Figures and data in Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD

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Tables
Additional files

4 figures, 3 tables and 2 additional files

Figures

Figure 1

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Structure of the YAP:TEAD complex.

The α-helix (A) and Ω-loop (B) binding interfaces. The surface of TEAD is represented and green ribbons indicate the α-helix (A, region 61–73) or the Ω-loop (B, region 85–99) of YAP. The different YAP residues that have been mutated are indicated. Interactions between hYAP Arg89 and TEAD (C) and between hYAP Ser94 and TEAD (D). The hydrogen bonds are represented by dotted purple lines. The TEAD α-helix (E) and Ω-loop (F) binding pockets. TEAD and YAP are represented by gray and green ribbons, respectively. The mutated TEAD residues for which a K_d^eq has been measured are represented in cyan. These figures are drawn from the pdb structure 3KYS (Li et al., 2010). TEAD residues are labeled according to hTEAD4 primary sequence.

https://doi.org/10.7554/eLife.25068.002

Figure 2 with 1 supplement

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Effect of the hTEAD4 Asp272Ala mutation in cells.

(A) Co-immunoprecipitation: N-terminally V5-tagged hTEAD4 (wild-type (wt) or Asp272Ala mutant) were co-transfected with wt hYAP into HEK293FT cells. YAP was immunoprecipitated, and co-immunoprecipitated nV5-TEAD4 was determined by anti-V5 Western Blot. (B) MCAT_Luc reporter assay: N-terminally V5-tagged TEAD4 (wt or Asp272Ala mutant) were co-transfected with wt hYAP into the HEK293::MCAT_Luc reporter model. Resazurin-normalized luciferase activity was measured and is plotted as fold induction over baseline. YAP and nV5-TEAD expression levels were determined in parallel by Western Blot. The expression level of hTEAD4 Asp272Ala mutant versus wt hTEAD4 was quantified by Image J software to be reduced by a factor of approx. 1.6, indicating that the approximate fourfold reduction in MCAT_Luc reporter signal cannot be solely attributed to expression differences, but does truly reflect a reduced activity of the hTEAD4 Asp272Ala mutant.

https://doi.org/10.7554/eLife.25068.005

Figure 2—figure supplement 1

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Analysis of the structure of the unbound hTEAD4 Asp272Ala mutant protein by Circular Dichroism (CD).

Proteins were dialyzed in 20 mM phosphate buffer pH 7.4, 100 mM KF, 0.25 mM TCEP and diluted in this buffer to 0.2 mg.mL⁻¹. CD spectra were recorded on a J-815 spectropolarimeter (Jasco (France); 1 mm path length quartz cell, ‘standard’ sensitivity, 0.5 nm bandwidth, 10 nm.min⁻¹ scanning, 2 s digital integration time, 1 nm step resolution). The measuring chamber was maintained in nitrogen (17 L.min⁻¹). Each spectrum was recorded as an average of 4 scans to reduce noise. After baseline correction, the mean residue ellipticity was calculated [θ]_MRW,λ = MRW.θ_λ/10.d.c, where MRW is the mean residue weight (MRW = M/(N-1)), M is the molecular mass of the protein, N is the number of amino acids in the protein, θ_λ is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm) of the cell, and c is the protein concentration (g.ml⁻¹). The figure represents the CD spectra of wt (white circles) and Asp272Ala (black circles) hTEAD4^217-434.

https://doi.org/10.7554/eLife.25068.006

Figure 3

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Coupling energies at the α-helix and Ω-loop binding pockets.

(A) α-helix interface. (B) Ω-loop interface. The gray and green ribbons represent the main polypeptide chain of TEAD and YAP, respectively. The C_α of the mutated amino acids are represented by spheres. The coupling energies (ΔΔG_int) between the different residues are symbolized by lines. Yellow lines: −1 kcal/mol < ΔΔG_int < −0.5 kcal/mol. Red lines: ΔΔG_int < −1 kcal/mol.

https://doi.org/10.7554/eLife.25068.008

Figure 4 with 1 supplement

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Double mutant cycle for the hYAP Arg89:hTEAD4 Asp272 interaction.

