Design of a photoswitchable cadherin

Article abstract of DOI:10.1021/ja404992r

There is a growing interest in engineering proteins whose function can be controlled with the spatial and temporal precision of light. Here, we present a novel example of a functional light-triggered switch in the Ca-dependent cell-cell adhesion protein E-cadherin, created using a mechanism-based design strategy. We report an 18-fold change in apparent Ca²⁺ binding affinity upon illumination. Our results include a detailed examination of functional switching via linked changes in Ca²⁺ binding and cadherin dimerization. This design opens avenues toward controllable tools that could be applied to many long-standing questions about cadherin's biological function in cell-cell adhesion and downstream signaling.

Full text of DOI:10.1021/ja404992r

Communication
pubs.acs.org/JACS
Design of a Photoswitchable Cadherin
Ryan S. Ritterson,*,†,‡ Kristopher M. Kuchenbecker,† Michael Michalik,‡,† and Tanja Kortemme†,‡
†Graduate Group in Biophysics & ‡California Institute for Quantitative Biomedical Research and Department of Bioengineering and
Therapeutic Sciences, University of California, San Francisco, San Francisco, California 94158, United States
S
* Supporting Information
ABSTRACT: There is a growing interest in engineering
proteins whose function can be controlled with the spatial
and temporal precision of light. Here, we present a novel
example of a functional light-triggered switch in the Ca-
dependent cell−cell adhesion protein E-cadherin, created
using a mechanism-based design strategy. We report an
18-fold change in apparent Ca2+ binding affinity upon
illumination. Our results include a detailed examination of
functional switching via linked changes in Ca2+ binding
and cadherin dimerization. This design opens avenues
toward controllable tools that could be applied to many
long-standing questions about cadherin’s biological
function in cell−cell adhesion and downstream signaling.
here has been considerable interest in light-based control of
1
Tbiological systems, and successful applications include
light-modulation of neuronal ion channels,2 light-switchable
signaling proteins,3 and light-controlled protein targeting.4
Light-based methods offer titratable, precise spatial and temporal
regulation that has been demonstrated in vitro,5 in cell culture,4,6
and in whole animals.7 Most examples of light-based control fall
into one of two categories: (a) those that are genetically encoded
using a recombinantly produced protein borrowed from nature,4
and (b) those created via targeted insertion of amino acids into a
protein sequence and subsequent reaction with them of an
exogenously introduced photoisomerizable small molecule,
typically azobenzene based.8 Azobenzene and related molecules
undergo a reversible cis−trans isomerization when exposed to
specific wavelengths of light, and this change in molecular shape
Figure 1. (A) A cartoon showing the basis of our design. As designed,
can be coupled to changes in protein function. While in (a) the
functional design is already provided naturally, one is limited
our photoswitch reduces Ca2+ binding affinity, which, in turn, reduces
homodimer affinity. (B) BSBCA undergoes a reversible cis/trans
both by the function (e.g., modulation of protein−protein
isomerization when illuminated with specific wavelengths of light. (C)
binding, tuning fluorescence intensity) already encoded by the
EC12 structure showing the region targeted for photoswitchability.
natural gene and by the requirement to fuse the natural protein
with the protein to be modulated. In contrast, the designs in (b)
allow many types of functional modulation, such as changes in
agonist binding, protein−protein binding, and protein folding. In
this work, we used a new strategy where changes in protein−ion
affinity couple to protein dimerization, in the cell−cell adhesion
protein cadherin (Figure 1A).
