WW Domains of the Yes-Kinase-Associated-Protein (YAP) Transcriptional Regulator Behave as Independent Units with Different Binding Preferences for PPxY Motif-Containing Ligands

Abstract

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.

Full text

RESEARCH ARTICLE
WW Domains of the Yes-Kinase-Associated-
Protein (YAP) Transcriptional Regulator
Behave as Independent Units with Different
Binding Preferences for PPxY Motif-
Containing Ligands
Manuel Iglesias-Bexiga
1¤
, Francisco Castillo
1
, Eva S. Cobos
1
, Tsutomu Oka
2
,
Marius Sudol
2
, Irene Luque
1
*
1Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of
Granada, 18071, Granada, Spain, 2Weis Center for Research, Geisinger Clinic, M.C. 26–08, 100 North
Academy Avenue, Danville, PA, 17822–2608, United States of America
¤. Current address: Rocasolano Physical Chemistry Institute, CSIC, Serrano 119, 28006, Madrid, Spain
*iluque@ugr.es
Abstract
YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the
object of heightened interest as a potent oncogene and stemness factor. YAP has two
major isoforms that differ in the number of WW domains they harbor. Elucidating the degree
of co-operation between these WW domains is important for a full understanding of the mo-
lecular function of YAP. We present here a detailed biophysical study of the structural stabil-
ity and binding properties of the two YAP WW domains aimed at investigating the
relationship between both domains in terms of structural stability and partner recognition.
We have carried out a calorimetric study of the structural stability of the two YAP WW do-
mains, both isolated and in a tandem configuration, and their interaction with a set of func-
tionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP
WW domains behave as independent units with different binding preferences, suggesting
that the presence of the second WW domain might contribute to modulate target recognition
between the two YAP isoforms. Analysis of structural models and phage-display studies in-
dicate that electrostatic interactions play a critical role in binding specificity. Together, these
results are relevant to understand of YAP function and open the door to the design of highly
specific ligands of interest to delineate the functional role of each WW domain in YAP
signaling.
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 1/22
OPEN ACCESS
Citation: Iglesias-Bexiga M, Castillo F, Cobos ES,
Oka T, Sudol M, Luque I (2015) WW Domains of the
Yes-Kinase-Associated-Protein (YAP) Transcriptional
Regulator Behave as Independent Units with Different
Binding Preferences for PPxY Motif-Containing
Ligands. PLoS ONE 10(1): e0113828. doi:10.1371/
journal.pone.0113828
Academic Editor: Haiwei Song, Institute of Molecu-
lar and Cell Biology, SINGAPORE
Received: February 4, 2014
Accepted: October 31, 2014
Published: January 21, 2015
Copyright: © 2015 Iglesias-Bexiga et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Funding: This work was supported by the Spanish
Ministry of Education and Science [grant BIO2009-
13261-CO2], the Spanish Ministry of Economy and
Competitivity [grant BIO2012-39922-CO2] including
FEDER (European Funds for Regional Development)
funds and the Governement of Andalusia [grant CVI-
5915]. Marius Sudol was supported by PA Breast
Cancer Coalition Grants (#60707 and #920093) plus
the Geisinger Clinic. The funders had no role in study
design, data collection and analysis, decision to pub-
lish, or preparation of the manuscript.

Introduction
The Yes kinase-associated protein, YAP, is a potent oncogene and stemness factor [1,2]. As a
transcriptional co-activator, YAP elicits its oncogenic action via a two-prong strategy: it up-
regulates genes that promote cell proliferation and also targets a set of genes that inhibit apo-
ptosis [3]. YAP is a transforming gene that is amplified in several human cancers including
breast, ovary, head and neck, and liver [4]. At present, the YAP gene and its products are stud-
ied intensively as YAP is one of the two main effectors of the newly delineated tumor suppres-
sor pathway known as Hippo. The activation of the Hippo pathway by cell-to-cell contacts
results in YAP phosphorylation on Serine 127 by Large Tumor Suppressor homolog (LATS) ki-
nases. This modification anchors YAP in the cytoplasm via its interaction with the 14–3–3 pro-
tein, preventing YAP from nuclear localization and impairing transcriptional activity. The
Hippo pathway was recently shown to crosstalk with a number of major signaling pathways in-
cluding Notch, Wnt and TGF beta [1].
Cloning of the YAP gene resulted in the identification of a small modular protein domain,
known as the WW domain, named after two conserved tryptophan residues spaced 20 to 22
residues apart within the sequence. WW domains are abundant and versatile protein-protein
interaction modules that recognize proline-rich motifs [5] and adopt a common three-stranded
antiparallel β-sheet fold [6,7]. The side chain of the first conserved tryptophan lies on one side
of the β-sheet in the hydrophobic core and is required for domain stability. The second trypto-
phan is located at one of the pockets at the binding site dedicated to proline recognition, the
xP pocket, and common to most of WW domains. A second pocket in the binding site is re-
sponsible for binding specificity within WW domains subfamilies. In the case of type-1 WW
domains, which preferentially recognize PPxY sequences, this specificity pocket is dedicated to
the recognition of the Tyr residue in the consensus core-motif [8,9].
YAP is characterized by a well differentiated modular architecture (Fig. 1A), containing a
transcriptional enhancer factor-binding domain (TB), a 14–3–3 binding-site, one or two type-
1 WW domains, an SH3 binding motif, a transcriptional activation domain (TAD), a PDZ
binding motif and several serine phosphorylation sites distributed throughout the sequence.
WW domains are critical for the interaction of YAP with LATS kinases (Fig. 1C) and these do-
mains also play a role in YAP ability to regulate transcription, cell transformation and tissue
growth [10,11]. There are two major isoforms of YAP that differ exclusively in the number of
WW domains: YAP1 (also named Yap1–1gamma; Uniprot code: P46937–6), which contains
only one WW domain and YAP2 (also known as Yap1–2 gamma; Uniprot code: P46937–1)
containing two WW domains. YAP2, which is predominantly expressed in neural tissues, is
considered the canonical sequence of YAP. Even though the precise signaling differences
among YAP splicing variants remain to be elucidated, some differences in the behavior of the
two isoforms have been reported. In this way, only YAP2 can interact with p73 [12] and
AMOTL1 (Angiomotin-Like-1) [13]. Also, YAP2 has been described to be a more potent tran-
scriptional co-activator of ErbB-4 than YAP1 [14].
Tandem repeats of modular domains are frequently found in signaling proteins, as a mecha-
nism of optimization of cellular signal transduction. Several instances of WW domain tandems
have been described, reporting different degrees of cooperativity between the constituting WW
domains [15]. The elucidation of the degree and mechanism of co-operation between the two
domains, the delineation of their binding specificity and the identification of proteins that asso-
ciate with each of them is of relevance for a full understanding of YAP function as a regulator
of the balance between proliferation and apoptosis within the Hippo signaling network. With
this aim we have performed a detailed thermodynamic analysis of the structural stability and
binding properties of the two WW domains of YAP, both as isolated domains and as domains
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 2/22
Competing Interests: The authors have declared
that no competing interests exist.