(A) The binding energies (kcal/mol) measured for the different pairwise interactions are indicated in boxes, and the differences between these binding energies (ΔΔG_1-4) are shown. The coupling energy (ΔΔG_int = ΔG_wt:wt + ΔG_R89A:D272A – ΔG_R89A:wt – ΔG_wt:D272A) is indicated (kcal/mol). Standard errors (SE) are given. (B) Representative sensorgrams of the Surface Plasmon Resonance experiments carried out to establish the double mutant cycle presented on A. wt or Asp272Ala hTEAD4 were immobilized on sensor chips and the binding of different concentrations of analyte (hYAP wt or Arg89Ala) was measured. K_d values were measured at equilibrium (K_d^eq). The contact time in the experiments was varied according to the affinity of each of the YAP proteins. (C) Binding isotherms obtained from the sensorgrams presented on B. The experiments were fitted with the Biacore T200 evaluation software using a one site binding with background model. The K_d^eq values and the standard error (SE) from the fit are indicated.

https://doi.org/10.7554/eLife.25068.009

Figure 4—source data 1 The double mutant cycles established in this study. The double mutant cycles have been grouped by hYAP residues. For explanations, see Figure 3 legend in the main text. The binding energies are in cal/mol and the standard errors (SE) are indicated.: https://doi.org/10.7554/eLife.25068.010
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Figure 4—figure supplement 1

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Position of hYAP Phe69 and hTEAD4 Phe337 at the α-helix binding pocket.

The α-helix (region 61–73) of YAP is represented by a green ribbon and hYAP Phe69 is indicated. The surface of TEAD is in gray and hTEAD4 Phe337 in orange. This figure was drawn from the pdb structure 3KYS (Li et al., 2010).

https://doi.org/10.7554/eLife.25068.011

Tables

Table 1

Binding affinities of the different YAP^50-151 proteins for wt hTEAD4^217-434. The affinities were measured at 298°K by Surface Plasmon Resonance in n ≥ 3 independent experiments. N-Avitagged hTEAD4^217-434 was immobilized on sensor chips. The K_d values were obtained from equilibrium data (K_d^eq). Averages and standard errors (SE) are given. ΔΔG = ΔG_mutant – ΔG_wt and SE_ΔΔG = (SE_ΔGmutant² + SE_ΔGwt²)^1/2.

https://doi.org/10.7554/eLife.25068.003

Mutation site	YAP^50-171	K_d^eq (nM)	ΔG (kcal/mol)	ΔΔG (kcal/mol)
Mutation site	Wild type	18 ± 0	−10.56 ± 0.01
α-helix	Leu65Ala	794 ± 4	−8.31 ± 0.01	2.24 ± 0.01
	Leu68Ala	464 ± 4	−8.63 ± 0.01	1.92 ± 0.01
	Phe69Ala	6447 ± 400	−7.08 ± 0.04	3.48 ± 0.04
Ω-loop	Met86Ala	2080 ± 69	−7.74 ± 0.02	2.81 ± 0.02
	Arg89Ala	27423 ± 381	−6.22 ± 0.01	4.34 ± 0.01
	Leu91Ala	30550 ± 2250	−6.15 ± 0.04	4.40 ± 0.05
	Ser94Ala	5623 ± 341	−7.16 ± 0.04	3.40 ± 0.04
	Phe95Ala	26045 ± 755	−6.25 ± 0.02	4.31 ± 0.02
	Phe96Ala	4755 ± 245	−7.25 ± 0.03	3.30 ± 0.03

