Structure, Volume 24

Supplemental Information

Adhesive Dimerization of Human P-Cadherin

Catalyzed by a Chaperone-like Mechanism

Shota Kudo, Jose M.M. Caaveiro, and Kouhei Tsumoto

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Supplemental Information

Adhesive dimerization of human P-cadherin catalyzed

by a chaperone-like mechanism.

Shota Kudo1, Jose M.M. Caaveiro2*, and Kouhei Tsumoto1,2,3,4*

1Department of Chemistry & Biotechnology, The University of Tokyo, Tokyo, 108-8639, Japan, 2Department

of Bioengineering, The University of Tokyo, Tokyo, 108-8639, Japan, and 3Institute of Medical Science, The

University of Tokyo, Tokyo, 108-8639, Japan.

*Correspondence should be addressed to JMMC (jose@bioeng.t.u-tokyo.ac.jp) or KT (tsumoto@bioeng.t.u-tokyo.ac.jp)

4Lead Contact

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Supplemental Table 1. Cadherin constructs employed in this study.a Related to Table 1 and 2.

Construct name Typeb Sequencec Mutation strand-swap dimer X-dimer

strand-swap dimer WT EC12 1-213 No mutation Yes Yes strand-swap dimer WT long EC12 1-241 No mutation Yes Yes strand-swap dimer K14E EC12 1-213 K14E Yes No strand-swap dimer Q101L EC12 1-213 Q101L Yes No strand-swap dimer EC1 EC1 1-129 No mutation Yes No strand-swap dimer EC1 K14E EC1 1-129 K14E Yes No strand-swap X-dimerII MEC12 Met0 + 1-213 Met0 No Yes strand-swap X-dimerII long MEC12 Met0 + 1-241 Met0 No Yes strand-swap X-dimerI MEC12 Met0 + 1-213 Met0/V4I/A5P/N22V No Yes int-X-dimer EC12 1-213 W2A No Yes enc-X-dimer MEC12 Met0 + 1-213 Met0/N22V No Yes monomer 1 MEC12 Met0 + 1-213 Met0/K14E No No monomer 2 REC12 Arg0 + 1-213 Arg0 No No monomer 3 MEC12 Met0 + 1-213 Met0/Q101L No No E-cad. strand-swap dimer WT EC12 1-213 No mutation Yes Yes aThis list does not include full-length constructs, or muteins encoding the non-natural amino acid pBPA. bComposition of the ectodomain. M and R refers the one-letter code of the additional residue incorporated at the N-terminus. cResidue numbering is based on the sequence of the mature protein. Supplemental Table 2. Kinetic characterization by SEC and fluorescence (FL).a,b Related to Figure 2.

Protein kobsSEC

(h-1) ∆∆G‡SEC (kcal mol-1)

kobsFL-assoc

(h-1) kobs

FL-dissoc

(h-1) ∆∆G‡FL-assoc (kcal mol-1)

X-dimer

strand-swap dimer WT ≥4.7 ± 2.1 NAd ≥102 ± 6 NDc NA Yes strand-swap dimer K14E 0.13 ± 0.01 ≥2.0 2.4 ± 0.1 0.78 ± 0.06 ≥2.2 No strand-swap dimer Q101L 0.18 ± 0.01 ≥1.8 5.1 ± 0.1 1.86 ± 0.06 ≥1.8 No strand-swap dimer WT (no Ca2+) 0.075 ± 0.002 ≥2.3 ND ND ND No strand-swap dimer EC1 0.092 ± 0.017 ≥2.2 ND ND ND No strand-swap dimer EC1 K14E 0.10 ± 0.01 ≥2.1 ND ND ND No

aKinetic constants are given as mean ± SD. b∆∆G‡ represents the difference of activation energy of dimerization between the muteins and WT calculated with the Arrhenius equation. cND, not determined. dNA, not applicable.

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Supplemental Table 3. Thermodynamic characterization by ITC.a,b Related to Figure 4.