in a tandem arrangement using calorimetric techniques and a set of functionally relevant li-
gands including peptide sequences derived from well-established functional partners of YAP,
such as LATS kinases [12] and from Patched protein homolog 1 (PTCH1), a protein from the
SHH pathway containing two PPxY motifs predicted as a cognate ligand of YAP WW domains
(Fig. 1D). In order to probe PTCH1 as a ligand of YAP, functional studies in a cell culture
model were performed.
Our work reveals that the WW domains of YAP behave as two independent units, both
in terms of structural stability and ligand recognition, with no overt evidence of cooperativity
between them. Binding studies show that the two WW domains exhibit different binding
preferences, showing high selectivity for some ligands, such as the second PPxY motif in
PTCH1. These results suggest that the presence of the second WW domain in YAP2 isoform
modulates YAP function through partner recognition. Phage display studies and analysis of
structural models of the different complexes suggest that electrostatic interactions play a key
role in determining individual binding specificity for the WW1 and WW2 domains in human
YAP.
Figure 1. A) Modular organization of the two isoforms of human YAP transcriptional regulator. Both forms are comprised of one TEAD transcription
factor-binding domain (TB), one (YAP-WW1) or two (YAP-WW1 and YAP-WW2) WW domains, one trans-activation domain (TAD) and one PDZ binding
motif. The symbol P indicates the proposed serine phosphorylation sites. B) Sequence alignment of the individual WW domains of human YAP1 and
YAP2 isoforms. Identical residues are shown on a black background and conservative changes on a gray background. Black arrows indicate those residues
constituting the xP and xY pockets at the binding site. Red numbers indicate their position in the sequence of the full protein. C) Modular organization of the
human LATS1 and LATS2 kinases containing an ubiquitin-associated domain (UBA), a C-terminal protein kinase domain (KIN-D) and one or two PY
sequences. D) Modular architecture of human PATCHED homologue 1 including a Sterol-sensing domain (SSD) and two PY sequences.
doi:10.1371/journal.pone.0113828.g001
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 3/22

Materials and Methods
Protein cloning, expression and purification
The actual boundaries of modular protein domains are not easy to define from their linear se-
quences (see Discussion in [16]). In the case of the WW domains, a demarcation of the func-
tional length of the domain was facilitated by the naturally occurring splicing event that added
one extra WW domain in the YAP2 isoform [17]. This second WW domain is 38 amino acids
long and it is encoded, in the human YAP gene, by a single exon [5,18]. In published reports,
WW domains of various lengths have been used for structural and functional studies, ranging
from 36 [19,20,21,22] to 56 amino acids [17,22]. Considering several studies that reported
expression problems for shorter WW domain sequences [20,23], we have expressed YAP WW
domains as 46 amino acids long domains with the two conserved tryptophan residues centered
in the sequence, as described in [6,24,25]. Accordingly, in the Uniprot P46937–1 entry the
constructs used in this study correspond to residues 165–209 for the first WW domain of
human YAP (YAP-WW1), residues 228–271 for the second WW domain (YAP-WW2), and
residues 165–271 for tandem construct (YAP-WW1-WW2). The corresponding amino acid
sequences for the two WW domains are shown in Fig. 1B).
The cDNAs for these constructs were synthesized by GENEART AG (Regensbug, Ger-
many). All gene fragments were subcloned into the pETM-30 vector (Protein Expression and
Purification Core Facility, EMBL, Heidelberg, Germany) for their expression as fusion proteins
containing an N-terminal poly-histidine tag together with a Glutathione-S-Transferase (GST)
tag and a Tobacco Etch Virus (TEV) protease cleavage site, for the removal of the affinity tags.
Plasmid-encoding WW domain constructs were expressed in a BL21 (DE3) strain of E. Coli
cells (Novagen). Cells were grown in Luria Bertani media at 37°C until OD
600nm
≈0.7. At this
point, expression was induced with 0.15 mM IPTG for 5 hours at 37ºC. All constructs were pu-
rified by Ni-NTA affinity chromatography as previously described [26]. Protein-containing
fractions were concentrated to 2 mg·mL
-1
in 50 mM sodium phosphate 300 mM sodium chlo-
ride pH 8.0 buffer, frozen in liquid nitrogen and stored at -80º C. Under these conditions, pro-
tein samples were stable for several months.
YAP2 WW domain mutants (W-P to A-A) and PTCH1 PPxY mutants (PPxA) were gener-
ated as described before for YAP2 and LATS1 respectively [12].
Peptide ligands
Synthetic peptide ligands were purchased from Peptide 2.0 Inc. (Chantilly, USA). All peptides
were acetylated and amydated at their N and C termini, respectively. They were synthesized in
the solid phase and their molecular mass was confirmed by mass spectrometry. Peptide purity
was assessed by analytical HPLC as greater than 98%.
Determination of protein and peptide concentration
Protein concentration was measured by absorbance at 280 nm using molecular weights of 5524
Da, 5410 Da, and 12529 Da and extinction coefficients of 12550 cm
-1
·M
-1
, 13960 cm
-1
·M
-1
and
26400 cm
-1
·M
-1
, for YAP-WW1, YAP-WW2 and YAP-WW1-WW2, respectively. Their mo-
lecular mass was confirmed by mass spectrometry and their extinction coefficients were deter-
mined as described by Gill & von Hippel [27]. Peptide concentration was determined by
absorbance at 278 nm using an extinction coefficient of 1450 M
−1
·cm
−1
per tyrosine residue for
ligands LATS1-a, PTCH1-a, and LATS1-b. For PTCH1-b, containing one tryptophan residue,
an extinction coefficient of 6990 M
−1
·cm
−1
at 280 nm was used.
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 4/22

