 2005, 280, 3832–3837.
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Selective Cross-Linking of Interacting Proteins
A R T I C L E S
levels found for endogenous proteins in mammalian cells.35,36
It should be possible to detect interactions of proteins expressed
at even lower expression levels by combining S-CROSS with
affinity purification to enrich the cross-linked proteins before
SDS-PAGE analysis. A limitation shared with other tag-based
approaches like FRET and protein complementation assays is
that the interpretation of S-CROSS results can be biased if the
geometry of the protein complex prevents the reaction of the
tags, leading to false negatives. However, S-CROSS has features
that make it attractive as compared to other techniques: (i) the
ease with which multiple homotypic and heterotypic interactions
can be simultaneously characterized, (ii) the ability to detect
strong and weak interactions, and (iii) the possibility to exploit
the versatility of chemical labeling to complement the cross-
linking studies with live cell imaging or other fluorescence-
based approaches.20 In summary, S-CROSS is a versatile,
sensitive, and simple method for the analysis of protein-protein
interactions.
expression of SNAP-p53∆, p53∆ (first 326 amino acids of p53)
was fused to SNAP-tag via a PAG linker. The gene encoding the
fusion protein was inserted between the EcoRI and BamHI sites of
the pSEMS1-SNAP26m plasmid (Covalys BioSciences). For
expression of CLIP-ALK5, the cytosolic domain of ALK5 (from
amino acid 162 to 503) was fused to the C-terminus of CLIP-tag
via a PAGYPYDVPDYA linker. The gene encoding the fusion
protein was inserted between the EcoRI and XhoI sites of pSEMS1-
SNAP26m plasmid (Covalys BioSciences). For expression of
SNAP-MEK1, MEK1 was fused to the C-terminus of SNAP-tag
via a PAGIGAPGSSTSLYKKAGT linker. The gene encoding the
fusion protein was inserted between the EcoRI and XhoI sites of
pSEMS1-SNAP26m plasmid (Covalys BioSciences). For expression
of ERK2-CLIP, ERK2 was fused to the N-terminus of CLIP-tag
via a DIEFAS linker. The gene encoding the fusion protein was
inserted between the ClaI and SbfI sites of the pCEMS1-CLIP10m
plasmid (Covalys BioSciences). For expression in E. coli and Ni-
NTA purification of (His)6-SNAP-FKBP and (His)6-CLIP-FRB,
FKBP and FRB were fused to the C-terminus of SNAP-tag and
CLIP-tag via a RSYPYDVPDYA linker. The genes encoding the
fusions proteins were inserted between the NdeI and BamHI sites
of the vector pET-15b (Novagen). Expression and purification of
the fusion proteins was done as previously described.39 All
constructs were verified by DNA sequencing.
Materials and Methods
Synthesis. Detailed synthetic procedures and characterizations
for all synthetic precursors are described in the Supporting
Information.
Cell Culture and Transfection. Human embryonic kidney
(HEK)-293 cells were cultured in suspension in ExCell-293 medium
(Sigma-Aldrich) supplemented with 4 mM L-glutamine (Lonza) at
37 °C in a 5% CO2 atmosphere. Adherent Chinese hamster ovary
(CHO)-K1 cells were cultured in Ham’s F12 (Lonza) supplemented
with 10% FBS (Lonza). HEK-293 cells were transiently transfected
with polyethylenimine as previously described.20 CHO-K1 were
transiently transfected with Lipofectamine (Invitrogen) according
to the manufacturer’s protocol.
General. SNAP-tag and CLIP-tag substrates were obtained from
Covalys BioSciences and New England Biolabs. Rapamycin and
FK506 were purchased from Sigma-Aldrich. AP1510 was obtained
from ARIAD. Nutlin-3a was purchased from Cayman Chemical.