Protein KDAUC

(µM) KD

ITC (µM)

∆Ga (kcal mol-1)

∆Ha (kcal mol-1)

−T∆Sa (kcal mol-1)

∆H / ∆G (%)

−T∆S / ∆G (%)

strand-swap dimer WT 1.95 ± 0.05 5.2 ± 0.6 −7.1 ± 0.1 −8.5 ± 0.6 +1.4 ± 0.7 120 −20 strand-swap X-dimerI 187 62 ± 10 −5.6 ± 0.1 −4.6 ± 0.4 −1.0 ± 0.5 82 18 enc-X-dimer 171 74 ± 20 −5.5 ± 0.1 +2.8 ± 0.1 −8.3 ± 0.2 −50 150 monomer 1 NDc ND ND ND ND ND ND E-cadherin strand-swap dimer WT

268 65 ± 17 −5.6 ± 0.1 −2.8 ± 0.1 −2.8 ± 0.2 50 50

aThermodynamic parameters were determined by ITC. bData are given as mean ± SD when possible. cNot determined. Supplemental Table 4. Protein-protein interactions in the X-dimer. Related to Figure 4.

Protein BSA (Å2) No. H-bondsa No. N-terminal H-bondsa,b enc-X-dimer 1,454 4 0 strand-swap X-dimerI 3,687 9 5

aH-bonds are calculated with respect to one protein chain not including Met0. bIntermolecular H-bonds in which residues 1-10 of the N-terminal region are participating. � Supplemental Table 5. Water-mediated interactions in strand-swap dimers. Related to Figure 5.

Protein Resolution (Å)

No. water molecules

No. water-mediated H-bonds

H-bonds per water moleculea

Average distance H-bonds (Å)a

P-cadherin WT 1.85 15 36 2.4 ± 0.5 2.9 ± 0.2 P-cadherin WT 2.05 11 26 2.4 ± 0.5 2.9 ± 0.2 E-cadherin WT 2.00 5 10 2.0 ± 0.5 3.0 ± 0.2 aData is given as mean ± SD.

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Supplemental Figure 1. Crystal structure of human P-cadherin. Related to Figure 1. (a) Comparison of the structures of the strand-swap dimer of human P-cadherin (residues 1-213, blue) and human P-cadherin long construct (residues 1-241) (orange). Residues 10-100 of EC1 were superposed. The value of RMSD correspond to that of EC1* (the asterisks indicate the second protein chain in the dimer), to reveal the differences in the structural conformation at the strand-swap dimer interface. (b) Comparison of strand-swap dimer of human P-cadherin (blue), mouse P-cadherin (PDB code 4NQQ, orange) and human E-cadherin (PDB code 2O72, green). RMSD was calculated as above. (c) Summary of interactions between residues 4-11 of the N-terminal strand and its own EC1 core in the crystal structure of monomer 1. Residues of the EC1 core and the N-terminal strand are depicted in black and magenta, respectively. The cyan spheres correspond to water molecules. The green dashed lines represent hydrogen bonds. The figure was prepared with LIGPLOT. (d) Summary of interactions between residues 4-11 of the N-terminal strand and its own EC1 core in the crystal structure of strand-swap dimer WT.

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Supplemental Figure 2. X-dimer accelerates strand-swap dimerization. Related to Figure 2. (a) Time course of the cleavage of EC12-SUMO constructs by Ulp1 protease. More than 90% of the constructs were processed by Ulp1 before 30 min (at 4 °C) or 1 min (at 25 °C). (b) SEC elution profiles and time-course of dimerization of strand-swap dimer EC1, or strand-swap dimer EC1 K14E (left panel). Both constructs lack the EC2 domain, which is strictly necessary for X-dimerization. Dimerization was monitored for up to 48 hours after addition of protease Ulp1. The time course represents the percentage of dimer calculated from the relative absorbance of the elution peaks (right panel). (c) Effect of Ca2+ in the dimerization of the construct termed strand-swap dimer WT was monitored by SEC. Protein was extensively dialyzed to remove traces of Ca2+ prior to the experiment. Dimerization was initiated by addition of Ulp1. SEC was carried out in a Ca2+-free buffer (10 mM HEPES, 150 mM NaCl, pH 7.5). (d) Distance between Ser201 residues in the dimer. This residue was selected to specifically cross-link the X-dimer but not the strand-swap dimer with pBPA. (e) Results of the photocrosslinking experiment. Purified strand-swap X-dimerII, monomer 1, and monomer 3 modified with the non-natural aminoacid pBPA at position 201 were exposed for five minutes to UV-light followed by western blot using a mouse anti-human P-cadherin antibody. Control experiments with samples not exposed to UV-light are also shown. (f) Western blot of the photocrosslinking experiment performed with samples of strand-swap dimer WT, strand-swap dimer K14E, and strand-swap dimer Q101L containing pBPA at position 201. Bands corresponding to the full-length mature protein, the SUMO-fusion protein, and protein truncated at the position of pBPA were also detected in the gel.