Differential Scanning Calorimetry (DSC)
The heat capacities of all samples were measured as a function of temperature using a high-
precision differential scanning VP-DSC micro-calorimeter (Microcal Inc., Northampton, MA).
Samples were prepared by extensive dialysis against a large volume of the appropriate buffers
(20 mM sodium phosphate at pH 7.0, 20 mM sodium acetate at pH 5.0, 20 mM sodium acetate
at pH 4.0 and 20 mM glycine at pH 3.0). All DSC experiments were performed at a scan rate of
1.5 K·min
-1
using a protein concentration around 1.0 mg·mL
-1
. Protein samples and reference
solutions were properly degassed according to manufacturer’s instructions and carefully loaded
into the cells to avoid bubble formation. Thermal denaturation scans were recorded from 5 ºC
to 110 ºC. Samples were cooled down inside the calorimeter and reheated to check the revers-
ibility of the unfolding process at each experimental condition. The DSC thermograms were
systematically corrected for the time-response of the calorimeter as well as for the instrumental
baseline obtained with both calorimeter cells filled with the corresponding dialysis buffer. After
normalization for protein concentration, the partial molar heat-capacity curves (C
p
) were cal-
culated from the resulting thermograms, assuming a value of 0.73 mL·g
−1
for the partial specific
volume of all proteins.
The resulting DSC traces for isolated WW domains at different pH values were fitted indi-
vidually and globally to a two-state (N⇄U) as described before [26,28]. Nonetheless, in this
case, the pH dependence of the unfolding heat capacity difference was explicitly included in
the model (see definition in Supporting Information), according to the following equation:
ΔCpNU¼CpU CpN þFpðpHÞΔCp;prot;ðequation1Þ
where ΔC
p,prot
is the protonation heat capacity change, defined here as the difference in heat
capacity at 100 ºC between the DSC traces at pH 7.0 and 3.0. ΔC
p,prot
was considered as a fixed
parameter in the global analysis. A value of 1.0 kJ·mol
-1
was estimated from the experimental
DSC traces for both individual domains (YAP-WW1 and YAP-WW2), which is in agreement
with theoretical estimates considering the maximum contribution of all charged amino acid
side chains in these domains [29]. F
p
is the protonation fraction at each pH and was considered
as an additional floating parameter for each pH condition. All DSC fittings and calculations
were performed with Sigma Plot 2000 (Systat Software Inc., Chicago, USA).
Isothermal Titration Calorimetry (ITC)
ITC experiments were carried out using a high-precision VP-ITC titration calorimeter (Micro-
cal Inc., Northampton, Massachusetts). The WW domains were extensively dialyzed against 40
mM sodium phosphate buffer, pH 7.0, with the exception of those experiments using the
LATS2 ligand, (containing a cysteine residue), for which the dialysis was performed in the pres-
ence of 10mM β-mercaptoethanol to reduce intermolecular di-sulphide bonds formed during
ligand storage. Protein and peptide solutions, ranging from 20 to 40 mM for the proteins and
from 0.8 to 2 mM for the ligands, were properly degassed to avoid bubble formation and equili-
brated to 25°C prior to the titration experiment. In all cases, a profile of injection volumes from
3to20ml was used to better define the titration curve. The heat evolved after each peptide in-
jection was obtained from the integral of the calorimetric signal. The heat associated with the
binding process was obtained as the difference between the heat of reaction and the corre-
sponding heat of dilution, as obtained from independent titrations of the peptides into the
buffer.
For the individual WW domains and those ligands with similar binding affinities for YAP-
WW1 and YAP-WW2 domains (LATS1-a and LATS2), the resulting binding isotherms were
analyzed by non-linear least-squares fittings to a model corresponding to a single set of
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 5/22

identical sites, according to the equation:
Q¼V0ΔH
2Ka
½1þKa½LtþnKa½Mtþ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð1þKa½LtþnKa½MtÞ24nK2
a½Mt½Lt
q;
ðequation2Þ
where Q is the net heat of binding, n is the number of binding sites, ΔH is the change in the en-
thalpy due to the binding process, K
a
is the association constant, V
0
is the active cell volume,
and [M
t
] and [L
t
] are the total concentrations of macromolecule and the ligand, respectively.
For those ligands characterized by different binding affinities for the two WW domains
(LATS1-b and PTCH1-a), analysis of the binding isotherms corresponding to the titration of
the tandem construct were also analyzed according to a model of two sets of independent bind-
ing sites, n
1
and n
2
. According to this model, the net heat of binding can be expressed as:
Q¼V0½MtΔH1
n1K1½Lt
1þK1½LtþΔH2
n2K2½Lt
1þK2½Lt

