-to- 3-amino acid mutations on the binding of ...

Supporting information

A study on the effect of synthetic α-to-β3-amino acid mutations on the

binding of phosphopeptides to 14-3-3 proteins

Sebastian A. Andrei, Vito Thijssen, Luc Brunsveld, Christian Ottmann, Lech-Gustav Milroy.

I. Supplementary Methods

- Peptide synthesis S2

- Protein expression S2

- Fluorescence Polarization (FP) S3

- Isothermal Titration Calorimetry (ITC) S3

- Crystallography S3

II. Supplementary Figures

- ITC thermograms S5

- FP curves S8

- Purified peptides LC-MS S10

III. References S24

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

S2

I. Supplementary methods

Peptide synthesis

All peptides were synthesized by standard Fmoc SPPS using an automated Intavis Multipep RSi peptide

synthesizer. Peptides containing a C-terminal amide were synthesized using a Rink Amide resin, whereas

a preloaded 2-Chlorotrityl resin was used for peptides with a C-terminal carboxylate. Fmoc protected β-

amino acids were used in the same manner as Fmoc protected α-amino acids. All reactions were

performed N-methyl-pyrrolidone (NMP). Fmoc deprotection was performed in 20% (v/v) piperidine and

acid activation in a solution of 1.2 M diisopropyl ethylamine (DIPEA) and 0.4 M (2-(1H-benzotriazol-1-yl)-

1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 8-fold excess). Each coupling reaction was

subsequently performed twice for one hour in 0.5 M amino acid solution (10-fold excess) and followed by

capping through acetylation (pyridine/acetic anhydride/DMF, 1/1/1, v/v/v). The last N-terminal amino

acid was either capped through acetylation or left as the free amine. The final peptides were cleaved off

the resin in trifluoracetic acid (TFA), H2O, triisopropylsilane (TIS) and ethanedithiol (EDT)

(92.5/2.5/2.5/2.5, v/v/v/v) for 5 hours at room temperature and precipitated in 20 mL diethyl ether (Et2O)

at -30 °C. The precipitated peptide suspension was stored at -30 °C for ten minutes, centrifuged at 2000

rpm and decanted. Twenty milliliters of fresh Et2O at -30 °C were added, the suspension stored at -30 °C

for another ten minutes and centrifuged again. The solvent was decanted and the remaining pellet dried

in the open air. Peptides were then purified by mass triggered reverse phase HPLC on an appropriate

gradient of H2O/acetonitrile + 0.1% TFA. The resulting peptide solutions were lyophilized and stored at -

30 °C until further use.

Protein expression

Proteins 14-3-3σΔC and full length 14-3-3σ & 14-3-3ζ were expressed according to the following general

protocol. Plasmids were transformed into NiCo21(DE3) cells (New England Biolabs) according to the

manufacturers protocol. Colonies were grown on LB agar plates + 100 μg/mL ampicillin overnight at 37

°C. One colony was picked and precultured in 25 mL sterile LB medium + 100 μg/mL ampicillin overnight

at 37 °C. 2 TB medium + 100 μg/mL ampicillin was then inoculated with the preculture and incubated at

37 °C until an OD600 of ~1.0 was reached. Protein expression was then induced with 0.4 mM IPTG and the

cells incubated overnight at 18 °C. Cells were then harvested by ultracentrifugation at 20000 rpm at 4°C,

flash frozen in liquid nitrogen and stored at -80°C until further purification.

Prior to purification, the cell pellets were thawed and resuspended in 10 mL/g pellet lysis buffer (25 mM

Tris, pH = 8.0, 150 mM NaCl, 5% v/v glycerol, 10 mM imidazole, 4 mM BME and 1 mM PMSF). The cells

were then lysed twice by homogenization using an EmulsiFlex-C3 homogenizer. The lysate was incubated

with benzonase (Merck Millipore) for 15 minutes and then centrifuged at 20000 g for 15 minutes. The

supernatant was applied in overnight circulation at 4 °C to a 5 mL HisTrap column pre-equilibrated with

20 column volumes (CV) lysis buffer. The column was then washed with 20 CV wash buffer (25 mM Tris,

pH = 8.0, 300 mM NaCl, 5% v/v glycerol, 25 mM imidazole and 4 mM BME) and the protein eluted with 40

mL elution buffer (20 mM HEPES, pH 8.0, 100 mM NaCl, 5% v/v glycerol, 250 mM imidazole and 4 mM