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Supplemental Figure 3. Cell adhesion mediated by P-cadherin. Related to Figure 2. (a) Cell aggregation of cells expressing full-length native P-cadherin and muteins K14E, W2A, and W2A-K14E. The experiment was conducted in a buffer containing Ca2+ for 120 minutes. Images were taken at the end of the incubation time. (b) Analogous experiment in the absence of Ca2+ but in the presence of 1 mM EDTA.

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Supplemental Figure 4. Detailed description of the X-dimer. Related to Figure 3. (a) Key interactions observed in four different crystal structures of the X-dimer. The coordinates of enc-X-dimer were used to illustrate them. (b) Insertion of Met0 and Trp2 each in separate hydrophobic pockets in the crystal structure of strand-swap X-dimerII. (c) Superposition of the coordinates of the strand-swap X-dimerII and strand-swap X-dimerII long. (d) BSA values of residues of human P-cadherin interacting in various dimers. The symbol # indicates non-polar residues of EC1 in the dimerization interface of the strand-swap X-dimerI. The BSA values corresponding to the enc-X-dimer, the int-X-dimer, the strand-swap X-dimerI, and the strand-swap dimer are shown in orange, green, blue and black, respectively. (e) Conformational changes of EC1* (surface representation) moving towards EC1 (ribbon representation) during dimerization. For the preparation of this figure only EC1 was superimposed. The structures of the enc-X-dimer (orange), the int-X-dimer (green), the strand-swap X-dimerI (blue), and the strand-swap dimer (gray) are shown. Residues 1-10 of EC1 are only depicted for the strand-swap X-dimerI and the strand-swap dimer WT. The yellow arrow illustrates the movement of EC1* with respect to EC1. (f) Pairwise distance between residues His79, Glu89, and Met92 of EC1 with the same residues of EC1*. These residues line the hydrophobic pocket accommodating Trp2. (g) RMSD plot for residues 1-20 of enc-X-dimer (orange), strand-swap X-dimerI (blue), and strand-swap dimer (black). RMSD values were calculated with respect to the coordinates of monomer 1. (h) Superposition of EC1 chain of the strand-swap X-dimerI and a similar dimer described in N-cadherin termed swapped plus X-dimer (Vendome et al., 2014). Residues 10-100 of EC1 were employed for the superposition. RMSD values for EC1 and EC1* were 0.97 and 8.3 Å, respectively. Two views rotated by about 90° in the y-axis are shown. The strand-swap X-dimerI and swapped plus X-dimer are depicted in blue and gray, respectively. The dotted line represents the approximate position of the two-fold axis.

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Supplemental Figure 5. Contribution of non-polar residues for the stepwise dimerization. Related to Figure 4. (a) Non-polar residues of EC1 interacting with the N-terminal strand as observed in the crystal structure of monomer 1 (residues 1-10 not depicted). The orange surface represents the contact area of these hydrophobic residues with the N-terminal strand. (b) BSA values of residues 4-10 of the N-terminal strand interacting with its own EC1 core in monomer 1 as calculated with PISA. (c) BSA of non-polar residues of EC1 involved in dimerization (dark gray) and other residues (light gray). The values are shown in Figure S4d. The fraction of non-polar surface contributing to the interaction surface is indicated. Supplemental Figure 6. Interaction surface in the strand-swap dimer. Related to Figure 5. (a) Schematic representation of the interaction interface of the strand-swap dimer of human P-cadherin. Residues belonging to domains EC1 and EC1* are depicted in black and magenta, respectively. Water molecules are shown in cyan. The green dotted lines indicate hydrogen-bonds. (b) Schematic view of the strand-swap dimer interface of human E-cadherin (PDB entry code 2O72). The figure was prepared with LIGPLOT.