;ðequation3Þ
where Q is the net heat of binding, n
1
and n
2
are the number of sites of each class, ΔH
1
and
ΔH
2
are the change in the enthalpy due to the binding process for each site, K
1
and K
2
are the
association constants for each site, V
0
is the active cell volume, and [M
t
] and [L
t
] are the total
concentrations of macromolecule and the ligand, respectively.
Both models were implemented in Origin 7.0 software (OriginLab Corporation, Northamp-
ton, MA). For the least-squares fit using the one set of sites model, the number of binding sites,
the association constant, and the binding enthalpy were considered as floating parameters,
while in the analysis with the two set of sites model, only the two association constants and the
two binding enthalpies were left to float. All experiments were performed at least twice. Typi-
cally, the variability of the experimental values was estimated to be about 1% in the number of
binding sites, 5% in the binding enthalpy and 10% in the binding affinity.
Cell Culture and Transfections
HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal calf serum. The semi-confluent cells we transiently transfected with DNA con-
structs using Lipofectamine (Invitrogen), as specified by the manufacturer.
Plasmids
YAP cDNA, wild type (WT) in pcDNA4/His-Max vector and p73 HA tagged expression vector
were as described previously [12]. Human PTCH1 cDNA (full length, 2172 bp) was sub-cloned
into pFLAG CMV2 vector into KpnI and XhoI sites. The FLAG-tagged PTCH1 was then sub-
cloned into pcDNA6/TR vector from Invitrogen. This vector encodes TET repressor and was
used to establish stable, inducible HEK293 cell line. The same method as the one used for the
establishment of YAP inducible HEK293 cells was used for the FLAG-tagged PTCH1 inducible
cells [12].
Cell counting assay
HEK293 cultivated in 1% FBS serum were induced by Tetracycline to express Flag-PTCH1
protein as described previously for YAP overexpression in HEK 293 cell [12]. Cells were trypsi-
nized and immediately counted using Beckman Coulter cell counter.
Role of WW Domains in Human YAP Isoforms
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Immunoprecipitation assay
Lysates of HEK 293 cells, the RIPA buffer used to lyse cells and FLAG M2 antibody from
Sigma company and YAP polyclonal rabbit antibody used in our assays, plus all other steps in
the analysis of immuno-precipitates on polyacrylamide gels and on western blots were exactly
as described previously [12].
Homology modeling of YAP complexes and calculation of electrostatic
potential
Homology modeling of isolated WW domains and their complexes with the different peptides
used in this study (LATS1-a, LATS1-b, LATS2, PTCH1-a and PTCH1-b) was performed using
the Discovery Studio Suite (Accelrys Inc., San Diego, USA). The homologue search and se-
quence alignment was performed against locally installed databases using BLAST and
PSI-BLAST. Multiple sequence alignments were calculated using sequence and structure infor-
mation of the protein family. A well-defined NMR structure of the complex between a
YAP-WW1 mutant and a PPPY containing peptide [6] (PDB code 1JMQ) was chosen as a tem-
plate for the modeling of YAP-WW1 and YAP-WW2 complexes with the different ligands.
The final 3D models were generated using the program MODELLER [30] and the lowest-
energy model was selected in each case. For each WW domain-peptide complex, the ligand
was built by mutation of the peptide in the 1JMQ template and was properly oriented by super-
position with the template using the Discovery Studio Suite. The protonation state of ionizable
residues in each model was determined using PDB2PQR software [31]. All models underwent
an energy minimization cycle in vacuum using the AMBER ff10 force field [32]. The quality of
the models was evaluated using PROCHECK [33]. The hydrogen bond interactions in the pro-
tein-ligand interfaces were evaluated with the MM-ISMSA software [34] considering that the
distance between the hydrogen and acceptor atoms is within 1.5 Å and 2.4 Å, the angle between
donor, hydrogen, and acceptor atoms varies within 130º and 165º; and the angle between the
hydrogen, acceptor, and the atom bound to the acceptor atom diverges between 115º and 145º.
The electrostatic potential of the protein and ligands was calculated from the model structures
using the DelPhi algorithm [35], as implemented in the Discovery Studio Suite. Before running
DelPhi, atom charges were assigned according to the CHARMm force-field parameters [36].
Phage-display study of WW domain binding preferences
The analysis of the binding preferences of the WW domains was performed by phage-display
as previously described [37]. Briefly, an N-term library in PIII was generated using a template
derived from the pS2202d phagemid, containing the Erbin-PDZ domain kindly provided by
Dr. Sachdev Sidhu (University of Toronto). The Erbin-PDZ sequence was removed by the in-
troduction of four stop codons between the secretion signal and the linker required for the cor-
rect display of the library variants in the phage. A randomized sequence corresponding to a
x
5
(L/P)PxYx
5
peptide library containing the L/PPxY core motif for type 1 WW domains was
subsequently introduced into the modified phagemid. The resulting library was then intro-
duced into the SS320 strain by electroporation. The subsequent phage propagation led to 10
11
library diversity, which was isolated at 5 x 10
13
phages/ml in PBS-Tween (0.05%) buffer. The
resulting library was then screened against immobilized GST-tagged YAP-WW2 domain
(GST-YAP-WW2) in PBS, carrying out seven rounds of an iterative selection process.
Single clones were chosen from round 3 to round 7 and tested for specific binding to the
GST-YAP-WW2 by phage ELISA in a Tecan Infinite M200 instrument [37,38]. In parallel, ad-
ditional binding tests were performed in the presence of p53BP2 peptide (EYPPYPPPPYPSG)
Role of WW Domains in Human YAP Isoforms
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[39], which was used as a competing agent for the selection for high-affinity sequences. Addi-
tionally, in order to evaluate the level of specificity of the selected sequences for the two WW
domains of YAP, the single clones derived from the GST-YAP-WW2 selection process were
also tested for their ability to displace p53BP2 from the YAP-WW1 domain. The DNA from
phages showing binding in the ELISA assay were used as PCR templates to amplify the pep-
tide-encoding regions, which were then sequenced and analyzed.
Results
YAP WW domains are marginally stable and behave as independent
folding units connected by an unstructured linker
The temperature dependence of the partial heat capacity of the isolated YAP-WW1,
YAP-WW2 domains and the YAP-WW1-WW2 tandem was measured by Differential Scan-
ning Calorimetry (DSC) at different pH values, ranging from pH 7.0 to pH 3.0. The reversibili-
ty of the unfolding transitions was checked and confirmed to be over 80% in all cases. No scan
rate or concentration effects were observed for any of the constructs, indicating that they be-
have as monomeric proteins that unfold in equilibrium under all studied conditions.
The two isolated domains were characterized by very broad calorimetric transitions
(Fig. 2A and B). To minimize the errors in the determination of the unfolding thermodynamic
parameters the denaturation curves for each WW domain at different pH values were
globally analyzed by non-linear least squares fitting to a two-state model [26] modified to in-
corporate ionization effects to the unfolded heat capacity to account for the progressive incre-
ment in C
p,U
with pH, which in these small and broad transitions become relevant (S1 Table).
The results of this analysis are summarized in S2 Table. At pH values between 4.0 and 7.0, both
YAP-WW1 and YAP-WW2 are fully folded below 20 ºC. YAP-WW2 shows a slightly higher
stability than YAP-WW1 (S1 Fig.). Even though the two-state model adequately describes the
experimental data (R and R
2
values of 0.99), the values of the thermodynamic parameters re-
sulting from the two-states analysis are not within the normal range expected for a standard
two-state protein (S2 Table). This suggests that, in as previously discussed for other WW do-
mains [26,40,41], the isolated WW domains of YAP are highly flexible and marginally stable
units in the limits of what can be considered cooperative folding.
The YAP-WW1-WW2 tandem construct (Figs. 2C and D) presents a single and broad tran-
sition that cannot be described by a two-state model. As illustrated in Fig. 2C and 2D, at all pH
values the DSC profile of the tandem can be perfectly reproduced by addition of the DSC
curves of the YAP-WW1 and YAP-WW2 domains plus the contribution of the heat capacity
of a disordered linker [24], calculated as the sum of the tabulated Cp contributions of the dif-
ferent amino acids in the sequence [42]. This indicates that the two WW domains in tandem
unfold independently, without any significant cooperative interactions between them or with
the connecting linker. In other words, the presence of the second WW domain in YAP2 does
not lead to the stabilization of the first WW domain nor to a conformational reorganization of
the tandem. We hypothesize, thus, that the presence of the second WW domain in the YAP2
isoform results in the modulation of the target recognition properties of YAP by inducing
changes in binding affinity and/or specificity towards their cellular partners.
Selection of ligands for YAP WW domains
To investigate this hypothesis, a thermodynamic analysis of the interactions of the isolated
WW domains and the YAP-WW1-WW2 tandem was performed using a set of PPxY contain-
ing ligands. LATS kinases play a key role in YAP functional regulation through Ser127
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 8/22

phosphorylation, and are well established as partners of YAP WW domains [12]. Accordingly,
three different peptide ligands corresponding to the PPxY containing sequences in LATS1 and
LATS2 were selected for the binding studies.
In order to widen the study, additional putative ligands of YAP WW domains were consid-
ered. Taking into account that YAP has been recently reported as a regulator of the Sonic
Hedgehog Pathway [43,44], we investigated if any members of this pathway contain PPxY mo-
tifs which could serve as targets for WW binding. Interestingly, we found that PATCHED1
(PTCH1) contained two PPxY motifs including one with four consecutive prolines (RYSPPP-
PYSSHS), which usually forms a core for high affinity peptide ligands to YAP WW domains
[39].
Figure 2. Differential Scanning Calorimetry thermal denaturation profiles of YAP1-WW1 (A) and YAP1-WW2 (B) domains. Symbols correspond
to experimental data for the temperature dependency of the partial molar heat capacity at different pH values (black circles for pH 7.0, green squares
for pH 5.0, orange triangles for pH 4.0 and red rhombi for pH 3.0). Solid lines correspond to the global fitting to the two-states model. The heat
capacities functions for the folded and unfolded states [C
p,N
(T) and C
p,U
(T)] resulting from the analysis are shown as short dashed lines. The dotted lines
show the C
p,N
(T) function calculated according to the molecular weight [28] and the C
p,U
(T) function estimated from the contributions of the amino acid
composition [41]. C) DSC profile for the YAP1-WW1-WW2 tandem. Shown are the DSC profiles for YAP-WW1 (dark blue circles), for YAP-WW2 (light blue
circles) and YAP-WW1-WW2 tandem (black circles) at pH 7.0. The dashed line corresponds to the additionof the heat capacity profiles of the individual WW
domains and the continuous black line represents the addition of the DSC experiments of the individual WW domains plus the contribution to the heat
capacity for the linker sequence in the tandem [41]. D) DSC profile for the YAP1-WW1-WW2 tandem at different pH values. Symbols correspond to
experimental data for the temperature dependency of the partial molar heat capacity of the tandem at differentpH values (black circles for pH 7.0, orange
triangles for pH 4.0 and red rhombi for pH 3.0). The continuous lines correspond to the curves resulting from the addition of the heat capacity profiles for the
individual WW domains and linker sequences under each condition.
doi:10.1371/journal.pone.0113828.g002
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 9/22