Cadherins are a key family of Ca-dependent cell−cell adhesion
proteins and are divided into several subtypes, including the most
commonly studied subtype, the classical cadherins. Classical
cadherins, which include E-, N-, P-, R-, and C-cadherin,9 are
composed of an intracellular domain, a transmembrane helix, and
five, repeated, extracellular domains labeled EC1 (N-terminal,
membrane distal) to EC5 (C-terminal, membrane proximal),
Labels indicate the design considerations. (D) Photoswitchability and
reversibility measured by absorbance after many cycles of illumination of
X-EC12.
along with three Ca2+ binding sites present in the loops at each
extracellular domain boundary.10,11 Calcium binding is required
for cadherin function, as depletion of Ca2+ disrupts cadherin-
mediated cell adhesion;12 the presence of Ca2+ is suggested to
rigidify the cadherin structure, allowing it to multimerize.13
Knockdowns of cadherin significantly slow cell−cell adhesion,14
Received: May 17, 2013
© XXXX American Chemical Society
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Journal of the American Chemical Society
Communication
and in a classic experiment, cadherin-free, nonadherent cells
transfected with cadherin acquire morphological similarities to
naturally adherent cells.15
subsequent illumination at 500−550 nm will reverse the
isomerization and produce a population that is >90% trans.
Illuminating our conjugated protein (X-EC12) at 365 nm with a
hand-held LED showed the characteristic reduction in
absorbance of the trans state. The reverse isomerization (pan-
visual illumination, including 500−550 nm bands) also behaved
as expected, leading to a reappearance of the absorbance band of
the trans state. We illuminated X-EC12 for 10 complete UV-
green illumination cycles without any apparent loss of
absorbance or switchability (Figure 1D); the switchability was
also titratable via shorter illumination times (Figure S3).
Our approach to creating a light-switchable cadherin aimed to
modulate its Ca2+ binding affinity. Because Ca2+ binding is
essential for cadherin multimerization, we reasoned that
reversibly changing Ca2+ binding affinity would be an effective
way to also modulate cadherin adhesive function (Figure 1A).
We designed cysteine residues into the protein to serve as
conjugation sites for an azobenzene-based photoisomerizable
chromophore, BSBCA (Figure 1B). BSBCA has been used in
previous applications,5,16 demonstrating reversible switching
between the cis and trans states when exposed to 370 nm (near
UV) and 550 nm (green) light, respectively.5 Our strategy
involved conjugating both ends of the chromophore to the Ca-
binding loops between cadherin domains EC1 and EC2, as these
Ca2+ sites have previously been shown to be most critical for
function.11 In addition, because the Ca2+ binding sites are located
in loop regions and bind Ca2+ with relatively weak affinities near
20 μM,17 we reasoned it would be easier to induce conforma-
tional changes affecting Ca2+ binding there than in more rigid
secondary structural elements or well-packed core regions of the
protein.
We next tested whether isomerization changes Ca2+ binding
affinity. To do so, we used a previously described mass
spectrometry based assay17 to directly measure the Ca2+ binding
affinity of WT C9A as well as trans and cis X-EC12 (Figures 2A
Because BSBCA spontaneously cross-links cysteine residues,18
the design challenge presented here can be generalized as the
problem of finding the best pair of residues to mutate to cysteine.
In practice, however, an enormous number of pairs are possible,
the overwhelming majority of which are likely to be nonfunc-
tional. We took a sequential and computational approach to
identifying likely functional pairs (Figure S1 and Supporting
Information [SI]). First, we used the program Rosetta19 to
computationally mutate all residues in four representative E-
cadherin structures (PDB identifiers: 1FF5, 1EDH, 2O72, and
1Q1P) to alanine (the simplest mutation) and then calculated
the predicted change in fold stability using a protocol we
developed previously.20,21 Residues with predicted destabiliza-
tion >1 kcal/mol were not considered, as mutations to these
residues they were presumed to be disruptive. Next, we narrowed
the pairs to those that would be geometrically compatible with
the small molecule. We calculated pairwise Cβ−Cβ distances
between the residues remaining using the 1FF5 structure and
kept those pairs that fell in the range 17−20 Å (appropriate for
the BSBCA trans isomer). Finally, the remaining pairs were
ranked for an additional set of structural and geometric
constraints (Figure 1C and SI). Eleven high-ranking pairs
(Table S1) were cloned, expressed, and tested for conjugability
(Figure S2), photoswitchability, and functionality (SI). One pair,
K129C/D138C (Figure 1C, red residues) showed the best
switchability and functionality, and was further characterized in
detail.