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Supplemental Figure 7. Comparison of classical cadherins. Related to Figure 6. (a) BSA (calculated with PISA) corresponding to the cis-surface of P-cadherin (blue) or E-cadherin (orange). Data corresponds to the strand-swap dimer. Residues engaging in H-bonds with partner residues are indicated with the letter “H”. (b) Sequence alignment of classical human E-, N-, P-, R- and M-cadherin (EC12 domain) using CLUSTALW2. Residues displaying high similarity are indicated with asterisks. Blue, pink and yellow boxes correspond to residues present at the interface of strand-swap dimer, X-dimer, and the cis-interface, respectively. (c) Sequence alignment of the N-terminal strand (residues 1-10) of human classical cadherins (hcad or hCDH), human desmocollins (hDSC), and human desmogleins (hDSG). Figure was prepared with JALVIEW with the Zappo color scheme (Waterhouse et al., 2009).

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Supplemental Movie 1. Motion picture of cell-adhesive dimerization of human P-cadherin. Related to Figure 7.

The animation illustrating the conformational change of P-cadherin molecule during cell adhesion was created with the

morph conformation script in CHIMERA (Pettersen et al., 2004).

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Crystallization solutions − The exact composition of the crystallization solution (and the concentration of glycerol

used as cryoprotectant) for each construct was: WT strand-swap dimer, HEPES 100 mM, pH 7.5, 31% PEG 400, 15

mM CaCl2, 5% glycerol (no additional cryoprotectant); strand-swap dimer WT (second), 100 mM HEPES, pH 7.5, 30%

PEG 400, 15 mM CaCl2, 5% glycerol (no additional cryoprotectant); strand-swap dimer WT long, 100 mM HEPES, pH

7.5, 20% PEG550-MME, 100 mM CaCl2 (15% glycerol); strand-swap dimer K14E, 100 mM TRIS pH 8.5, 26% PEG

4,000, 170 mM Li2SO4, 15% glycerol (no additional cryoprotectant); strand-swap dimer Q101L, 100 mM HEPES, pH

7.5, 30% PEG 400, 150 mM CaCl2, 5% glycerol (no additional cryoprotectant); strand-swap X-dimerII, HEPES, pH 7.5,

28 % PEG 400, 250 mM CaCl2 (no additional cryoprotectant); strand-swap X-dimerII long, 20% PEG 3350, 200 mM

MgCl2 (20% glycerol); strand-swap X-dimerI, 100 mM TRIS pH 8.5, 1.2 M Li2SO4, 10 mM NiSO4 (25% glycerol); int-

X-dimer, 100 mM HEPES pH 7.5, 25% PEG 4,000, 10% 2-propanol (20% glycerol); enc-X-dimer, 100 mM TRIS, pH

8.5, 1.15 M LiSO4, 7.5 mM NiSO4 (25% glycerol); monomer 1, 100 mM TRIS, pH 8.5, 20% PEG 4,000, 100 mM

CaCl2 (20% glycerol); monomer 2, 100 mM HEPES, pH 7.5, 28% PEG 400, 200 mM CaCl2 (5 % glycerol). Protein

crystals were harvested and stored in liquid N2 until data collection at the synchrotron. Details of the data collection and

refinement procedure, and the methods to analyze the structural data are given in the main text and Table 1 and 2.

Kinetics of strand-swap dimerization monitored by SEC − Because the mature form of P-cadherin spontaneously

dimerizes even in the absence of Ca2+, the protein was purified with a N-terminal SUMO-tag. Dimerization was

initiated by cleaving off the SUMO-tag (20 μM) with 2 μM Ulp1 in assay buffer for one hour at 4 °C. Samples were

analyzed at 4 °C instead of room temperature to slow the dimerization kinetics so the dimer/monomer could be

monitored by SEC. The monomer/dimer mixtures were separated in a Superose12 10/300 GL column (GE Healthcare,

Piscataway, NJ) at a flow rate of 0.5 mL min-1. The fraction of dimer was estimated from the absorbance of the

corresponding elution peak according to the following equation:

Fraction dimer = Abs dimer / (Abs monomer + Abs dimer)

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Because a defined kinetic model for the dimerization of P-cadherin is not available, we have estimated the kinetic

parameters from the observed rate constant (kobsSEC), which was calculated by fitting an exponential decay equation to

the time course with ORIGIN8.

Kinetic of strand-swap dimerization monitored by intrinsic fluorescence − The dimerization of P-cadherin was

monitored by intrinsic fluorescence in a spectrofluorometer F-2500 (Hitachi, Japan) at 25 °C under constant stirring.