The interaction between PTCH1 and YAP was examined using PTCH1 and YAP mutants
in a cell culture model. PTCH1 mutants in which the signature tyrosine residue (Y) in each
PPxY motif was substituted with alanine were generated. Such mutation has been shown to ab-
rogate binding of PPxY-containing ligands to WW domains [14,22]. The resulting PTCH1
variants (the wild type protein, the single mutants labeled PY1and PY2and the double mu-
tant labeled PY1&2) were fused to FLAG tags and transiently co-expressed with YAP in
HEK293 cells, followed by immunoprecipitation and immunoblotting (Fig. 3A). In this experi-
mental setting PTCH1 WT bound strongly to YAP (Fig. 3A, upper panel). Binding became
weaker when the first PPxY motif was mutated (PY1or PY1&2), while no significant effect
was observed for the mutation of the second PPxY motif (PY2). These data suggest that, in
fact, PTCH1 and YAP interact and that the first PPxY motif in PTCH1 plays a critical role in
the formation of the complex.
Figure 3. YAP interacts with PTCH1 Through its WW Domains. A) Intact PPxY motifs in PTCH1 are
required for binding to YAP. Two PPxY sequences in PTCH1 were mutated to PPxA. Wild type (WT) or single
(PY1*or PY2*) or double (PY1*&2*) mutants of PTCH1 in Flag-tag vector were transiently co-transfected
with YAP2 (in pcDNA4/HisMax) into HEK293 cells. Cell lysates were immunoprecipitated with Flag
antibodies, resolved on SDS-PAGE and immunoblotted with YAP antibody or Flag antibody. B) Intact WW
domains in YAP are required for binding to PTCH1. Two WW domains in YAP were mutated to render them
inactive in terms of ligand binding. Wild type (WT) or double (1&2 WW*) mutant of YAP2 in pcDNA4/HisMax
vector were co-transfected with Flag-PTCH1 or Flag vector alone. Cells were lysed and analyzed as in A.
doi:10.1371/journal.pone.0113828.g003
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 10 / 22

In order to test whether the PTCH1-YAP interaction is actually mediated by the YAP WW
domains, two highly conserved amino acids in each of the two WW domains (i.e. the second
signature W and the conserved, carboxy-terminal P) were mutated to A. The WQDP sequence
of amino acids 199–202 in the first WW domain of YAP was changed to AQDA, while the
WLDP sequence of amino acids 258–261 in the second WW domain of YAP was changed to
ALDA. Such substitutions in WW domains render the mutated domains inactive in terms of li-
gand binding [14]. The double mutant was labeled as 1&2 WW. This mutant and the YAP
WT were co-transfected with Flag-PTCH1 into HEK293 cells, followed by immunoprecipita-
tion and immunoblotting (Fig. 3B). Relatively strong binding was detected in the case of YAP
WT. However, this binding was barely detectable when both WW domains of YAP were mu-
tated. Together, the results suggest that the binding between YAP and PTCH1 is mediated by
the WW domains of YAP (Fig. 3A and B). Experiments with a cell-apoptotic model also sup-
port the YAP-PTCH1 interaction (S3 Fig.), revealing that, in the presence of PTCH1, the effect
of YAP expression in the cell growth is reduced and YAP also failed to stabilize p73, in agree-
ment with the observed effects for the interactions with other YAP Partners [12].
Even though the cellular studies were performed with overexpressed proteins and do not
provide conclusive evidence of the physiological relevance of this interaction, they provide vali-
dation to the idea that, at least in this setup, the PTCH1 PPxY sequences can interact with YAP
WW domains. Consequently, we decided to incorporate the PTCH1 sequences to our binding
studies. In summary, a set of five different peptide ligands containing 12 amino acids with the
PPxY motif centered within the sequences corresponding to the PPxY motifs in LATS1,
LATS2 and PTCH1, were selected for the binding studies (see Table 1 for sequences).
The two WW domains of YAP2 act as independent docking sites with
different binding properties
The binding energetics of all peptide ligands to each YAP WW domain and to the tandem con-
struct were measured by Isothermal Titration Calorimetry (ITC). The upper panels in Fig. 4
show, as an example, the calorimetric titrations of the LATS1-b ligand (YQGPPPPYPKHL)
with each construct. The corresponding binding isotherms are shown in the lower panels. The
results of the thermodynamic analysis for the isolated domains are summarized in Table 2 and
illustrated in S2 Fig. The ITC experiments confirmed that all five PPxY-containing ligands
bind to at least one of the WW domains of human YAP with K
d
values in the low mM range. It
Table 1. Peptide ligands derived from YAP functional targets.
Ligand
1
Sequences
Sonic Hedgehog Pathway
PTCH1 “Protein Patched Homolog 1”
G.lycosylated membrane receptor
PTCH1-a
572
R
-7
Y
-6
S
-5
P
-4
P
-3
P
-2
P
-1
Y
0
S
1
S
2
H
3
S
4583
PTCH1 “Protein Patched Homolog 1”
Glycosylated membran e receptor
PTCH1-b
1243
E
-7
G
-6
L
-5
W
-4
P
-3
P
-2
P
-1
Y
0
R
1
P
2
R
3
R
41254
Hippo pathway
LATS1 “Large Tumor Suppressor Homolog 1”
Serine/threonine kinase
LATS1-a
369
N
-7
R
-6
Q
-5
P
-4
P
-3
P
-2
P
-1
Y
0
P
1
L
2
T
3
A
4380
LATS1 “Large Tumor Suppressor Homolog 1”
Serine/threonine kinase
LATS1-b
552
Y
-7
Q
-6
G
-5
P
-4
P
-3
P
-2
P
-1
Y
0
P
1
K
1
H
1
L
563
LATS2 “Large Tumor Suppressor Homolog 2”
Serine/threonine kinase
LATS2
511
R
-7
R
-6
C
-5
P
-4
P
-3
P
-2
P
-1
Y
0
P
1
K
2
H
3
L
4522
1
Numbers indicate the position of each ligand in the context of the full-length protein sequence.
doi:10.1371/journal.pone.0113828.t001
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 11 / 22

