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. 2023 Jan 1;32(1):e4545. doi: 10.1002/pro.4545

N‐terminal β‐strand in YAP is critical for stronger binding to scalloped relative to TEAD transcription factor

Fedir Bokhovchuk 1, Yannick Mesrouze 1, Marco Meyerhofer 1, Patrizia Fontana 1, Catherine Zimmermann 1, Frédéric Villard 2, Dirk Erdmann 1, Joerg Kallen 2, Scheufler Clemens 2, Camilo Velez‐Vega 3, Patrick Chène 1,✉
PMCID: PMC9798255  PMID: 36522189

Abstract

The yes‐associated protein (YAP) regulates the transcriptional activity of the TEAD transcription factors that are key in the control of organ morphogenesis. YAP interacts with TEAD via three secondary structure elements: a β‐strand, an α‐helix, and an Ω‐loop. Earlier results have shown that the β‐strand has only a marginal contribution in the YAP:TEAD interaction, but we show here that it significantly enhances the affinity of YAP for the Drosophila homolog of TEAD, scalloped (Sd). Nuclear magnetic resonance shows that the β‐strand adopts a more rigid conformation once bound to Sd; pre‐steady state kinetic measurements show that the YAP:Sd complex is more stable. Although the crystal structures of the YAP:TEAD and YAP:Sd complexes reveal no differences at the binding interface that could explain these results. Molecular Dynamics simulations are in line with our experimental findings regarding β‐strand stability and overall binding affinity of YAP to TEAD and Sd. In particular, RMSF, correlated motion and MMGBSA analyses suggest that β‐sheet fluctuations play a relevant role in YAP53‐57 β‐strand dissociation from TEAD4 and contribute to the lower affinity of YAP for TEAD4. Identifying a clear mechanism leading to the difference in YAP's β‐strand stability proved to be challenging, pointing to the potential relevance of multiple modest structural changes or fluctuations for regulation of binding affinity.

Keywords: correlated motions, molecular dynamics, molecular recognition, TEAD, YAP

Short abstract

PDB Code(s): 8a8q and 8a8r;

1. INTRODUCTION

The precise modulation of the transcriptional activity of the TEAD (TEA/ATTS domain) transcription factors is important for the function of the Hippo pathway, which is essential in organ morphogenesis (Davis & Tapon, 2019; Ma et al., 2019; Wu & Guan, 2021; Zheng & Pan, 2019). Several proteins that modulate the transcriptional activity of TEAD have been identified which bind to an overlapping region at the surface of TEAD, perhaps competing to gain access to this transcription factor. The current biochemical and structural data show that the TEAD‐binding domains (TBD) of these proteins have common features, but they also highlight differences between them (Figure 1). The TBD of VGLL1 (vestigial‐like 1) is made up of a β‐strand and an α‐helix. The α‐helix contains the well‐conserved Φ‐D/E‐D/E‐H‐F motif (Φ is any hydrophobic residue) that is key for the interaction with TEAD (Pobbati et al., 2012; Simon et al., 2016). Although this secondary structure element makes a large contribution to the binding of VGLL1 to TEAD, the presence of the β‐strand is required for nanomolar affinity (Mesrouze et al., 2014). VGLL4 possesses two different TBDs (Jiao et al., 2014). The N‐terminal TBD is formed only of a single α‐helix containing a Φ‐D/E‐D/E‐H‐F motif, while the C‐terminal TBD is similar to the VGLL1 TBD with an additional α‐helix at its C‐terminus. The two TBDs can be separated by more than 300 amino acids in some members of this protein family (Mesrouze, Meyerhofer, et al., 2021a). The C‐terminal TBD, in particular its two α‐helices, plays a major role in the interaction between VGLL4 and TEAD (Jiao et al., 2014; Mesrouze, Meyerhofer, et al., 2021a). The TBDs of YAP (Yes‐associated protein) and of its paralog TAZ (transcriptional co‐activator with PDZ motif) are made up of a β‐strand, an α‐helix and an Ω‐loop (Chen et al., 2010; Kaan et al., 2017; Li et al., 2010). The β‐strand and the α‐helix, which contains a conserved L‐x‐x‐L/M‐F (x is any amino acid) motif (Mesrouze, Bokhovchuk, et al., 2021b), bind to the same area at the surface of TEAD as the corresponding secondary structure elements of VGLL1/4. In YAP and TAZ, the hot spot of the interaction is the Ω‐loop, but the α‐helix is required for nanomolar affinity (Hau et al., 2013). The β‐strand is not thought to contribute much to the YAP/TAZ:TEAD interaction (Hau et al., 2013; Li et al., 2010). The presence of an Ω‐loop in the TBD of TEAD interactors was initially only observed for YAP/TAZ, but recent findings have shown that other proteins that bind to these transcription factors also contain this secondary structure element. For instance, VGLL2, a paralog of VGLL1, possesses a β‐strand:α‐helix region similar to the one found in VGLL1, but in addition it has an Ω‐loop which interacts with TEAD in the same way as the Ω‐loop present in YAP/TAZ (Mesrouze et al., 2020). The Ω‐loop enhances the affinity of VGLL2 for TEAD, but a large part of the binding energy comes from the interactions made by the β‐strand:α‐helix region (Mesrouze et al., 2020). Finally, FAM181A and FAM181B contain an Ω‐loop (FAM181B also possesses an L‐x‐x‐L/M‐F motif) (Bokhovchuk, Mesrouze, Delaunay, et al., 2020a). The interaction between fragments of these two proteins and TEAD has been demonstrated in vitro, but its relevance in an in vivo context is not yet known (Bokhovchuk, Mesrouze, Delaunay, et al., 2020a).