Excitation and emission wavelenths were set at 295 and 337 nm, respectively. To monitor the kinetics of association,

dimerization of P-cadherin was initiated by cleaving off the SUMO-tag construct (3 μM) with Ulp1 protease. To

monitor the dissociation kinetics of the strand-swap dimer, 67 μM P-cadherin in assay buffer was quickly diluted to 1

μM. The observed rate constants (kobsFL-assoc, kobs

FL-dissoc) were obtained using the same exponential decay equation as that

employed in the SEC experiment ORIGIN8.

Trapping X-dimer using a genetically encoded photoreactive probe − To trap the X-dimer during dimerization

without inhibiting the strand-swap dimerization, we introduced the genetically encoded non-natural aminoacid p-

benzoyl phenylalanine (pBPA) in the sequence of EC12 (Dorman and Prestwich, 1994; Sato et al., 2011). The pBPA

moiety was incorporated in the sequence of EC12 at position 201 following the protocols described previously (Abe et

al., 2012; Farrell et al., 2005; Young et al., 2010). Briefly, the expression of protein in LB medium containing 1 mM

pBPA was induced by the addition of 0.02 % arabinose and 0.5 mM IPTG. Protein purification was carried out as

above. pBPA was incorporated in the following constructs: MEC12, MEC12 K14E, MEC12 Q101L, EC12, EC12

K14E, and EC12 Q101L. The photocrosslinking reaction was carried out with 50 μL of the MEC12 constructs (5 μM),

or with EC12 constructs (40 μM) under UV-light using a B-100AP lamp (UVP Inc., CA) at 4 °C for 5 min in PCR

tubes. Samples were separated by SDS-PAGE. Identification of the photocrosslinked dimers was performed by western-

blot using mouse anti-human P-cadherin antibody (BD Biosciences) and goat anti-mouse IgG-HRP (Santa Cruz

Biotechnology, CA). Images were obtained with a LAS-4000 image analyzer (GE Healthcare).

Determination of affinity constants of self-dimerization by ITC − The affinities of P-cadherin and E-cadherin

were examined by the dilution method in an iTC200 instrument (GE Healthcare) (Burrows et al., 1994). The cell of the

calorimeter was filled with assay buffer, and the syringe with concentrated solution of protein. The protein was diluted

in the buffer solution of the cell by injecting ~2 µL aliquots 18 consecutive times every 2 minutes at 20 °C under

constant stirring (1,000 rpm). Data was analyzed with ORIGIN7 software using the dissociation analysis module. The

concentration of P-cadherin in the syringe was 170 µM for strand-swap dimer WT, 590 µM for strand-swap X-dimerI,

580 µM for enc-X-dimer, and 640 µM for monomer 1. The concentration of E-cadherin EC12 in the syringe was 600

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µM. The selection of these concentrations was based in the dissociation constant determined in the AUC measurements

(see below). Each measurement was performed at least twice.

Determination of affinity constants of self-dimerization by AUC − Equilibrium AUC was performed using a

Beckman XL-I ultracentrifuge with a Ti60An rotor (Beckman Coulter, CA). Samples (120 μL) in assay buffer at Abs280

of 0.2, 0.3, or 0.5 were loaded in standard cells (12 mm) with double-sector centerpieces and quartz windows. Samples

were equilibrated at 10,000 rpm for 36 hours at 20 °C after which an absorbance scan at 280 nm was performed ten

times from 6.7 cm to 7.2 cm at a radial step-size of 0.001 cm. The rotation speed was increased stepwise to 14,000 rpm

and to 24,000 rpm. In each step the samples were equilibrated for 16 hours and a scan of absorbance was performed

analogously. The density of buffer and v-bar were calculated with SEDNTERP. The value of KD was determined with

ORIGIN6 using global fitting to all the dataset following a simple monomer-dimer equilibrium. The experiment

corresponding to the strand-swap dimer of P-cadherin was performed in duplicate.

Preparation of CHO cells expressing P-cadherin − To evaluate the ability of P-cadherin and several of its

muteins to induce cell-adhesion, CHO cells stably expressing full-length P-cadherin (WT, K14E, W2A, and W2A-

K14E) were prepared with the Flp-In-CHO system (Life technologies) following the instructions of the manufacturer.