We focused on, and expressed, the first two domains of E-
cadherin (EC12), because they contain the homodimeric
binding interfaces22 and are the specificity determining
domains,23 making them most principally responsible for
cadherin’s function. Additionally, the shortened EC12 construct
can be readily produced in high yields in E. coli. EC12 contains a
single native cysteine residue, which we mutated to alanine
(C9A), previously shown not to affect cadherin function.24
We first sought to show that K129C/D138C conjugated with
trans BSBCA (termed X-EC12) could undergo isomerization to
cis. Unconjugated BSBCA in the trans state has an absorbance
peak near 370 nm that decreases when illuminated at this
wavelength, resulting in a population that is 80−90% cis;
Figure 2. Characterization of photoswitchable Ca2+ binding affinity. (A)
Ca2+ binding as monitored by mass spectrometry. While WT and trans
X-EC12 bound three Ca2+ ions specifically, cis X-EC12 showed
considerably weaker, predominantly nonspecific binding (Figure S4
and SI). Fits are based on a model of a single class of binding site for a
maximum of three specifically bound Ca2+ ions. (B) The half-life of the
cis state as a function of Ca2+ concentration, as measured by absorbance.
Error bars are +1 SD from three independent experiments.
and S4; these assays used a cadherin concentration of 2 μM,
significantly below a previously measured homodimeric Kd of
WT cadherin (98.6
15.5 μM),22 to avoid potential
complications due to cadherin dimerization). If isomerization
alters Ca2+ binding, the cis X-EC12 should have weaker affinity
than trans. In addition, because EC12 binds three Ca2+ ions, any
of which could be interfered with, a decrease in apparent
cooperativity as measured by the Hill coefficient (Nh) would be
expected. WT C9A cadherin specifically bound three Ca2+ ions
with a dissociation constant Kd = 28.5 1.9 μM (throughout the
text, errors are the boundaries of a 95% confidence interval unless
otherwise indicated) and extensive cooperativity with Nh = 2.85
0.47, close to previously reported numbers17 of Kd = 20 0.7
μM and Nh = 2.6 0.2. Trans X-EC12 showed 2-fold weaker
affinity and less cooperativity, with Kd = 55.2 5.8 μM and N h=
1.80 0.35, but also bound three Ca2+ ions. In contrast, Ca2+
binding to cis X-EC12 was dominated by nonspecific binding. By
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Communication
using quadruple and higher Ca-bound states from trans X-EC12
(Figure S5) as a reference for nonspecific binding, we subtracted
the estimated contribution of nonspecific Ca2+ binding from the
measured average Ca2+ occupancy for cis X-EC12 (SI). The
resulting line shows significantly reduced binding compared to
trans. (A quantitative fit was not possible due to required Ca2+
concentrations being higher than the dynamic range of the
assay.)
To more directly measure the Ca2+ binding of cis X-EC12, we
turned to a different assay that determined the cis half-life as a
function of Ca2+ concentration. One general caveat inherant to
azobenzene-based strategies is that switching to the cis state is
generally incomplete; i.e., the cis state always contains a minor
trans population.8,18 However, the entirety of any change
observed in half-life experiments is due only to the cis
subpopulation, allowing measurement of pure cis properties
unaffected by the small fraction that remains trans. Therefore, if
chromophore isomerization significantly affects Ca2+ binding in
our conjugated constructs, with stronger binding of Ca2+ to trans
X-EC12, by thermodynamic coupling we would expect to see a
change in the cis X-EC12 half-life with Ca2+ (Figure 2B). The cis
state is thermodynamically unstable, and cis BSBCA relaxes back
to the stable trans state in the dark with a half-life of ∼20 min at
25 °C,18 although conjugation to proteins can alter chromophore
half-lives.5,25 By observing the increase in absorbance at 370 nm
during relaxation of our conjugated constructs back to trans, one
can compute the half-life of the process (Figures S6 and S7; SI;
these assays used a protein concentration of 12 μM to minimize
potential changes in half-life due to protein dimerization). The
half-life should decrease with increasing Ca2+ concentration as
trans X-EC12 becomes stabilized by Ca2+ binding. As expected,
we observed a half-life decrease from ∼72 to 28 min, with an
EC50 of 996 135 μM Ca2+. This represents a nearly 18-fold
change in apparent Ca2+ binding affinity from the 55 μM for trans
X-EC12 (mass spectrometry analysis, Figure 2A).