FIGURE 1.

FIGURE 1

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Schematic representation of the TBD of different TEAD interactors. The secondary structure elements of the TBD from the TEAD interactors are represented. The length of the different elements is arbitrary. The amino acids indicated are described in the text. By homology with YAP/TAZ, the L‐x‐x‐L/M‐F motif present in FAM181B has been arbitrarily placed in an α‐helix. VGLL3, a paralog of VGLL1 which also binds to TEAD, is not indicated because the structure of its TBD is not known. TBD, TEAD‐binding domain

In this report, we show that the β‐strand present in the TBD of YAP, which was shown to play only a minor role in the interaction with human TEAD, significantly increases the affinity of YAP for scalloped (Sd), the Drosophila homolog of TEAD. We combined biophysical, structural, and molecular dynamics methods to study molecular features contributing to this surprising finding.

2. RESULTS AND DISCUSSION

2.1. YAP binds more tightly to scalloped than to TEAD4

In a recent report, we studied the interaction between scalloped (Sd), the ortholog of TEAD in Drosophila melanogaster, and Yorkie (Yki, ortholog of YAP) (Mesrouze et al., 2020). While the corresponding human proteins were included in these experiments, we made a surprising observation: YAP, and also Yki, has a higher affinity for Sd than for TEAD4 (Table 1). Since the YAP fragment used in these experiments, YAP50‐171, contains regions that could contribute to this difference that lie outside the TBD, we repeated the measurements with a peptide, YAP50‐99, that mimics only the YAP TBD (β‐strand:α‐helix: Ω‐loop region). YAP50‐99 also binds more tightly to Sd than to TEAD4, indicating that the higher affinity of YAP50‐171 for Sd is due to its TBD (Table 1). In an earlier report, we measured the affinity of the three other human TEAD proteins for YAP50‐99: they bind to this peptide with similar affinity as TEAD4 (K d TEAD1‐3 between 44 and 77 nM) (Bokhovchuk et al., 2019). We also show here that a TEAD protein derived from the fish Anabas testudineus (Mesrouze, Bokhovchuk, et al., 2021b) (TEADA.t.) binds less tightly to YAP50‐99 (K d = 29 ± 3 nM, Table 1) than Sd. Altogether, this reveals that Sd has a unique affinity for YAP when compared to these five TEAD proteins.

TABLE 1.

Affinity measurements by surface plasmon resonance

YAP50‐171 (K d eq nM) Yki30‐146 (K d eq nM) YAP50‐99 (K d eq nM) YAP61‐99 (K d eq nM)
TEAD4217‐434 15 ± 1 78 ± 3 31 ± 2 59 ± 1
Sd223‐440 2.0 ± 0.1 10 ± 1 3.8 ± 0.2 44 ± 3
K d TEAD4/K d Sd 7.5 7.8 8.2 1.3
TEADA.t. n.d. n.d. 29 ± 3 n.d.
Lys350AlaSd 3.6 ± 0.2 n.d. n.d. n.d.
Mut‐7TEAD4 12 ± 1 n.d. n.d. n.d.
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Note: The TEAD4 and Sd proteins were immobilized on sensor chips and the affinity of the different ligands was measured at equilibrium, K d eq, using a 1:1 interaction model. The data represent the mean ± standard error of n ≥ 2 independent experiments. The measurements were conducted at 298 K. n.d. not determined.

Since the β‐strand present in the YAP TBD plays only a marginal role in the interaction with TEAD (Hau et al., 2013; Li et al., 2010), we designed a peptide, YAP61‐99, that contains only the α‐helix: Ω‐loop region of the TBD and we measured its affinity for TEAD4/Sd. In agreement with previous results (Hau et al., 2013), the deletion of the β‐strand had little effect (less than two‐fold change) on the affinity of YAP for TEAD (compare YAP50‐99 and YAP61‐99 Table 1). However, it induced a 10‐fold decrease in affinity for Sd, although YAP61‐99 binds with a similar K d to TEAD4 and Sd (Table 1). This is an unexpected finding, which shows for the first time that the β‐strand located in the YAP TBD can make a significant contribution to the interaction with a TEAD protein.

2.2. Nuclear magnetic resonance measurements show that the β‐strand region of YAP is more rigid once bound to Sd than to TEAD4

To study the binding of YAP to TEAD4 and Sd by a different method, we collected 2D 15N‐1H‐SOFAST‐HMQC spectra of 15 N‐labeled YAP50‐171 in its unbound form and in a complex with unlabeled TEAD4/Sd. In accordance with earlier NMR studies (Feichtinger et al., 2018; Feichtinger et al., 2022; Tian et al., 2010), the binding of YAP to TEAD4 resulted in the appearance of 25–30 much more dispersed and less intense cross‐peaks (Figure 2a), indicating that YAP adopts a more ordered conformation upon binding to TEAD4. The same is observed when YAP binds to Sd (Figure 2b). The comparison of the signals between the two complexes (Figure 2c) reveals the presence in the spectrum obtained with Sd of four distinct and disperse peaks scattered within the 8.5–9 ppm region in the 1H dimension and the 118–130 ppm region in the 15 N dimension, which is characteristic for a β‐strand conformation. Considering that the only β‐strand present in the YAP TBD is located between residues 53 and 57, the presence of these peaks could indicate that this region of YAP adopts a more rigid and less dynamic conformation once bound to Sd than to TEAD4. These results, together with the data obtained by surface plasmon resonance (SPR), reveal a difference in the way the β‐strand region of the YAP TBD interacts with TEAD4 and Sd.