SNAP-tag and CLIP-tag used in this work are 182-amino-acid
mutants of the wild-type human O6-alkylguanine-DNA alkyltrans-
ferase in which the last 25 amino acids were deleted and the
following mutations were introduced: for SNAP-tag,37 K32I, L33F,
C62A, Q115S, Q116H, K125A, A127T, R128A, G131K, G132T,
M134L, R135S, C150Q, S151G, S152D, G153L, A154D, N157G,
and S159E; and for CLIP-tag,20 K32I, L33F, M60I, C62A, Y114E,
Q115S, Q116H, A121 V, K125A, A127T, R128A, G131N, G132T,
M134L, R135D, C150Q, S151G, S152D, G153S, A154D, N157P,
and E159L. The fluorescence gel images were recorded with a
Pharos FX molecular imager (Bio-Rad) and analyzed with Quantity
One software (Bio-Rad). If not mentioned, experimental data were
fitted with Prism software package (GraphPad Software). Simula-
tions were performed with the DYNAFIT software.38
Plasmid Constructions. For mammalian expression of SNAP-
FKBP and CLIP-FRB, FKBP and FRB were fused to the C-
terminus of SNAP-tag or CLIP-tag via a RSYPYDVPDYA linker.
The genes encoding the fusion proteins were inserted between the
NheI and BamHI sites of the pECFP-Nuc plasmid (Clontech). For
the expression of SNAP-FKBP-F36M, FKBP-F36M was generated
by PCR from FKBP and then fused to the C-terminus of SNAP-
tag via a RSYPYDVPDYA linker. The gene encoding the fusion
protein was inserted between the NheI and BamHI sites of the
pECFP-Nuc plasmid (Clontech). For expression of SNAP-Mdm2
and CLIP-Mdm2, full length Mdm2 was fused to the C-terminus
of SNAP-tag and CLIP-tag via a RS linker. The genes encoding
the fusion proteins were inserted between the NheI and BamHI sites
of the pECFP-Nuc plasmid (Clontech). For expression of SNAP-
p53 and Clip-p53, full length p53 epitope-tagged at the C-terminus
with a Flag-tag (DYKDDDD) was fused to the C-terminus of
SNAP-tag or CLIP-tag via a PAG linker. The genes encoding the
fusion proteins were inserted between the EcoRI and BamHI sites
of the pSEMS1-SNAP26m plasmid (Covalys BioSciences). For
S-CROSS in Cell Extracts. At 24 h after transfection, samples
of one million HEK-293 cells transfected with CLIP-tag and/or
SNAP-tag constructs were lysed in 50 µL of buffer 1 (100 mM
KH2PO4, 150 mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100, pH
7.0, protease inhibitor cocktail) or buffer 2 (50 mM HEPES, 25
mM NaCl, pH 7.2, protease inhibitor cocktail) supplemented with
the cross-linker (see figure legends for concentrations) by perform-
ing three cycles of freezing and thawing. Total protein concentra-
tions in cell extracts were determined by Bradford assay. Cell
extracts were incubated for 1 h at room temperature before addition
of SDS loading buffer and boiling at 95 °C. Samples were then
analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
and in-gel fluorescence scanning. The concentrations of the fusion
proteins in cell extract were estimated by labeling an aliquot of
lysate with 5 µM BG-647, a SNAP-tag substrate based on the
Dyomics dye DY-647, and 5 µM BC-647, a CLIP-tag substrate
based on the Dyomics dye DY-647, and comparison of the
fluorescence intensity of the labeled monomers with that of a known
quantity of recombinant SNAP-tagged protein labeled with BG-
647. For each experiment, the lysis buffer, the quantity of extract
loaded on gel, and the concentration of each fusion proteins in nM
and pmol/(mg of total protein) are listed in Table S1 (Supporting
Information). The concentration in nM gives the molar concentration
of fusion protein in the lysate, while the concentration in pmol/
(mg of total protein) is a measure of the expression level of the
fusion protein. The cross-linking efficiency (CLE) was determined
using the following equations and assuming that the different labeled
species have the same fluorescence properties.
(35) Mourant, J. R.; Yamada, Y. R.; Carpenter, S.; Dominique, L. R.;
Freyer, J. P. Biophys. J. 2003, 85, 1938–47.