is interesting to note that each of the LATS kinases contain at least one ligand with
dissociation constants close to or below 10 mM for YAP-WW1. This is the case for the only
PPxY-containing sequence in LATS2 and for one of the two PPxY-containing peptides derived
from LATS1 and PTCH1 (LATS1-b and PTCH1-b), with K
d
values of 7.1, 3.7 and 13.2 mM, re-
spectively. These values are close to those previously reported for the p53BP2 ligand, character-
ized by the highest binding affinity measured to date for YAP WW domains (K
d
= 1.8 mM for
YAP-WW1 and 12 mM for YAP-WW2) [39]. It is interesting to note that, in vitro, the two iso-
lated PPxY-containing sequences in this protein can interact with YAP-WW1 with dissocia-
tions constants in the low micromolar range, being the binding affinity of the PTCH1-b
ligands slightly higher than the PTCH1-a. Nonetheless, in the cellular assays, mutation of the
second PPxY motif (PTCH1-b) in the context of the full-length proteins had a much smaller ef-
fect on PTCH1-YAP binding. For all ligands studied, the K
d
values for the tandem construct
do not differ significantly from those obtained for the individual domains, suggesting that the
WW domains in YAP behave as two independent binding sites for these peptide ligands.
In addition to binding affinity, ITC provides relevant information about the enthalpic and
entropic components to the Gibbs energy of binding, which report on the type and magnitude
of the forces driving the binding affinity [45], providing, thus, a valuable insight into the nature
of the interactions. Binding of the WW domains of YAP with the five peptide ligands is driven
by favorable enthalpy contributions, ranging from -53 to-80 kJ·mol
-1
, partially compensated
for by unfavorable entropic contributions. This energetic signature is similar to that reported
for other WW domain-mediated interactions [19,46,47] and for other proline-rich recogni-
tion domains such as SH3 [48,49,50] or UEV domains [51]. In the case of SH3 domains, this
Figure 4. Isothermal titration calorimetry experiments for YAP1-WW1, YAP1-WW2 and YAP-WW1-WW2 tandem with the LATS1-b ligand at 25ºCin
20 mM sodium phosphate pH 7.0. Upper panels show the heat effects associated with the injection of LATS1-b ligand into the calorimetric cell containing
the protein. Lower panels show the corresponding binding isotherm ligand normalized by protein concentration corrected for the heat of dilution. Solid lines
correspond to the best fit of data to a one-site model (equation 2) in the experiments with individual WW domains and to a two sets of independent binding
sites model (equation 3) in the experiment with the tandem (right panel).
doi:10.1371/journal.pone.0113828.g004
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 12 / 22

thermodynamic pattern has been associated to the interplay of several factors, including the re-
distribution of the native state conformational ensemble upon ligand binding [50,52] and the
presence of interfacial water molecules [53].
Interestingly, the analysis of the binding energetics of the five chosen ligands of YAP WW
domains indicates that, in spite of the high sequence similarity between the binding sites of the
two domains, they behave differently with respect to ligand recognition. Some YAP ligands,
such as LATS1b, bind more tightly to YAP-WW1 than to YAP-WW2 (See Table 2 and
S2 Fig.). These data are in agreement with co-immunoprecipitation assays of LATS and YAP
using mutants of individual WW domains rendered inactive in terms of ligand binding [12].
These assays, which involved full-length proteins, also showed a more prevalent role of YAP-
WW1 than YAP-WW2 domain in the formation of the YAP - LATS complex. This selectivity
between the two WW domains is particularly evident for the PTCH1-b ligand, for which no in-
teraction was detected with YAP-WW2.
Moreover, even for those ligands, such as PTCH1-a, LATS2 or LATS1-a, which do not seem
to distinguish between the two WW domains of YAP, significant differences, up to 20 kJ·mol
-1
,
are observed in their enthalpic and entropic contributions to the binding affinity. Even though
these contributions are not translated into changes in binding affinity due to enthalpy/entropy
compensation effects [54], they indicate that the balance of intermolecular forces driving the
binding of these ligands is different for each WW domain. In sum, our biophysical data suggest
that YAP-WW1 and YAP-WW2 are not equivalent modules with respect to ligand
recognition.
Table 2. Binding thermodynamics of peptide ligands to YAP WW domains.
Protein Ligand n K
d
(μM) ΔG
ap
(kJ·mol
-1
)ΔH
ap
(kJ·mol
-1
) -T·ΔS
ap
(kJ·mol
-1
)
YAP-WW1 PTCH1-a RYSPPPPYSSHS 0.95 27.6 -26.1 -65.0 38.9
PTCH1-b EGLWPPPYRPRR 1.09 13.2 -27.9 -75.2 47.3
LATS1-a NRQPPPPYPLTA 0.96 21.5 -26.7 -67.0 40.3
LATS1-b YQGPPPPYPKHL 0.91 3.7 -31.0 -69.0 38.0
LATS2 RRCPPPPYPKHL 1.05 7.1 -28.8 -78.5 49.7
YAP-WW2 PTCH1-a RYSPPPPYSSHS 0.91 45.8 -24.8 -65.9 41.1
PTCH1-b EGLWPPPYRPRR n.b. —— — —
LATS1-a NRQPPPPYPLTA 1.09 27.0 -26.1 -76.2 50.1
LATS1-b YQGPPPPYPKHL 0.94 33.9 -25.6 -70.2 44.6
LATS2 RRCPPPPYPKHL 1.08 8.2 -29.1 -57.8 28.7
YAP-WW1-WW2 PTCH1-a
2
RYSPPPPYSSHS 1
3
24.7 -26.4 -52.9 26.5
1
3
47.4 -24.7 -75.2 50.5
PTCH1-b
1
EGLWPPPYRPRR 1.23 16.9 -27.3 -80.2 52.9
LATS1-a
1
NRQPPPPYPLTA 2.04 33.4 -25.6 -72.0 46.4
LATS1-b
2
YQGPPPPYPKHL 1
3
4.8 -30.4 -70.8 40.4
1
3
31.9 -25.7 -51.8 26.1
LATS2
1
RRCPPPPYPKHL 2.02 9.2 -28.8 -68.8 40.0
The variability in the experimental values was estimated to be about 1% for the number of sites, 5% for the binding enthalpy and 10% for the dissociation
constant. n.b.: no binding detected.
1
Data obtained using a binding model for one set of sites with n = 2.
2
Data obtained using a binding model for two different sets of sites with n = 1 for each set of sites. (see methods for details).
3
Number of binding sites fixed to 1 in fitting procedure.
doi:10.1371/journal.pone.0113828.t002
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 13 / 22