FIGURE 2.

FIGURE 2

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Nuclear magnetic resonance study of the interaction between YAP and TEAD4/Sd. (a) Superposition of the 15N‐1H‐SOFAST‐HMQC spectra of the apo 15N‐YAP and the 15N‐YAP:Sd complex ([YAP]/[Sd] = 1), cross‐peaks are shown in black and blue, respectively. (b) Superposition of the 15N‐1H‐SOFAST‐HMQC spectra of the apo 15N‐YAP and the 15N‐YAP:TEAD4 complex ([YAP]/[TEAD4] = 1), cross‐peaks are shown in black and red, respectively. (c) Superposition of the 15N‐1H‐SOFAST‐HMQC spectra of the 15N‐YAP:Sd and 15N‐YAP:TEAD4 complexes, cross‐peaks are shown in blue and red, respectively. The YAP backbone amide cross‐peaks that appear only upon interaction with Sd are indicated by black arrows

2.3. YAP dissociates more slowly from Sd than from TEAD4

To analyze the binding of YAP in more detail, we carried out kinetic studies to determine the association (k on) and dissociation (k off) rate constants for the YAP:Sd and YAP:TEAD4 interactions. The observed binding rate constants, k obs, were measured in stopped‐flow binding experiments monitoring the change in fluorescence triggered by the binding of YAP to TEAD4/Sd. The changes in fluorescence obtained with Sd were smaller than the ones measured for TEAD4 (Figure S1), leading to a higher variability in the measurements (Figure S1). Therefore, we only used the concentrations of YAP that showed the lowest variability. The average traces obtained in the stopped‐flow experiments could be fitted to a single‐exponential equation for both interactions (Figure S1). This is in agreement with our earlier results, indicating that the association between YAP and TEAD4 follows an apparent one‐step binding mechanism (Bokhovchuk, Mesrouze, Meyerhofer, et al., 2020b). The k on values determined from these experiments were 8 ± 1 and (6.1 ± 0.1) μM−1 s−1 for the YAP:TEAD4 and YAP:Sd complexes, respectively (the value for YAP:Sd is an estimate). A displacement assay using a dansylated form of YAP was employed to measure the dissociation rate constants (Bokhovchuk, Mesrouze, Meyerhofer, et al., 2020b) (Figure S1). The k off values were 0.41 ± 0.01 and 0.06 ± 0.01 s−1 for the YAP:TEAD4 and YAP:Sd complex, respectively. Taken together, these results show that the k on values are similar for both interactions while the k off values are very different: YAP dissociated much more slowly from Sd than from TEAD4. In line with this finding, the sensorgrams obtained by SPR with YAP50‐171 and YAP50‐99 showed a much longer dissociation phase from Sd than from TEAD4, while the dissociation phase obtained with YAP61‐99 (missing the β‐strand) was similar for both proteins (Figure S2). This reveals that the enhanced affinity of YAP for Sd comes predominantly from a slower dissociation of the YAP:Sd complex.

2.4. The β‐strand binding interface is similar in the YAP:Sd and YAP:TEAD4 Complexes

The β‐strand present in the YAP TBD interacts upon binding with a β‐strand located at the surface of TEAD (Li et al., 2010). The analysis of the amino‐acid sequence corresponding to this region shows that there is only one change between Sd and TEAD1‐4/TEADA.t.. Ser353Sd is replaced by a threonine in these TEAD proteins (Figure 3a). Since this analysis showed no large differences between Sd and TEAD4, we determined the structure of the YAP50‐99:Sd and YAP50‐99:TEAD4 complexes by X‐ray crystallography (Table S1). The two protein complexes have an analogous global structure (Figure S3) and the β‐strand binding interface is conserved (Figure 3b). The amino acids from the β‐strand of YAP are located at the same position in both protein complexes; this also applies to the residues from TEAD4/Sd. Ser353Sd and its TEAD4 counterpart, Thr347TEAD4, are at the same position and do not interact with YAP in our structures (Figure 3b). Altogether, these data indicate that the binding site for the β‐strand from the YAP TBD is very similar in the two protein complexes.

FIGURE 3.