Table 2

Properties of the different hTEAD4^217-434 proteins. The melting temperatures (T_m) of the proteins were determined in n ≥ 3 independent experiments in a fluorescence-based thermal shift assay. Averages and standard errors (SE) are given. ΔT_m = T_m^mutant – T_m^wt and SE_ΔTm = (SE_Tmmutant² + SE_Tmwt²)^1/2. For dissociation constant measurements, the different N-Avitagged hTEAD4^217-434 proteins were immobilized on sensor chips and their affinity for wt hYAP^50-171 was measured at 298°K by Surface Plasmon Resonance in n ≥ 3 independent experiments. K_d values were obtained from equilibrium data (K_d^eq). Averages and standard errors (SE) are given. ΔΔG = ΔG_mutant – ΔG_wt and SE_ΔΔG = (SE_ΔGmutant + SE_ΔGwt)^1/2. n. d.: not determined.

https://doi.org/10.7554/eLife.25068.004

Mutation site	hTEAD4^217-434	T_m (°C)	ΔT_m (°C)	K_d^eq (nM)	ΔG (kcal/mol)	ΔΔG (kcal/mol)
Mutation site	Wild type	54.0 ± 0.1		18 ± 0	−10.56 ± 0.01
α-helix	Phe337Ala	53.1 ± 0.1	−0.9 ± 0.1	202 ± 8	−9.12 ± 0.02	1.43 ± 0.02
	Tyr369Ala	49.9 ± 0.2	−4.2 ± 0.3	n. d.
	Phe373Ala	53.0 ± 0.3	−1.0 ± 0.3	12 ± 0	−10.79 ± 0.00	−0.24 ± 0.01
	Lys376Ala	53.4 ± 0.1	−0.7 ± 0.1	203 ± 9	−9.13 ± 0.03	1.43 ± 0.03
	Leu377Ala	49.8 ± 0.1	−4.3 ± 0.2	n. d.
	Leu380Ala	52.8 ± 0.1	−1.3 ± 0.2	162 ± 7	−9.26 ± 0.03	1.30 ± 0.03
	Val389Ala	53.0 ± 0.0	−1.0 ± 0.1	304 ± 5	−8.88 ± 0.01	1.67 ± 0.01
Ω-loop	Glu263Ala	53.0 ± 0.2	−1.1 ± 0.2	220 ± 9	−9.07 ± 0.02	1.48 ± 0.03
	Val265Ala	52.0 ± 0.0	−2.0 ± 0.1	106 ± 3	−9.51 ± 0.02	1.05 ± 0.02
	Asp272Ala	54.1 ± 0.1	0.1 ± 0.1	6995 ± 317	−7.03 ± 0.03	3.53 ± 0.03
	Lys273Ala	48.1 ± 0.2	−5.9 ± 0.3	n. d.
	Leu295Ala	47.4 ± 0.1	−6.6 ± 0.1	n. d.
	Lys297Ala	48.4 ± 0.2	−5.7 ± 0.3	n. d.
	Val414Ala	50.9 ± 0.1	−3.2 ± 0.1	14 ± 1	−10.73 ± 0.02	−0.17 ± 0.02
	Tyr429Phe	52.3 ± 0.1	−1.7 ± 0.1	71 ± 6	−9.75 ± 0.05	0.81 ± 0.05

Table 3

Summary of the coupling energy (ΔΔG_int) measured for each pair of residues. ΔΔG_int values are in kcal/mol and were calculated according to the description given in the Materials and methods section. Average and standard errors (SE) are shown. The gray cells indicate mutations of YAP and TEAD in the same binding pocket. n. m. (not measured) indicates that ΔΔG_int could not be determined experimentally because the K_d values for the mutant YAP:mutant TEAD interaction in the corresponding double mutant cycles were above our assay limit (>200 µM).