Electrostatic interactions play a key role in determining binding
specificity between the two WW domains in human YAP
The most characteristic residues at the binding site of class I WW domains are conserved in
both, YAP-WW1 and YAP-WW2 (see Fig. 1B), with the exception of Leu30 and Gln35 in
YAP-WW1 that are substituted by Ile and Lys respectively in YAP-WW2. Accordingly, as illus-
trated in Fig. 5A and S4 Fig., the modeled structures for YAP-WW1 and-WW2 domains
bound to the different peptide ligands are very similar and reproduce the main binding features
characteristic of class I WW complexes. Hydrogen bond interactions and their proton-donor
and acceptor distances for all complexes are summarized in S3 Table. All modeled complexes
share a highly conserved pattern of hydrogen bond interactions within the canonical binding
site, differing only with respect to the interactions established with polar and positively charged
residues at the N-terminal regions of the ligands.
Electrostatic potential calculations carried out with the modeled structures for YAP-WW1
and YAP-WW2 (Fig. 5B), show that the Q35K and E25Q substitutions at the xY pocket and β2
strand respectively have a significant impact on the electrostatic potential of the two domains
in the binding site area, leading to a marked polarization in the binding site of YAP-WW2,
with a region of strongly positive electrostatic potential located over the ‘xY pocket’and the
loop connecting the β2 and β3 strands (including lysine 35) and a region of markedly
negative potential corresponding to the ‘xP pocket’. However, this polarization is not observed
in YAP-WW1, suggesting a relevant role of electrostatic interactions in the determination of
binding specificity between these two domains.
In fact, analysis of the ligand sequences known to interact preferentially with YAP-WW1,
including the LATS and PTCH1 peptides used in this study, and the recently described se-
quences from SMAD7 (ELESPPPPYSRYPM) and AMOT p130 (MRYQHPPEYGAARP)
[55,13], shows a clear preference for positively charged residues C-terminal to the PPxY core
motif (Fig. 5D). These sequences present a region of positive potential, resulting in repulsive in-
teractions with the ‘xY pocket’at the YAP-WW2 binding site (See Fig. 5C). The PTCH1-b li-
gand constitutes an extreme situation, with a stretch of three positively charged arginines that
would completely preclude interaction with YAP-WW2. Moreover, considering that electro-
static interactions contribute mostly to the binding entropy, the idea that electrostatic interac-
tions play a prevalent role in determining binding specificity between these domains is in
agreement with the fact that the differences in binding affinity found in this study between the
two WW domains are of entropic origin (see Table 2).
In order to further investigate thisidea, the binding preferences of YAP-WW2 were studied
by phage display. From these experiments, a set of 42 peptides, with the ability to displace the
p53BP2 peptide ligand (EYPPYPPPPYPSG) from the YAP-WW2 domain, were identified.
These sequences are, thus, “high affinity”peptides expected to bind YAP-WW2, with dissocia-
tion constants below 12 mM for this domain.Two types of sequences were obtained. The first
was a neutral proline-rich sequence, AGRPPPPPYPGPPL, which was selecteda total of 14 times
in the set of 42 peptides. This sequence is reminiscent of the p53BP2 ligand, although it includes
an arginine at position 3, which is frequent in YAP ligands (Fig. 5D). As observed for the
p53BP2 peptide ligand (unpublished data), ELISA binding experiments showed that this pro-
line-rich peptide seems to interact similarly with thetwo WW domains of YAP. The second type
of sequences obtained included peptides characterized by a relatively variable N-terminal region
and, most interestingly, by the invariant presence of negatively charged residues C-terminal to
the PPxY core motif. These sequences, shown in Fig. 5E, are markedly different from those de-
rived from natural ligands binding preferentially to YAP-WW1 (Fig. 5D). From the ELISA assay
results, contrary to the above-mentioned AGRPPPPPYPGPPL sequence, the second set of
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 14 / 22

Figure 5. A) Cartoon representation of the YAP-WW1 (left) and-WW2 (right) domains in complex with PTCH1-b. YAP-WW1 and—WW2 domains are
shown as green and blue surfaces respectively. Peptide ligands and protein residues defining the canonical xP and xY pockets at the binding sites are shown
as sticks. Non-conserved residues at the binding site are labelled in red. Hydrogen bonds interactions are shown as discontinuous black lines. B) Surface
representation of the electrostatic potential calculated for the free YAP-WW1 and YAP-WW2 domains. Regions of strong negative potential are shown
in red and positively charged regions are shown in blue. C) Surface representation of the electrostatic potential calculated for the YAP-WW1 domain/
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 15 / 22

sequences seems to be highly selective for YAP-WW2, not being able to displace the p53BP2 li-
gand from the YAP-WW1 domain. These results indicate that the second set of sequences have
a reduced binding affinity for YAP-WW1, in comparison to the YAP-WW2 domain. Interest-
ingly, this is in agreement with the fact that p73 protein, which contains the sequence
SHCTPPPPYHADPS, has been described to interact exclusively with the YAP2 isoform [12].
Discussion
YAP, as one of the main effectors in the Hippo tumor suppressor pathway, is currently the sub-
ject of great interest and intense study. Elucidating how YAP regulates signaling in the context
of this pathway is key to understand cell proliferation, differentiation and apoptosis. Further-
more, as information accumulates for Hippo and other cancer-related signaling cascades, the
need to integrate this knowledge, elucidating the interplay between different pathways grows
even more apparent.
The Hippo pathway has been recently reported to cross-talk with other signaling cascades,
including TGFB, Wnt [56] and Notch [57,58], controlling proliferation in different types of
cells and acting as a central node integrating signals originated in other pathways (see [59] for
a review). With respect to the interaction between Hippo and Sonic Hedgehog pathways, re-
cent studies suggest that SHH signaling acts downstream of YAP in regulation of neuronal dif-
ferentiation [44], being YAP either amplified or up-regulated in human SHH-associated
medulloblastomas [43]. The results presented here suggest that PTCH1 might function as a
cognate ligand of YAP, providing a first hint for a molecular link between these two pathways.
Clearly, a more detailed analysis performed under physiological conditions and out of the
scope of this article is needed to fully establish the physiological relevance of this potential
cross-talk via PTCH1 and YAP that, if confirmed, could help identify new signaling events that
regulate Hippo-YAP pathway.
In any case, we have shown that PTCH1 is able to interact with YAP in a cellular context
and that this interaction is mediated by YAP WW domains, showing a marked preference for
the WW1 domain. In vitro, the two isolated PPxY-containing sequences in PTCH1 can interact
with YAP-WW1 with dissociation constants in the low micromolar range, which can be con-
sidered as a tight interaction in view of the range of binding affinities typically found for WW
domains. Nonetheless, according to the results of the in vivo assays, in the context of full-length
proteins the PTCH1-YAP interaction seems to be mostly driven by binding of the first PPxY
motif in PTCH1 to the WW1 domain in YAP. Even though it is feasible that the second
PTCH1-PPxY motif could act as a docking site for different signaling proteins in higher-level
assemblies, the actual role of this PTCH1 sequence and whether it is implicated at all in the
Hippo-SHH interplay remains to be elucidated. In any case, the discrepancies between in vitro
and in vivo results highlight the importance of contextual effects in protein-protein interactions
mediated by modular domains [60,61] and the need to be cautious in the interpretation of in
vitro results obtained with isolated peptides and domains in terms of cellular events.
With all these caveats, the thermodynamic study presented here provides, nonetheless, very
valuable information in terms of the interplay between the two WW domains in YAP and their
LATS1-b complex and free LATS1-a, PTCH1-a and PTCH1-b ligands. Regions of strong negative potential are shown in red and positively charged
regions are shown in blue. D) Frequency of occurrence of amino acids in natural ligands binding preferentially to YAP1-WW1. Web-logo [67] showing
the frequency of occurrence of the different amino acids for a set of peptide ligands corresponding to naturalproteins binding preferentially to YAP-WW1. E)
Frequency of occurrence of amino acids in phage-display selected ligands binding preferentially to YAP1-WW2. Web-logo showing the frequency of
occurrence of the different amino acids for the set of charged sequences selected by YAP-WW2 in phagedisplay experiments, excluding the neutral, proline-
rich sequence AGRPPPPPYPGPPL.
doi:10.1371/journal.pone.0113828.g005
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 16 / 22