For the cross-linking between SNAP-tagged and CLIP-tagged
monomers within a heterodimer:
(36) Fujioka, A.; Terai, K.; Itoh, R. E.; Aoki, K.; Nakamura, T.; Kuroda,
S.; Nishida, E.; Matsuda, M. J. Biol. Chem. 2006, 281, 8917–26.
(37) Gronemeyer, T.; Chidley, C.; Juillerat, A.; Heinis, C.; Johnsson, K.
Protein Eng. Des. Select. 2006, 19, 309–316.
(39) Sielaff, I.; Arnold, A.; Godin, G.; Tugulu, S.; Klok, H.-A.; Johnsson,
(38) Kuzmic, P. Anal. Biochem. 1996, 237, 260–273.
K. ChemBioChem 2006, 7, 194–202.
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Gautier et al.
CLEhetero ) Fcross-linked heterodimer/(Fcross-linked heterodimer
+
The reaction progress curve (fluorescence intensity of the cross-
linked dimer vs time) was fitted to a pseudo first-order reaction
model. Second-order rate constants were then obtained by dividing
the pseudo first-order constants by the concentration of free protein.
Rate constants are given with a 95% confidence interval.
qFlabeled SNAP-monomer
)
where Fcross-linked heterodimer is the fluorescence intensity of the band
corresponding to the cross-linked heterodimer, Flabeled
SNAP-monomer
Determination of the Rate Constants k′1, k′2, k′3, and k′4 of
the Intramolecular Cross-Linking Process. Recombinant (His)6-
SNAP-FKBP and (His)6-CLIP-FRB (each 0.2 µM) were preincu-
bated with 5 µM rapamycin to form the ternary complex, and then
incubated with different concentrations of SC-Cy5 (1, 2, 4, 6, and
10 µM) in reaction buffer (50 mM HEPES, 1 mM dithiothreitol,
0.1 mg/mL bovine serum albumin, pH 7.2) at 24 °C. Aliquots were
taken at different times (2.5, 5, 10, 15, 20, 40, and 80 min), boiled
at 95 °C for 5 min in SDS loading buffer containing 100 µM
benzylguanine and 100 µM bromothenylcytosine (SNAP-tag and
CLIP-tag blockers) to stop the reaction, and analyzed by SDS-
PAGE. Experiments were done in duplicates. The fluorescence
intensities of all of the Cy5-labeled species (labeled SNAP-FKBP,
labeled CLIP-FRB, and cross-linked SNAP-FKBP and CLIP-FRB)
were determined for the different times and the five concentrations
of cross-linker by in-gel fluorescence scanning. The reaction
progress curves (fluorescence intensity vs time) of the cross-linked
dimer formation and the direct labeling of SNAP- and CLIP-tagged
monomers for the five cross-linker concentrations (set of 210
experimental points) were fitted with DYNAFIT software38 to
determine the rate constants using the model given in Figure 3b.
This kinetic model assumes a tight ternary complex FKBP-
rapamycin-FRB that does not dissociate during the reaction. The
script used for fitting is given in the Supporting Information. Rate
constants are given with a 95% confidence interval.
Microscopy. At 24 h after transfection, CHO-K1 cells seeded
in µ-Dish (Ibidi) and transfected with CLIP-tag and/or SNAP-tag
constructs were labeled with 10 µM BC-505, a CLIP-tag substrate
based on the Dyomics dye DY-505, and/or 2 µM TMR-star, a
SNAP-tag substrate based on tetramethylrhodamine, in culture
medium for 30 min. After three washings with culture medium,
cells were fixed with 4% formaldehyde solution for 20 min and
washed twice with phosphate buffered saline. Cells were imaged
with a confocal Leica SP5 white laser equipped with an oil
immersion objective HCX PL APO 63×/1.40-0.60. Fluorescence
emission was measured between 500-560 nm for DY-505 (excita-
tion wavelength: 488 nm) and 560-660 nm for TMR (excitation
wavelength: 540 nm).