FIGURE 3

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Structure of the YAP:Sd and YAP:TEAD4 complexes. (a) Sequence alignment of the residues located in the β‐strand of the YAP‐binding domain of different TEAD/Sd proteins. TEADA.t.: TEAD from Anabas testudineus. Ser353Sd is in red. UniProtKB Seq. Id. Sd: P30052. TEAD1: P28347; TEAD2: Q15562; TEAD3: Q99594; TEAD4: Q15561; TEADA.t.: XP_026221540.1. (b) Superimposition of the YAP:Sd (PDB 8a8q) and YAP:TEAD4 (PDB 8a8r) structures. The figure represents only the region where the β‐strand from the YAP TBD binds to TEAD4/Sd. The figure was drawn with PyMOL (Schrödinger Inc., Cambridge, MA)

Although the structure and the amino‐acid content of the region where the β‐strand of YAP binds are highly consistent between TEAD4 and Sd, we observed that Lys350Sd is acylated while the corresponding residue, Lys344TEAD4, is not (Figure 3b). TEAD and Sd are known to be acylated (myristoylated or palmitoylated) on a conserved cysteine (Cys367TEAD4 or Cys373Sd) and this modification is important for the function of these transcription factors (Chan et al., 2016; Noland et al., 2016). Recently, it has been shown that Lys350Sd can be acylated: the effect of this modification on Sd function has been studied (Mesrouze et al., 2022). Only 50% of the Sd protein used in our assays was acylated on Lys350AlaSd (the rest is acylated on Cys373Sd) (Mesrouze et al., 2022), but this acylation may nevertheless enhance the binding of YAP to Sd. To check this possibility, we measured the affinity of YAP50‐171 for the Lys350AlaSd mutant, which is only acylated on Cys367Sd (Mesrouze et al., 2022). The affinity of Lys350AlaSd for YAP50‐171, K d = 3.6 ± 0.2 nM (Table 1), is similar to the one measured for wtSd (Table 1), suggesting that the presence of acylated Lys350Sd is not required for YAP to bind more tightly to Sd. However, it cannot be excluded that the effects triggered by the loss of a lysine lateral chain and the absence of an acyl at position‐350 compensate each other so that the affinity of Lys350AlaSd for YAP is the same as for wtSd.

The structural data show that the binding of YAP to Sd/TEAD4 expands the antiparallel β‐sheet present in these proteins. Two YAP:Sd heterodimers interact with each other via the bound β‐strand of YAP to form an antiparallel β‐sheet that stabilizes this heterotetrameric complex (Figure S4). These interactions are not observed in our structure of the YAP:TEAD4 complex (Figure S4) or in a published structure of the YAP:TEAD1 complex (PDB 3kys, Li et al., 2010). The formation of heterotetramers may enhance the stability of the YAP:Sd complex and, as a consequence, the overall affinity of YAP for Sd. To check this hypothesis, we conducted SEC/MALS (size exclusion chromatography/multi‐angle light scattering) experiments. Under our experimental conditions, YAP50‐171 only forms heterodimeric complexes with TEAD4 and Sd (Figure S4). Therefore, the formation of the heterotetramers observed in the YAP:Sd structure might be triggered by the crystallization conditions.

2.5. Molecular dynamics simulations showed that the β‐strand from the YAP TBD was more stable when bound to Sd than when bound to TEAD4, and that the binding free energy of the YAP:Sd complex is more favorable than that of YAP:TEAD4

We next explored whether the difference in stability of the β‐strand of YAP when bound to TEAD4 and Sd would also be observed in silico. MD simulations were carried out starting from both complexes in non‐acylated, Lys350Sd/Lys344TEAD4 acylated, and Cys373Sd/Cys367TEAD4 acylated forms.

Analysis of the simulations with the non‐acylated proteins showed that the β‐strand region of YAP remains stable when bound to Sd (Figure 4a), but it dissociates from TEAD4 after ~20 ns of run time (200 total/replica; Figure 4b), readily losing its β‐strand character once unbound from TEAD4. Representative plots of the YAP53‐57 β‐strand RMSD vs. MD simulation frame number have also been included for reference in Figure 4g for all complexes studied. No other relevant differences in the structure of Sd or TEAD4 (relative to the corresponding X‐ray data) were detected. Other than the β‐strand dissociation, YAP predominantly remains close to its initial state. We did observe higher flexibility around the region YAP75‐83, in line with the fact that this segment was unresolved in both co‐crystal complexes.

FIGURE 4.

FIGURE 4

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Molecular dynamics simulations. Representative time‐averaged structures (40–200 ns) extracted from molecular dynamics simulations of YAP:non‐acylated Sd (a), YAP:non‐acylated TEAD4 (b), YAP:acyl‐Lys344TEAD4 (c), YAP:acyl‐Cys367TEAD4 (d), YAP:acyl‐Lys350Sd (e), and YAP:acyl‐Cys373Sd (f) Representative plots of the YAP53‐57 β‐strand RMSD (referenced to the initial state) versus MD frame number, for all six complexes shown in a‐f (g). MD simulations were performed using the YAP:Sd (PDB 8a8q) and YAP:TEAD4 (PDB 8a8r) structures, and figures a–f were drawn with PyMOL (Schrödinger Inc., Cambridge, MA)

The simulations with TEAD4 acylated on Lys344TEAD4 or Cys367TEAD4 were different. The N‐terminal region of YAP (specifically YAP53‐57) remained bound to TEAD4 and, for much of the simulation time, adopted a β‐strand conformation (Figure 4c,d). Conversely, the simulations carried out with Sd acylated on Lys350Sd or Cys373Sd show comparable stability around YAP's β‐strand region to that observed in the non‐acylated Sd simulations (Figure 4e,f). Comparing the dynamics of the complexes formed with acylated TEAD4 and acylated Sd suggested that YAP's β‐strand region is less structured when in complex with TEAD4. Antiparallel β‐sheet structure content plots for the five residues found to adopt a β‐strand conformation in both crystals (YAP53‐57) indicated that YAP was more likely to adopt this conformation with Sd than with TEAD4 (Figure S5A,B). Moreover, these five YAP residues were found to have consistently lower average per‐residue fluctuation in Sd than in TEAD4 (Figure S5C,D). The clear structural shifts observed via MD between the complexes formed by YAP with non‐acylated TEAD4/Sd, as well as the simulations with the acylated proteins, support the notion that the β‐strand region is more stable in the YAP:Sd complex.