https://doi.org/10.7554/eLife.25068.007

			hYAP^50-171
			α-helix			Ω-loop
			Leu65	Leu68	Phe69	Met86	Arg89	Leu91	Ser94	Phe95	Phe96
hTEAD^217-434	α-helix	Phe337	−0.34 ±0.04	−0.65 ±0.04	−0.96 ±0.04	0.17 ±0.04	−0.44 ±0.03	n. m.	−0.09 ±0.05	n. m.	0.02 ±0.06
		Lys376	−0.65 ±0.04	−0.41 ±0.05	−1.28 ±0.06	0.13 ±0.04	−0.62 ±0.03	−0.41 ±0.06	−0.22 ±0.06	−0.30 ±0.04	−0.14 ±0.05
		Leu380	−0.36 ±0.05	−0.29 ±0.04	−1.01 ±0.06	0.07 ±0.03	−0.29 ±0.03	−0.26 ±0.05	−0.08 ±0.05	−0.18 ±0.03	−0.02 ±0.06
		Val389	−0.52 ±0.06	−0.29 ±0.05	−1.11 ±0.05	0.16 ±0.03	n. m.	n. m.	0.03 ±0.05	n. m.	−0.06 ±0.04
	Ω-loop	Glu263	−0.05 ±0.05	−0.09 ±0.03	−0.10 ±0.06	0.07 ±0.05	−0.59 ±0.06	−0.62 ±0.05	−0.96 ±0.05	−0.51 ±0.05	−0.04 ±0.07
		Val265	0.26 ±0.03	0.15 ±0.03	0.13 ±0.05	0.09 ±0.03	−0.58 ±0.02	−0.28 ±0.06	−0.06 ±0.06	−0.61 ±0.04	−0.22 ±0.06
		Asp272	n. m.	−0.02 ±0.04	n. m.	−1.85 ±0.04	−3.49 ±0.05	−2.74 ±0.06	−1.65 ±0.05	−2.50 ±0.07	−1.67 ±0.06

Additional files

Supplementary file 1 Dissociation constants for the different YAP:TEAD complexes. The N-Avitagged hTEAD4^217-434 proteins were immobilized on sensor chips and their affinity for the hYAP^50-171 proteins was measured at 298°K by Surface Plasmon Resonance in n ≥ 3 independent experiments. K_d values (in nM) were obtained from equilibrium data (K_d^eq). Averages and standard errors (SE) are given.: https://doi.org/10.7554/eLife.25068.012
Download elife-25068-supp1-v1.docx
Supplementary file 2 Calculated K_d for pairs of hYAP and hTEAD4 mutants. K_d values were calculated (K_d^calc) assuming ΔΔG_int = −500 cal/mol between the YAP:TEAD pairs. A calculation example for the hYAP Arg89:hTEAD4 Val389 pair is given in the following. The difference in binding energy between the two interactions wt hYAP:wt hTEAD4 and hYAP Arg89Ala:wt hTEAD4 is ΔΔG = 4338 cal/mol (Figure 4—source data 1, ΔΔG₁ double mutant cycle for hYAP Arg89:hTEAD4 Val389). With ΔΔG_int = −500 cal/mol, the difference in binding energy between the interactions wt hYAP:hTEAD4 Val389Ala and hYAP Arg89Ala:wt hTEAD4 Val389Ala should be ΔΔG = 3838 cal/mol. As ΔG = −8881 cal/mol for wt hYAP:hTEAD4 Val389Ala, the binding energy of the interaction between hYAP Arg89Ala and wt hTEAD4 Val389Ala should be ΔG = −5043 cal/mol. Using K_d^calc = e^ΔG/RT the dissociation constant for the interaction hYAP Arg89Ala:wt hTEAD4 Val389Ala should be K_d^calc = 199 µM. The last column gives the calculated K_d values for the interaction between the two mutant proteins (K_d^add) if no coupling exists between the two residues (ΔΔG_int = 0 cal/mol in the above calculation). All K_d^add values are above 200 µM in agreement with our experimental data.: https://doi.org/10.7554/eLife.25068.013
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