BME). The protein was then pipetted into a SpectrumLabs Spectra/Por 10000 Da MWCO dialysis bag and

S3

dialysed overnight at 4 °C against dialysis buffer (25 mM HEPES, pH = 8.0, 100 mM NaCl, 4 mM BME, 2

mM MgCl2). For the σΔC protein, 1:500 mg/mg TEV protease was added to the dialysis bag. The full length

proteins were then concentrated to ~50 mg/mL using 10000 Da MWCO Amicon spinfilters, aliquoted, flash

frozen in liquid nitrogen and stored at -80 °C until further usage. The σΔC protein was instead applied to

a 5 mL HisTrap column pre-equilibrated with 20 CV dialysis buffer. The flowtrough was captured and

concentrated to ~50 mg/mL using 10000 Da MWCO Amicon spinfilters. The concentrated σΔC protein was

then applied to a HiLoad superdex 75 16/60 SEC column using an Äkta FPLC apparatus. The fractions

containing protein were then pooled, concentrated to ~50 mg/mL protein, flash frozen in liquid nitrogen

and stored at -80°C until further use.

Fluorescence Polarization

The YAP peptides were dissolved to a concentration of 5 mM in pure MilliQ water. A mastermix containing

the labeled reporter peptide (FITC-ERα) at 100 nM and 14-3-3 protein (1 µM in case of 14-3-3ζ & 5 µM in

case of 14-3-3σ and 5 µM when comparing the various isoforms) was made in FP buffer (10 mM HEPES,

pH = 7.50, 150 mM NaCl, 0,1% (v/v) Tween 20). Then a 2-fold dilution series starting from 250 uM (in case

of 14-3-3ζ) or 500 uM (in case of 14-3-3σ) of the unlabeled YAP peptides was made in the mastermix in

black Corning low volume, round bottom 384-well assay plates (Corning #4514). Fluorescence polarization

was then measured in a Tecan Safire II at room temperature (excitation wavelength 485 (20) nm and

emission wavelength 535 (25) nm). All data points are measured in triplicate. The IC50-values were then

determined by plotting the data in Origin scientific graphing software and fitting the curves with a 3-

parameter sigmoidal dose response curve.

Isothermal titration calorimetry

All ITC measurements were performed on a MicrocalTM iTC200 calorimeter. The device cell was filled with

a solution of 80 µM of the 14-3-3 protein in ITC buffer (100 mM NaCl, 25 mM HEPES, 10 mM MgCl2, 0.5

mM TCEP and PH 7.40). The syringe was filled with a 1 mM solution of the peptide in ITC buffer. Reference

cell was filled with MilliQ water. The peptide was then titrated to the protein at 37 °C though 19 injections

of 2 l with 180 seconds spacing between each injection. Additional settings: Reference power: 5 μCal/s;

initial delay: 60 s and stirring speed: 750 rpm. Data was processed using Origin 7 (Originlab) software

containing an automated module for calculation and fitting of ITC data.

Crystallography Crystals of the binary complex of the YAP peptides and 14‐3‐3σΔC were grown by mixing 10 mg/mL 14‐3‐

3σΔC with the appropriate YAP peptides in a molar ratio of 1 : 2 in 10 mM HEPES pH 7.4, 150 mM NaCl

and 2 mM BME and incubating overnight at 277K. The formed complex was then set up for crystallization

by mixing 1 : 1 (v/v) with 0.095 M HEPES pH 7.5, 26% PEG400, 0.19 M CaCl2, 5% glycerol and incubating

in a sitting drop at 277 K. Crystals grew within a week. X-ray diffraction data was collected at 100 K at the

p11 beamline of the PETRA-III synchrotron of the DESY facility in Hamburg, Germany using a Pilatus 6M-F

detector.1,2 The data was indexed and integrated using iMosflm and scaled and merged using Aimless.3

Phasing was done by molecular replacement using Phaser4,5 and 3MHR as a starting model and was

S4

followed by iterative rounds of refinement and manual model building using Phenix.Refine6 and Coot7

respectively. Model validation was performed using MolProbity.8 Figures were created using PyMol.

Table S1: Crystallography data collection and refinement statistics of βYAP3, βYAP7 and βYAP3.2 in complex with 14-3-3σΔC.