We then investigated whether the loss of stability of the YAP53‐57 β‐strand observed in simulation (along with any other varying interactions) translates into a difference in the predicted affinity of YAP to Sd and TEAD4. For that purpose, an estimate of the binding free energy for the complex (ΔG bind) was obtained by means of MMGBSA calculations (Kollman et al., 2000). Figure 5 shows that ΔG bind for both Lys‐acylated and Cys‐acylated Sd complexes is more favorable than for either of the YAP:TEAD4 complexes, in line with our SPR results. Note that the predicted binding free energy of each Lys‐acylated complex seems to be more favorable than that of its corresponding Cys‐acylated complex. The latter result challenges our experimental findings with the Lys350AlaSd mutant indicating that Lys350‐acylation is not essential for tighter binding of YAP to Sd. Rationalizing this disconnect is an appealing topic for future work.

FIGURE 5.

FIGURE 5

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Binding free energies (ΔGbind) computed via the MMGBSA method for both Cys‐acylated and Lys‐acylated complexes of YAP bound to TEAD4 and Sd

Although all of our in silico observations are consistent with our experiments, no key structural shifts/changes were identified from the MD simulations that would provide a well‐defined mechanistic picture rationalizing YAP's β‐strand unbinding/instability when bound to TEAD4.

2.6. Effect of regions surrounding the β‐strand binding site

In an effort to narrow down explicit sources of YAP's β‐strand instability in TEAD4 relative to Sd, we identified seven residues that are not conserved between both proteins within 8 Å of YAP53‐73 (Figure S6) and generated a TEAD4 mutant, Mut‐7TEAD4, containing the seven residues from Sd. The thermal stability of Mut‐7TEAD4 was compared to that of TEAD4 in a fluorescence thermal shift assay (Figure S7). The thermograms obtained with Mut‐7TEAD4 showed that this protein is not unfolded and its melting temperature, T m = 56.0 ± 0.0°C, is even slightly higher than that of wtTEAD4, T m = 53.0 ± 0.1°C. As this result suggests that the overall structure of TEAD4 is preserved in Mut‐7TEAD4, we measured its affinity for YAP50‐171. Mut‐7TEAD4 has a similar affinity for YAP50‐171 (K d = 12 ± 1 nM, Table 1) as wtTEAD4 (Table 1), suggesting that the difference in amino acids observed between Sd and TEAD4 in the region surrounding bound YAP53‐73 does not cause the higher affinity of Sd for YAP.

Given our unfruitful attempts to isolate key mutations—or evident structural changes—that could act as the trigger for YAP's β‐strand instability, we hypothesized that it could be the result of a concerted effect involving multiple subtle changes in intra and inter‐molecular interactions between YAP and TEAD4/Sd. We therefore turned back to modeling to study differences in root mean square fluctuations (RMSF) and correlated motions between both acylated complexes. Correlated motions are small collective movements of residues that can lead to allosteric effects without triggering major conformational changes (Xu et al., 2021). They are a fundamental property of β‐sheets (Fenwick et al., 2014), and can play a key role in molecular recognition involving these secondary structure elements (Fenwick et al., 2011).

An RMSF analysis of our MD trajectories strongly supports the involvement of TEAD4 /Sd β‐sheet fluctuations in YAP binding. Figure 6 shows that the major differences in RMSFs (protein main chain heavy atoms) between acylated TEAD4 and Sd when bound to YAP come from β‐sheets contiguous to YAP's β‐strand (highlighted in orange in Figure 6a,b) and to a lesser extent from the loop–helix–loop motif formed by residues 60–75 (highlighted in slate blue in Figure 6a,b). Keeping in mind that we found no significant differences in the interactions formed in both complexes, it is likely that higher fluctuations in the TEAD4 regions adjacent to YAP's β‐strand promote destabilization of YAP's β‐strand motif. Yet, the question remains as to whether the more stable β‐sheets in Sd effectively transfer information through correlated motions between individual β‐strands, ultimately holding YAP's β‐strand more strongly.

FIGURE 6.

FIGURE 6

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RMSF analysis of the MD trajectories. Per‐residue root mean square fluctuations (RMSF) of Cys‐acylated and Lys‐acylated YAP:TEAD4 and YAP:Sd complexes, computed from MD frames collected for the last 160 ns (total 200 ns) of protomer A and B simulations for each case (a). The regions with highest differences in RMSF between TEAD4 and Sd are highlighted in shaded ovals. The shaded regions have been mapped onto a representative structure of TEAD4 (B) and colored according to (a). Specifically, a loop‐helix‐loop motif (Glu276‐Ala292TEAD4) is colored in slate, whereas β1‐β4 sheets plus two other β‐strand rich regions (Val316‐Ile327TEAD4 and Glu348‐Leu366TEAD4) are colored in orange. YAP is colored in magenta and TEAD4 in green