Figure 3. Characterization of photoswitch homodimeric binding. (A)
Homodimeric binding monitored in SPR as a function of Ca2+
concentration. The data were fit to a Hill equation. Faded points
contain significant nonspecific binding and were not used in the fits.
Responses between flow cells were scaled to minimize a least-squares
difference, and then mean values were normalized such that the fit value
at [Ca2+] = ∞ was 1.0 (SI). Error bars are 1 SD of the three active flow
cells in the instrument after scaling and normalization. Inset shows fits at
low Ca2+ concentrations. (B) Homodimeric binding monitored in SPR
at 1 mM Ca2+, after repeated illumination cycles. Responses between
flow cells were scaled to minimize a least-squares difference. Error bars
are 1 SD of the three active flow cells in the instrument after scaling.
2 SD error, as measured by a bootstrapping analysis of the data,
of 71.2 14 μM; see SI and Figures S10, S11) and Nh = 2.24
(2.45 1.7). In comparison, trans X-EC12 has an EC50 of 156
μM (170 33 μM), with Nh = 1.38 (1.28 0.28). These EC50
values are higher than those measured in the mass spectrometry
assay (Figure 2A), which is due to cadherin binding multiple Ca2+
ions to function, causing any measured EC50 to necessarily be, at
a minimum, a multiple of the protein concentration used, which
here was 40 μM. Strikingly, cis X-EC12 showed substantially
weakened binding (Figure 3A), with EC50 = 619 μM (611 180
μM) and Nh = 0.76 (0.77 0.15), demonstrating a nearly 4-fold
change in Ca2+ affinity under these conditions. (Note: non-
specific protein binding to the SPR chip appeared at Ca2+
concentrations >2 mM for WT C9A and trans X-EC12; see
SI.) The change in protein−protein binding was also reversible as
measured over multiple illumination cycles with 40 μM protein
and 1 mM Ca2+ (Figure 3B).
An alternative explanation for the observed decrease in the
SPR signal upon isomerization to the cis state could be an
increase in cis homodimerization in solution, reducing the
effective concentration of X-EC12 cadherin monomers available
to bind to the WT cadherin immobilized on the chip. To exclude
that possibility, we analyzed X-EC12 cadherin homodimerization
via gel filtration. The observed decrease in the dimer/monomer
ratio after UV illumination additionally confirms the expected
weaker cis homodimerization upon illumination (Figure S12).
Questions remain about the structure of the functional
cadherin multimers, including evidence that cadherin forms
strand-swapped dimers.22,26,27 While we cannot directly
determine the structure of the interacting species formed in
our SPR experiments, each set of SPR traces for a given cadherin
variant can be fit to a single off rate returning to baseline levels,
even at higher Ca2+ concentrations (Figure S13). This behavior is
We also observed a cooperative transition in half-life duration,
with a measured Nh of 2.4 0.74. In showing interdependence
between isomerization and Ca2+ binding, these results indicate
that, as expected, isomerization of the chromophore significantly
weakens Ca2+ binding. In addition, the observed cooperative
nature indicates multiple Ca2+ ions are binding simultaneously
during the transition from cis to trans, hinting that the cis state
likely weakens multiple binding sites.