βYAP2 βYAP2.2 βYAP3 βYAP3.2 βYAP4 βYAP5 βYAP7

PDB code 6G6X 6G8P 6G8J 6G8Q 6G8K 6G8L 6G8I

Data collection

Resolution (Å)a 1.13 (1.13-

1.15) 1.90 (1.94-

1.90) 1.47 (1.50-

1.47) 1.85 (1.89-

1.85) 1.25 (1.27-

1.25) 1.37 (1.39-

1.37) 1.60 (1.63-

1.60)

Space group C2221 C2221 C2221 C2221 C2221 C2221 C2221

Cell parameters (Å)

82.118, 111.849, 62.630

α=β=γ=

90°

82.037, 111.499, 62.460

α=β=γ=

90°

82.149, 112.019, 62.680

α=β=γ=

90°

82.108, 111.900, 62.620

α=β=γ=

90°

82.260, 11.730, 62.510

α=β=γ=

90°

82.258, 111.969, 62.650

α=β=γ=

90°

82.118, 112.170, 62.550

α=β=γ=

90°

Rmergea,b 0.082 (1.36) 0.085 (0.18) 0.099 (0.97)

0.126 (0.665)

0.101 (0.853)

0.133 (0.640)

0.161 (1.53)

Average I/σ(I)a,b 12.1 (1.6) 18.6 (9.7) 12.1 (2.2) 11.4 (2.6) 10.8 (1.8) 10.0 (2.0) 10.2 (2.0)

CC1/2 (%)a,b,c 0.998

(0.767) 0.992

(0.983) 0.994

(0.627) 0.995

(0.770) 0.995

(0.669) 0.992

(0.708) 0.979

(0.708)

Completeness (%)a,b

100 (99.9) 100 (96) 97.8 (94.4) 99.3 (90.8) 99.9 (98.8) 99.8 (96.0) 100 (100)

Redundancya,b 11.2 (8.1) 11.2 (8.2) 12.3 (11.1) 11.1 (7.3) 11.7 (7.6) 9.9 (4.4) 11.5 (11.5)

Refinement

Number of protein/solvent/li

gand atoms 2309/399/- 2122/349/- 2150/375/- 2071/357/- 2181/387/- 2203/426/- 2103/383/-

Rwork/Rfree (%) 14.96/16.78 13.3/15.9 15.89/17.26 14.40/17.54 14.10/15.97 13.57/15.72 14.96/17.34

Unique reflections used in

refinement 107651 22861 48296 24785 79638 60814 38473

R.m.s. deviations from ideal values bond lengths (Å) /

bond angles (°)

0.008/0.95 0.004/0.67 0.004/0.63 0.009/0.89 0.009/1.0 0.008/0.98 0.010/0.89

Average protein/solvent/ligand B-factor (Å2)

111.9/-/- 98.6/-/- 78.6/-/- 85.4/-/- 112.6/-/- XX 65.4/-/-

Ramachandran favored (%)

98.3 98.3 98.3 98.3 98.3 98.3 98.3

Ramachandran allowed (%)

1.7 1.7 1.7 1.7 1.7 1.7 1.7

Ramachandran outliers (%)

0 0 0 0 0 0 0

S5

II. Supplementary Figures

A B

C D

Peptide Protein N (sites) Ka (M-1) ΔH (cal/mol) ΔS (cal/mol/deg)

A YAP1.1 14-3-3ζ 1.42 ± 0.00884 1.02E6 ± 1.23E5 -6229 ± 62.80 7.40

B YAP2.2 14-3-3ζ 1.37 ± 0.00741 1.26E6 ± 1.44E5 -9484 ± 79.56 -2.67

C YAP1.1 14-3-3σ 1.45 ± 0.0204 5.05E5 ± 1.02E5 -5791 ± 115.2 7.42

D YAP2.2 14-3-3σ 1.41 ± 0.00453 1.26E6 ± 8.61E4 -8918 ± 44.54 -0.851

Figure S1: ITC data of YAP peptides A: YAP1.1 against 14-3-3ζ B: YAP2.2 against 14-3-3ζ C: YAP1.1 against 14-3-3σ D: YAP2.2 against 14-3-3σ

S6

E F

G H

Figure S2: ITC data of βYAP peptides E: βYAP2 against 14-3-3ζ F: βYAP2 against 14-3-3ζ G: βYAP2 against 14-3-3σ H: βYAP2 against 14-3-3σ.