We built dynamic cross correlation maps (DCCMs) from Cys‐acylated and Lys‐acylated MD simulations (see Section 4), focusing our analysis on correlations between sheets β1‐β4 (see Figure S8 for β‐sheet nomenclature and residue differences between TEAD4 and Sd along the β‐sheet) following our RMSF results. Overall, discrepancies were found between the DCCMs computed for protomers with the same acylation type (Figure 7), indicating that additional sampling time would be needed to achieve convergence. Despite not reaching full convergence (which would require a higher computational cost since it entails convergence of relative motions of atom pairs, as opposed to changes in position of individual atoms with respect to their reference state), our analysis points to differences in β1‐β4 cross‐correlations between both complexes. Specifically, two of the DDCMs—corresponding to Cys‐acylated and Lys‐acylated YAP:Sd simulations (Figure 7c,g)—show appreciably stronger correlation between these β‐sheets relative to the remaining simulations. Conversely, one of the Cys‐acylated YAP:TEAD4 DCCMs (Figure 7a) displays noticeably weaker correlations between β‐sheets compared to the other runs. Altogether, these results are consistent with the notion that stronger through β‐sheet communication in YAP:Sd versus YAP:TEAD4 likely contributes to the YAP53‐57 β‐strand stability. It is worth noting that other regions of TEAD4 and Sd are also probably involved in allosteric communication with YAP and could play a role in YAP's β‐strand stability and overall affinity to YEAD4/Sd.

FIGURE 7.

FIGURE 7

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Per‐residue dynamic cross‐correlation maps for β1–β4 sheets of Cys‐acylated TEAD4 (a and b) and Sd (c and d), and Lys‐acylated TEAD4 (e and f) and Sd (g and h), computed from MD simulations. The coloring scale ranges from white (pairs are fully correlated, a value of 1) to black (pairs are fully anticorrelated, a value of −1); fully uncorrelated pairs are colored green (a value of 0). β‐Strands 1, 2, 3, and 4 comprise TEAD4 residues Val341‐Glu346, Ile328‐Cys335, Ile395‐Thr400, and Leu408‐Tyr413, respectively

3. CONCLUSION

The TEAD transcription factors interact with different proteins that regulate their transcriptional activity, amongst them the YAP protein. The region of YAP that binds to TEAD is largely unstructured in solution (Feichtinger et al., 2022; Tian et al., 2010), but upon binding to TEAD it adopts a well‐defined structure formed by three different secondary structure elements: a β‐strand, an α‐helix, and an Ω‐loop (Li et al., 2010). Structure–function studies have shown that the α‐helix and the Ω‐loop are essential for the interaction, while the β‐strand plays only a minor role (Hau et al., 2013; Li et al., 2010). In this study we show that human YAP binds about 10 times more tightly to the Drosophila TEAD analog, Sd, than to human TEAD4 and that this difference comes surprisingly from the β‐strand present in the YAP TBD. NMR revealed that this β‐strand is more rigid once bound to Sd than to TEAD4 and pre‐steady state kinetic measurements indicate the YAP:Sd complex dissociates more slowly than the YAP:TEAD4 complex. However, these differences cannot be explained when the structures of the YAP:Sd and YAP:TEAD4 complexes are compared. This prompted us to hypothesize that a combination of several small structural differences between the YAP:Sd and the YAP:TEAD4 complexes might cause the higher affinity of YAP for Sd. Molecular dynamics simulations are in line with our experimental findings regarding tighter binding of YAP to Sd relative to TEAD4, as well as contrasting stability of YAP53‐57 between both complexes in non‐acylated and acylated states. Furthermore, modeling suggests decreased flexibility in Sd's β1‐β4 strands as well as in YAP53‐57 relative to YAP:TEAD4, with a concomitant increase in correlated motions across the β‐sheet. This implies that allosteric interactions can have an impact on the binding of a TEAD regulator to this transcription factor. Interestingly, several compounds binding to the myristate/palmitate‐binding pocket of TEAD, located next to the binding site of the β‐strand region of YAP, affect the interaction with YAP (e.g., Kaneda et al., 2020; Tang et al., 2021). Since no clear understanding on how these molecules inhibit the YAP:TEAD interaction resulted from the analysis of the available structures, one may speculate on the basis of our current results, that they could perturb communication pathways between YAP and TEAD without triggering any large observable structural changes.

4. MATERIALS AND METHODS

4.1. Peptides

The synthetic peptides (both N‐acetylated and C‐amidated) were purchased from Biosynthan (Germany). The peptides YAP50‐99 (Ac‐AGHQIVHVRGDSETDLEALFNAVnNPKTANVPQTVPnRLRKLPDSFFKPP‐NH2), YAP51‐99 (Ac‐GHQIVHVRGDSETDLEALFNAVnNPKTANVPQTVPnRLRKLPDSFFKPP‐NH2), YAP61‐99 (Ac‐SETDLEALFNAVnNPKTANVPQTVPnRLRKLPDSFFKPP‐NH2) and YAP50‐100 (Ac‐AGHQIVHVRGDSETDLEALFNAVnNPKTANVPQTVPnRLRKLPDSFFKPPE‐NH2) (n is norleucine) are derived from the sequence of human YAP (UniProtKB P46937). Their purity and chemical integrity has been determined by liquid chromatography–mass spectrometry (LC–MS) from 10 mM stock solutions in 90:10 (v/v) dimethyl sulfoxide: water.