is the fluorescence intensity of the band corresponding to the labeled
SNAP-tagged monomer, and q is the ratio [CLIP-tagged protein]/
[SNAP-tagged protein] when [CLIP-tagged protein] < [SNAP-
tagged protein], and is equal to 1 when [CLIP-tagged protein] g
[SNAP-tagged protein]. q was determined by measuring the ratio
of the fluorescence intensities of the monomers labeled with BG-
647 and BC-647 in a separate experiment (see above), assuming
that the two labeled monomers have the same fluorescence
properties.
For the cross-linking of SNAP-tagged monomers within a
homodimer:
CLEhomo ) 2Fcross-linked homodimer/(2Fcross-linked homodimer
Flabeled SNAP-monomer
+
)
homodimer
where Fcross-linked
is the fluorescence intensity of the
band corresponding to the cross-linked homodimer, and
SNAP-monomer
Flabeled
is the fluorescence intensity of the band
corresponding to the labeled SNAP-tagged monomer.
Kinetic Analysis. Determination of the Rate Constants k1
and k3 of the First Step of the Intermolecular Cross-Linking
Process. Recombinant (His)6-SNAP-FKBP and (His)6-CLIP-FRB
(each 0.2 µM) were incubated with 2 µM SC-Cy5 in reaction buffer
(50 mM HEPES, 1 mM dithiothreitol, 0.1 mg/mL bovine serum
albumin, pH 7.2) at 24 °C. Aliquots were taken at different times,
boiled at 95 °C for 5 min in SDS loading buffer containing 100
µM benzylguanine and 100 µM bromothenylcytosine (SNAP-tag
and CLIP-tag blockers) to stop the reaction, and analyzed by SDS-
polyacrylamide gel electrophoresis and in-gel fluorescence scanning.
Experiments were done in duplicate. The reaction progress curve
(fluorescence intensity of the labeled protein vs time) was fitted to
a pseudo first-order reaction model. Second-order rate constants
were then obtained by dividing the pseudo first-order constants by
the concentration of cross-linker. Rate constants are given with a
95% confidence interval.
Determination of the Rate Constants k2 and k4 of the
Second Step of the Intermolecular Cross-Linking Process.
SNAP-FKBP-Cy5-BC and CLIP-FRB-Cy5-BG were prepared by
incubating 1 µM of recombinant SNAP-FKBP and CLIP-FRB with
5 µM SC-Cy5 for 30 min at room temperature. The labeled proteins
were then purified from excess SC-Cy5 using a centrifugal filter
device (Microcon YM-50, Millipore). The washing step was
repeated twice with reaction buffer (50 mM HEPES, 1 mM
dithiothreitol, 0.1 mg/mL bovine serum albumin, pH 7.2) to remove
all unreacted cross-linker. Solutions containing about 0.25 µM of
SNAP-FKBP-Cy5-BC and CLIP-FRB-Cy5-BG were then incubated
with either 5 µM CLIP-FRB or 5 µM SNAP-FKBP at room
temperature. Aliquots were taken at different times, boiled at 95
°C for 5 min in SDS loading buffer containing 100 µM benzylgua-
nine and 100 µM bromothenylcytosine (SNAP-tag and CLIP-tag
blockers) to stop the reaction, and analyzed by SDS-PAGE and
fluorescence gel imaging. Experiments were done in duplicates.
Acknowledgment. We thank Dr. D. Maurel for technical
assistance, Prof. C. H. Heldin (Uppsala, Sweden) for ALK5 cDNA,
and ARIAD Pharmaceuticals for AP1510. This work was supported
by the European Research Training Network PROSA (AG), a JSPS
Research Fellowship for Research Abroad (EN), a FEBS long-term
fellowship (GL), and the Swiss National Science Foundation.
Supporting Information Available: Additional methods and
materials, Figures S1, S2, S3, and S4, Table S1, and complete
refs 1 and 3. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA907818Q
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