E βYAP2 14-3-3ζ 1.11 ± 0.0139 7.52E4 ± 6.37E3 -8955 ± 169.8 -6.57

F βYAP2 14-3-3 ζ 1.09 ± 0.0133 8.23E4 ± 6.87E3 -8946 ± 162.0 -6.36

G βYAP2 14-3-3σ 1.04 ± 0.00517 5.63E4 ± 1.61E3 -9404 ± 72.19 -8.59

H βYAP2 14-3-3 σ 1.11 ± 0.0127 5.22E4 ± 3.56E3 -9557 ± 175.6 -9.23

S7

I J

K

Figure S3: ITC data of βYAP peptides I: βYAP4 against 14-3-3ζ J: βYAP4 against 14-3-3σ K: βYAP4 against 14-3-3σ.


I βYAP4 14-3-3ζ 1.01 ± 0.00812 1.49E5 ± 9.59E3 -7569 ± 83.55 -0.741

J βYAP4 14-3-3σ 1.34 ± 0.00606 9.24E4 ± 3.77E3 -7397 ± 51.51 -1.13

K βYAP4 14-3-3σ 1.07 ± 0.00567 1.22E5 ± 5.06E3 -7978 ± 59.19 -2.45

S8

Figure S4 Competitive FP results of YAP peptides against 14-3-3ζ and 14-3-3σ. (1 µM 14-3-3ζ and 100 nM FITC-ERα).

Figure S5 Competitive FP results of βYAP peptides against 14-3-3ζ. (5 µM 14-3-3ζ and 100 nM FITC-ERα).

Figure S6 Competitive FP results of βYAP peptides against 14-3-3σ. (5 µM 14-3-3σ and 100 nM FITC-ERα).

S9

Figure S7 Competitive FP results of βYAP peptides against 14-3-3η. (5 µM 14-3-3η and 100 nM FITC-ERα).

Figure S8 Competitive FP results of βYAP peptides against 14-3-3ε. (5 µM 14-3-3ε and 100 nM FITC-ERα).

Figure S9 Competitive FP results of βYAP peptides against 14-3-3τ. (5 µM 14-3-3τ and 100 nM FITC-ERα).

S10

YAP 1.1 H2N-R A H pS S P A S L Q-COOH Expected mass: [M+H]+ =1133.5

[M+2H]2+= 567.3

[M+3H]3+ = 378.5

RT: 0.00 - 15.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

so

rba

nce

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

10.73803.25

10.84803.17

10.97803.17

2.35567.50 9.58

282.3311.14803.08

9.69282.33

12.16362.83

9.40256.33

8.70328.08

7.81282.33 12.47

391.172.52

567.426.20

795.004.92

376.921.03

182.754.73

985.00

10.650.00 11.98

0.00

11.730.00

9.730.00

9.500.008.62

0.008.200.007.62

0.007.200.00

6.780.00

6.230.005.72

0.005.200.004.70

0.004.050.003.52

0.002.270.00

0.620.00

NL:5.83E5

TIC MS 150316VTYAP11

NL:3.23E4

Total Scan PDA 150316VTYAP11

150316VTYAP11 #273-296 RT: 2.27-2.46 AV: 24 NL: 5.73E3T: ITMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

567.42

378.67

391.25

396.58586.42

755.83485.33358.67 428.83147.08 856.58560.33 617.17 781.33313.33187.00 889.33661.67227.25 689.33 954.33 997.25

S11

YAP 1.2 Ac-R A H pS S P A S L Q-COOH Expected mass: [M+H]+ = 1175.5

[M+2H]2+ = 588.3

[M+3H]3+ = 392.5

RT: 0.00 - 15.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

so

rba

nce

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

10.75803.17

2.86588.50

10.81803.17

10.86803.1710.96803.25

9.57282.33

11.16803.17

9.69282.33

12.18413.42

9.51282.338.77

328.087.81

300.083.00

588.422.58

622.9212.46391.08

4.91377.00

6.23966.25

0.55158.00

10.680.00 11.98

0.00

11.730.00

9.270.008.75

0.008.300.00

7.920.007.17

0.006.900.006.23

0.005.650.005.13

0.004.700.002.78

0.004.130.00

0.620.00

13.630.00

NL:5.39E5

TIC MS 150316VTYAP12

NL:3.21E4

Total Scan PDA 150316VTYAP12

150316VTYAP12 #338-353 RT: 2.81-2.94 AV: 16 NL: 2.48E4T: ITMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