4.2. Protein cloning, expression, and purification

Human TEAD4 (a.a. 217–434), Sd from Drosophila melanogaster (a.a. 223–440), TEAD (TEADA.t.) from Anabas testudineus (a.a. 211–428), human YAP (a.a. 50–171), and Yki from D. melanogaster (a.a. 30–146) were purified as previously described (Mesrouze et al., 2020; Mesrouze, Bokhovchuk, et al., 2021b; Mesrouze, Meyerhofer, et al., 2017a). The Lys350AlaSd, and Ser342AlaSd mutations were carried out with the same methodology as previously reported (Mesrouze et al., 2018). TEAD4 with seven mutated residues, Mut‐7TEAD4, was generated with four consecutive rounds of mutagenesis. First round: Thr329ValTEAD4 and Lys333IleTEAD4. Second round: Thr347SerTEAD4. Third round: His375GlnTEAD4 and His379AsnTEAD4. Finally, fourth round: His362GlnTEAD4 and Leu366MetTEAD4. The purity, concentration, and identity of the proteins was determined by RP‐HPLC and LC–MS (Figure S9).

4.3. Nuclear magnetic resonance spectroscopy

All NMR spectra were acquired at 298 K on a Bruker 800 MHz AVII spectrometer equipped with CryoProbe (Bruker, Billerica, MA). 15 N‐labeled YAP50‐171 was obtained as previously described (McCoy et al., 2007; Vonrhein et al., 2011). Samples contained 100 μM 15 N‐labeled YAP50‐171 without or with an equimolar amount of unlabeled TEAD4 or Sd in NMR buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA in H2O∕D2O 90∶10 [vol/vol]). The proteins were dialyzed overnight against the NMR buffer. The protein concentration was determined by HPLC and the proteins were mixed before measurement to form the YAP:TEAD4/Sd complexes. The binding of YAP to TEAD4/Sd was monitored by 2D 15 N‐1H‐SOFAST‐HMQC experiments (Schanda et al., 2005) by comparing with the spectra obtained with apo‐YAP. NMR data processing and analysis were performed using TopSpin (v. 4.0.7; Bruker Billerica, MA).

4.4. X‐ray crystallography

YAP:Sd complex structure. Co‐crystals of Sd223‐440 with YAP51‐99 were grown at 293 K using the sitting drop vapor diffusion method. Sd223‐440 (8.3 mg/ml) in 50 mM Tris pH 8.0, 250 mM NaCl, 2 mM MgCl2, 1 mM TCEP, and 5% glycerol was pre‐incubated with 0.5 mM YAP51‐99 ([Sd]/[YAP] ~ 1.5). For crystallization, the protein peptide complex was mixed with an equal volume (0.3 μl + 0.3 μl) of the reservoir solution (0.1 M MES pH 6.0, 20% PEG8000 and 0.2 M sodium acetate trihydrate). Prior to shock cooling in liquid nitrogen, the crystal was soaked in reservoir solution with 30% glycerol for a few seconds. X‐ray diffraction data were collected at the Swiss Light Source (SLS, beamline X10SA) using an Eiger pixel detector. Raw diffraction data from one crystal were analyzed and processed using the autoPROC (Vonrhein et al., 2011) STARANISO (v 3.3 Global Phasing Ltd, Cambridge, UK) toolbox. The structure was determined by molecular replacement with PHASER (McCoy et al., 2007) using the coordinates of previously solved in‐house structures of Sd223–440 as a search model. The software programs COOT (Emsley & Cowtan, 2004) and BUSTER (v 2.11.8; Global Phasing Ltd., Cambridge, UK) were used for iterative rounds of model building and structure refinement (Table S1). The refined coordinates of the complex structure have been deposited in the Protein Data Bank (www.wwpdb.org) under accession code 8a8q.

YAP:TEAD4 complex structure. Co‐crystals of TEAD4217‐434 with YAP50‐100 were grown at 293 K using the sitting drop vapor diffusion method. TEAD4217‐434 (5.0 mg/ml) in 20 mM Tris pH 8.5, 100 mM NaCl, 1 mM TCEP, 5% glycerol was pre‐incubated with 0.5 mM YAP50‐100 ([YAP]/[TEAD4] ~ 2.5). Diffraction quality crystals were grown using 50 mM sodium acetate trihydrate pH 4.6, 50 mM magnesium acetate tetrahydrate, and 25% PEG400 as the reservoir solution. Prior to shock cooling in liquid nitrogen, the crystal was soaked in reservoir solution with 20% glycerol for a few seconds. X‐ray diffraction data were collected at the Swiss Light Source (SLS, beamline X10SA) using a Pilatus pixel detector. X‐ray data processing and structure refinement were performed as described for the YAP:Sd complex (Table S1). The refined coordinates of the complex structure have been deposited in the Protein Data Bank (www.wwpdb.org) under accession code 8a8r.

4.5. Size exclusion chromatography/multi‐angle light scattering

The protein samples were analyzed using size exclusion chromatography coupled to a multi‐angle light scattering detector (SEC/MALS). Briefly, an Agilent 1100 series HPLC system (Agilent, Santa Clara, CA) was equipped with a Superdex 200 Increase 5/150 GL column (Cytiva, Marlborough, MA), an Optilab T‐rEX refractive index detector, and a DAWN Heleos‐II light‐scattering detector (Wyatt, Santa Barbara, CA). The protein samples (20 μl—concentration of 0.3–0.4 mM) were injected onto the column equilibrated with 50 mM Tris, 100 mM NaCl, 2 mM MgCl2, 1 mM TCEP, pH 8.0. Data collection and processing was performed with ASTRA software version 7.1 (Wyatt, Santa Barbara, CA). Protein concentration was calculated using the refractive index signal (assuming dn/dc = 0.185). Molecular mass moments at peak max were calculated using the Zimm fit method (Wyatt, 1993).