Cloning was performed by the limiting dilution-culture method. Cells with similar expression levels were selected for

analysis. The DNA sequence of monomeric GFP was fused at the C-terminal of some of the human P-cadherin

constructs. CHO cells stably expressing P-cadherin were cultured in Ham’s F12 medium (Life technologies)

supplemented with 10 % fetal bovine serum (FBS), and a cocktail of three antibiotics (1% penicillin-streptomycin, and

0.5 mg mL-1 hygromycin).

Cell aggregation assay − The ability of P-cadherin to induce cell adhesion was evaluated according to the reported

methodology (Urushihara et al., 1979). Briefly, cells were first treated with 0.1 % trypsin in HMF buffer (10 mM

HEPES, 137 mM NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 1 mM CaCl2, 5.5 mM glucose, pH 7.4) termed TCA

treatment. In the presence of Ca2+, P-cadherin is resistant to trypsinization whereas other molecules are digested (Nose

and Takeichi, 1986). Stop buffer (HMF containing 10 % FBS) was added and trypsin removed after extensive wash

with HMF buffer. After washing cells with Ca2+-depleted HMF (HCMF), 500 μL of a cell suspension at 1 × 105 cells

mL-1 was placed on a 24 well plate pre-treated with 1% (w/v) BSA. Cell aggregation was initiated by adding Ca2+ at a

final concentration of 1 mM, after which the plate was placed on a shaker at 80 rpm for 60 minutes at 37 °C. Images of

the cells were taken at different times with an EVOS XL Core Imaging System (Life technologies).

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Cell attachment assay − The cell attachment assay was adapted from a previous study (Ko et al., 2000). A volume

of 100 μL of acceptor cells at a concentration of 3 × 105 cells mL-1 were incubated in a 96-well plate (Greiner) coated

with poly-D-lysine to 95% confluency. Donor cells were subjected to TCA treatment after which they were labeled with

the dye calcein AM (Life technologies). After washing the cells three times with solution, donor cells were added to

acceptor cells and incubated at 37 °C for 15 min at concentrations ranging from 1.4 × 103 cells mL-1 to 2 × 105 cells mL-

1. After washing the cells gently with HMF four times, the fluorescence intensity of calcein AM was monitored using

excitation and emission wavelengths of 480 and 520 nm, respectively.

Immunofluorescence − Expression and localization of human P-cadherin proteins on the surface of CHO cells

were monitored by immunofluorescence. Cells (100 μL) at a concentration of 1 × 105 cells mL-1 were added to a 96-

well plate (Greiner) pre-coated with poly-D-lysine and incubated for 12 hours until confluency was 70%. After washing

the cells with HMF, the cells were fixed with 4 % p-formaldehyde for 10 min at room temperature. Cells were washed

with HMF buffer 4 times and blocked with Ham’s F12 medium for another 30 min. Cells were subsequently treated

with a solution containing mouse anti-human P-cadherin antibody (BD Biosciences) at 1 μg mL-1 for one hour at R.T.

After washing with HMF buffer four times, a mixture of 1 μg mL-1 goat anti-mouse IgG-Alexa488 and 0.5 μg mL-1

Hoechst 33342 was loaded (Life technologies). Cells were washed several times with HMF after which images were

recorded with an In-Cell analyzer 2000 instrument (GE Healthcare) using the FITC filter (490/50 excitation, 525/36

emission) or DAPI filter (350/50 excitation, 455/50 emission) with 20 × 0.45 NA objective.

Live cell imaging − Assembly of P-cadherin molecules was quantitatively monitored by live-cell imaging with an

In Cell Analyzer 2000 instrument. A total of 104 cells expressing human P-cadherin WT-mGFP or K14E-mGFP were

seeded on each well of a 96-well plate (Greiner) and incubated for 2 days at 37 °C. Images of cells were directly taken

at 37 °C using 60 × 0.7 NA objective. Data was analyzed with the In Cell Developer Toolbox 1.9 using four

independent images for each condition. Dense fluorescence spots were automatically detected by the software with

segmentation parameter set as “object” (value of kernel size and sensitivity were 5 and 60, respectively). Subsequently

the intensity of every spot was integrated, and added to calculate the total intensity per cell. Values are expressed as

average ± standard deviation.

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