588.50

405.33 783.92599.50410.58392.75 552.92 623.92 809.67 903.50731.58 931.50680.33459.00304.17 971.58167.00111.00 227.08

S12

YAP 2.1 H2N-R A H pS S P A S L Q-CONH2

Expected mass: [M+H]+ = 1132.5

[M+2H]2+ = 566.8

[M+3H]3+ = 378.2

RT: 0.00 - 15.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

so

rba

nce

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

10.78803.08

10.86413.42

2.16566.92

10.94413.42

12.21413.42

2.02566.92 11.13

803.0810.38427.33

0.60158.00

9.99391.00

12.35391.08

6.26822.172.37

566.838.94

391.088.52

391.005.35

805.927.47

149.003.87

600.250.75

401.25

12.000.00

10.700.00

11.720.00

9.250.008.20

0.007.720.00

7.420.00

6.980.006.45

0.005.680.005.25

0.004.750.004.23

0.003.720.00

0.620.00 3.22

0.002.080.00

NL:3.85E5

TIC MS 22042016YAP21

NL:2.73E4

Total Scan PDA 22042016YAP21

22042016YAP21 #160-178 RT: 2.02-2.24 AV: 19 NL: 6.46E3T: ITMS + p ESI Full ms [100.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

566.92

378.33

390.92

1132.58577.92485.33

358.42 755.08 1154.67129.08 856.50616.75 908.67 1034.58 1300.08 1576.831365.67 1655.75 1986.671795.25

S13

YAP 2.2 Ac-R A H pS S P A S L Q-CONH2


[M+2H]2+ = 587.8

[M+3H]3+ = 392.5

RT: 0.00 - 15.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

so

rba

nce

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

2.81588.00

10.76413.333.00

588.42 12.20413.33

12.000.00

10.680.00

11.750.00

2.730.00 8.67

0.008.450.007.75

0.006.980.00

6.850.006.25

0.005.780.005.27

0.004.750.004.23

0.000.620.00

3.700.00 13.87

0.00

NL:2.55E6

TIC MS 22042016YAP22

NL:2.73E4

Total Scan PDA 22042016YAP22

22042016YAP22 #218-230 RT: 2.75-2.90 AV: 13 NL: 8.35E4T: ITMS + p ESI Full ms [100.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

588.00

1175.00

783.42616.92

392.25 1468.081196.67802.58 1566.33 1761.92552.33 894.33628.92 1011.42199.25 1957.33256.25 1327.92

S14

βYAP 1 Ac-R A H pS βS P A S L Q-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S15

βYAP 2 Ac-R A H pS S βP A S L Q-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S16

βYAP 3 Ac-R A H pS S P βA S L Q-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S17

βYAP 4 Ac-R A H pS S P A βS L Q-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S18

βYAP 5 Ac-R A H pS S P A S βL Q-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S19

βYAP 6 Ac-R A H pS S P A S L βQ-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S20

βYAP 7 Ac-βR A H pS S P A S L Q-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S21

βYAP 8 Ac-R βA H pS S P A S L Q-CONH2


[M+2H]2+ = 594.8

[M+3H]3+ = 396.8

S22

βYAP 2.2 Ac-R A H pS S βP A S βL Q-CONH2


[M+2H]2+ = 601.8

[M+3H]3+ = 401.5

S23

βYAP 3.2 Ac-R A H pS S P βA S L βQ-CONH2


[M+2H]2+ = 601.8

[M+3H]3+ = 401.5

S24

III. References

1 A. Burkhardt, T. Pakendorf, B. Reime, J. Meyer, P. Fischer, N. Stübe, S. Panneerselvam, O. Lorbeer, K. Stachnik, M. Warmer, P. Rödig, D. Göries and A. Meents, Eur. Phys. J. Plus, 2016, 131, 56.

2 A. Meents, B. Reime, N. Stuebe, P. Fischer, M. Warmer, D. Goeries, J. Roever, J. Meyer, J. Fischer, A. Burkhardt, I. Vartiainen, P. Karvinen and C. David, International Society for Optics and Photonics, 2013, vol. 8851, p. 88510K.

3 H. R. Powell, O. Johnson and A. G. W. Leslie, Acta Crystallogr. D Biol. Crystallogr., 2013, 69, 1195–1203. 4 A. J. McCoy, Acta Crystallogr. D Biol. Crystallogr., 2007, 63, 32–41. 5 A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni and R. J. Read, J. Appl.

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