4.6. Surface plasmon resonance, fluorescence thermal shift assay, and stopped‐flow fluorescence binding experiments

The SPR and FTSA experiments and their analysis were conducted as previously described (Mesrouze et al., 2020; Mesrouze, Bokhovchuk, et al., 2017b). Representative sensorgrams and thermograms are represented in Figures S2 and S7, respectively. Binding kinetics were studied using a model SX‐20 stopped‐flow spectrometer (Applied Photophysics, Leatherhead, UK) by recording changes in fluorescence. The experiments and the analyses of the data were carried out as previously reported (Bokhovchuk, Mesrouze, Meyerhofer, et al., 2020b).

4.7. Molecular dynamics simulations

The YAP:TEAD4 (PDB 8a8r) and YAP:Sd (PDB 8a8q) complexes were prepared via Schrödinger's Protein Preparation Wizard (Release 2022‐1, Schrödinger LCC, New York, NY). Missing loops were built in this step. The resulting structures were further processed by separating the two protomers comprising the crystallographic unit of each complex, and changing the rotameric and protonation states selected to achieve full consistency of the hydrogen‐bonding network. This resulted in four monomeric systems to be modeled for the non‐acylated form, two for YAP:TEAD4 and two for YAP:Sd. Each system was then solvated in TIP3P water, preserving crystallographic waters. 3DRISM (Gusarov et al., 2006) was used to find waters in buried pockets, which were included in the simulations. Four Lys‐acylated and four Cys‐acylated systems were also prepared from their respective apo systems, by covalently attaching a myristoyl group to the corresponding residue and re‐solvating.

Two 200‐ns explicit solvent unrestrained MD runs were carried out for each system (both wild‐type and mutant MD simulations). All runs were given a random seed for the initial assignment of atomic velocities, to perturb the dynamics and bolster sampling. Time‐averaged structures were obtained in accordance with previous work (Velez‐Vega et al., 2014), for the 40–200 ns period of each production MD simulation. The MD preparation, minimization, and equilibration protocol followed that described in a prior publication (Velez‐Vega et al., 2014).

The Amber MD package (Case et al., 2005) was used for simulation on our GPU cluster. AmberTools (Case et al., 2005), VMD (Humphrey et al., 1996), and Placevent (Sindhikara et al., 2012) were used to post‐process MD trajectories, and Pymol (v 2.4 Schrödinger Inc., Cambridge, MA) was employed for visualization. MMGBSA calculations were performed using Schrödinger's Prime module (Kollman et al., 2000; Release 2022–1, Schrödinger LCC, New York, NY), whereas RMSF and RMSD calculations were respectively enacted via the atomicfluct and rmsd functions available through AmberTools (Case et al., 2005).

To compute dynamic cross‐correlation maps (DCCMs) for both complexes, we extended all MD simulations of Lys‐acylated and Cys‐acylated Sd and TEAD4 to 400 ns/replica, to obtain better cross‐residue statistics. For each case, both replicas were merged after removing the first 100 ns of each replica (cutoff based on how fast RMSFs equilibrated). A DCCM analysis of the resulting MD trajectories was carried out via the matrix correl function in AmberTools (Case et al., 2005; Hunenberger et al., 1995).

AUTHOR CONTRIBUTIONS

Fedir Bokhovchuk: Investigation (equal); methodology (equal). Yannick Mesrouze: Investigation (equal); methodology (equal). Marco Meyerhofer: Investigation (equal); methodology (equal). Patrizia Fontana: Investigation (equal); methodology (equal). Catherine Zimmermann: Investigation (equal); methodology (equal). Frederic Villard: Investigation (equal); methodology (equal). Dirk Erdmann: Formal analysis (equal); supervision (equal). Joerg Kallen: Formal analysis (equal); supervision (equal). Clemens Scheufler: Formal analysis (equal); supervision (equal). Camilo Velez‐Vega: Conceptualization (equal); formal analysis (equal); writing – original draft (equal); writing – review and editing (equal). Patrick Chene: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); supervision (equal); writing – original draft (equal); writing – review and editing (equal).

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Supporting information

Appendix S1: Supporting Information

Click here for additional data file. (1.7MB, pdf)

Appendix S2: Supporting Information

Click here for additional data file. (885.8KB, pdf)

ACKNOWLEDGMENTS

We thank Dr. Cesar Fernandez and Dr. Wolfgang Jahnke for their support in the NMR experiments.

Fedir B, Yannick M, Marco M, Patrizia F, Catherine Z, Frédéric V, et al. N‐terminal β‐strand in YAP is critical for stronger binding to scalloped relative to TEAD transcription factor. Protein Science. 2023;32(1):e4545. 10.1002/pro.4545

Bokhovchuk Fedir and Mesrouze Yannick contributed equally to this study.

Review Editor: Jeanine Amacher

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Associated Data

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Supplementary Materials

Appendix S1: Supporting Information

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Appendix S2: Supporting Information

Click here for additional data file. (885.8KB, pdf)

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