After successfully demonstrating photoswitchable Ca2+ bind-
ing in our engineered cadherin, we next asked whether the
change in Ca2+ binding affinity also results in the expected change
in protein binding activity. We used surface plasmon resonance
(SPR) to measure protein homodimerization as a function of
Ca2+ concentration (Figures 3, S8, and S9; SI). In this assay,
similar to that of Harrison et al.,22 biotinylated WT C9A cadherin
was immobilized to the SPR chip and WT C9A, trans or cis X-
EC12 were flowed over it. Direct measurements of both Ca2+
affinity and homodimeric protein affinity in SPR are difficult due
to solution homodimers competing with those on the surface,
reducing the effective protein concentration; to minimize
solution homodimerization, we used a protein concentration
(40 μM) below the Kd for homodimerization of WT EC12
cadherin.22 Additionally, cis measurements are of mixed
populations due to the inability of reaching full conversion to
the cis state and some thermal relaxation to trans during the
experiment, limiting the observable fold change in affinity (SI).
We observed a Ca2+ binding EC50 for WT C9A cadherin, as
measured by a single Hill fit, of 72.0 μM (with mean fit values and
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Journal of the American Chemical Society
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cadherin variant.
Taken together, our results demonstrate the successful design
of a reversibly photoswitchable cadherin. When illuminated with
light, its Ca2+ binding affinity changes from 56 to 996 μM, a
nearly 18-fold change, and this change in affinity is coupled to a
change in protein−protein binding. One constraint on our
current design is the inability of BSBCA and other currently
available chromophores to switch completely to cis. Several new
chromophores have become available25,28,29 that possess either
more complete isomerization or longer half-lives that could allow
for isolation and use of the pure cis state.
When applied in cell culture experiments, the light-
modulatable cadherin could help answer several outstanding
questions about cadherin’s function. One way to introduce this
engineered molecule into a cellular context is via cadherin-coated
substrata. Coated substrata have been used to study cell−cell
adhesion30 and stem cell pluripotency.31 Creation of coated
surfaces allows spatial control of cadherin patterning and fine
control over cadherin concentration, which could help maximize
the switchability of cadherin-mediated adhesion.30
Although interest in photoswitchable proteins has increased in
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perhaps because finding good cysteine attachment points
remains difficult. Compared to high-throughput and other
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that may have a lower activation energy barrier to transition
compared to more rigid parts of protein domains. While not all
proteins could be modified in this way, we believe our combined
rational/computational selection method and our focus on loops
can be generalized to create other photoswitchable designs.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Data for other library members; detailed experimental methods;
descriptions of, and figures for, data analysis methodology;
additional figures for the unconjugated mutant. This material is
available free of charge via the Internet at http://pubs.acs.org.
(27) Haussinger, D.; Ahrens, T.; Aberle, T.; Engel, J.; Stetefeld, J.;
̈
Grzesiek, S. EMBO J. 2004, 23, 1699.
AUTHOR INFORMATION
Corresponding Author
ryan.ritterson@cal.berkeley.edu
■
(28) Blanco-Lomas, M.; Samanta, S.; Campos, P. J.; Woolley, G. A.;
Sampedro, D. J. Am. Chem. Soc. 2012, 134, 6960.
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Int. Ed. 2012, 51, 6452.
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Tanaka, T.; Okabe, M.; Fukuda, K.; Akaike, T. PLoS ONE 2006, 1, e15.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Laura Lavery and Rebeca Choy
for contributing to the original idea, Elizabeth Tanner, Drs.
Matthew Banghart, Andrew Woolley, James Nelson, and Nicolas
Borghi for helpful discussions, Dr. Woolley separately for a gift of
BSBCA, Alec Nielsen for assistance with assay development and
the Mass Spectrometry Resource at UCSF (funded by NIH
P41GM103481, PI A.L. Burlingame). R.R. and K.K. were
partially funded by NIH Grant T32 GM008284. Research
funding for this project came from NSF Grant 1134127 to T.K.
and the Synthetic Biology Engineering Research Center (NSF
EEC-0540879, PI J. Keasling).
REFERENCES
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