binding properties. Differential scanning calorimetry studies reveal that, both isolated and in a
tandem disposition, these domains show marginal stability, with thermodynamic properties
typical of downhill proteins, characterized by a poor cooperativity and, thus, high conforma-
tional flexibility [25]. This thermodynamic scenario set the best framework for a high plasticity
in ligand recognition, in agreement with the functional diversity reported for these YAP WW
domains [47,55,62]. Furthermore, no evidence of functional cooperativity between the two
YAP WW domains was found, either in terms of structural stability, or with respect to the rec-
ognition of peptide ligands. This indicates that, in every aspect, these two domains function as
independent docking sites. This independent behavior contrast with previous studies reporting
on tandems of WW domains that function as a single unit connected by a structured linker,
WW domains chaperoning each other by increasing their stability and physiological function,
etc. [13,63,64]. According to the evidence presented here, the WW domains of YAP1 behave
as two independent, fully folded, although marginally stable units, connected by an unstruc-
tured linker.
Most interestingly, the analysis of the binding energetics of the selected set of peptide li-
gands revealed that, in spite of the high similarity within the binding site region, the two WW
domains in YAP interact differently with their ligands, as hinted by previous studies reporting
modest differences (up to 5.3 kJ·mol
-1
) in binding affinity with other ligands [6,22,47,55].
Nonetheless, to our knowledge, this is the first report of a high selectivity between the two WW
domains in YAP. This selectivity becomes more relevant considering that differences in partner
recognition have been reported for the two YAP isoforms (only YAP2 can interact with p73
[12] and AMOTL1 (Angiomotin-Like-1) [65]. Moreover, it has been proposed that YAP may
affect different signaling pathways in a cell-specific manner, interacting with different tran-
scription factors in different cell types, and functioning differently depending on its binding
partners [44]. In this context, the different specificity profile revealed by our binding study sug-
gests that the specific partner recognition by each WW domain might be an important factor
for YAP functional modulation.
These results also suggest that high level of intrinsic specificity can be encoded within rela-
tively short peptide sequences for these domains. Analysis of the structural models of the com-
plexes, together with the phage display study of the binding preferences of these domains,
indicate that electrostatic interactions play a very relevant role in determining binding specific-
ity within the YAP WW domains. The significance of electrostatics in protein folding, binding
and function is well established. In fact, it has been reported that the electrostatic potential is a
distinguishing feature of WW domains, according to which they can be organized in four dif-
ferent categories [66]. In this sense, the two WW domains of YAP can clearly be classified in
two different groups regarding to the degree of polarization of the binding site region. In the
case of the PTCH1-b sequence, the high level of specificity has been achieved, not only by max-
imizing favorable interactions with the target, but also making unfavorable the interactions
with off-target molecules, fine-tuning the electrostatic potential of the ligand. This observation
is of interest, not only for the understanding of YAP function, but also from the design point of
view, revealing the possibility of designing small ligands showing high specificity for one of
these domains, of interest to delineate the functional role of each WW domain in YAP signal-
ing and, eventually, of potential therapeutic value.
Supporting Information
S1 Fig. Stability curves for the individual WW domains of YAP. Gibbs energy changes for
the thermal unfolding of YAP-WW1 (left panel) and YAP-WW2 (right panel) isolated do-
mains resulting from the global fit of the DSC curves at several pH values. Solid lines with
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 17 / 22

symbols represent the temperature dependence of the Gibbs energy function ΔG
N-U
(T) as a
function of the pH: circles for pH 7.0, squares for pH 5.0, triangles for pH 4.0 and rhombi for
pH 3.0. The arrows indicate the pH dependence of the T
S
values.
(TIF)
S2 Fig. Binding energetics of peptide ligands to YAP-WW1 (black bars) and YAP-WW2
(white bars). A) Dissociation constants for the LATS1-a, LATS1-b, LATS2, PTCH1-a and
PTCH1-b peptide ligands for their interaction with the isolated WW domains of YAP. B)
Enthalpic and entropic contributions to the binding affinity.
(TIF)
S3 Fig. Induction of YAP Expression Results in Reduced Cell Attachment, which is Rescued
by PTCH1 (top panel). HisMax-YAP or control vector were transfected into HEK293 cells
that express Flag-PTCH1 WT in an inducible system. 24 hrs post transfection, the cells were
distributed into new plates and the expression of Flag-PTCH1 WT was induced by tetracycline.
O hr or 96 hrs post induction, cells were trypsinized and their numbers were counted. The
growth rates in this 96 hrs are shown in the graph. The expression of induced Flag-PTCH1 and
transfected YAP was monitored by immunoblotting. PTCH1 impairs the ability of YAP to
stabilize p73 (lower panel). HEK293 cells that express Flag-PTCH1 WT or Flag-PTCH1
PY1&2mutant in an inducible system were transfected with HA-p73 and HisMax-YAP
WT. 24hrs later, the cells were plated in fresh DMEM containing 1% FBS. Tetracycline was
added to the medium to induce the expression of Flag-PTCH1 WT or mutant. 96hrs after in-
duction, the cells were harvested, followed by immunoblotting using indicated antibodies.
(TIF)
S4 Fig. Modelled structures of YAP-WW1 (left panels) and YAP-WW2 (right panels) in
complex with A) PTCH1-a, B) PTCH1-b, C) LATS1-a, D) LATS1-b and E) LATS2 peptide
ligands. YAP-WW1 and YAP–WW2 domains are shown as green and blue surfaces respec-
tively. Peptide ligands and protein residues defining the canonical xP and xY pockets at the
binding sites are shown as sticks. Non-conserved residues at the binding site are labelled in red.
Hydrogen bonds interactions are shown as discontinuous black lines.
(TIF)
S1 Table. Nature and identity of ionisable groups and contribution to the heat capacity
(Fp·ΔC
p,prot
) of YAP WW domains.
(DOC)
S2 Table. Thermal denaturation parameters for the isolated YAP WW domains at different
pH values.
(DOC)
S3 Table. Hydrogen-bonding interactions in modeled complexes of YAP WW domains.
(DOCX)
Acknowledgments
We thank Dr. Sachdev Sidhu and Dr. Javier Murciano for their invaluable assistance in setting
up phage display experiments and for many helpful discussions. We thank Dr. Carles Corbi for
his assistance with modeling and electrostatic calculations. We thank Antonio Barbachano
(IIB-CSIC) for providing experimental data to understand the YAP-PATCH network. Support
from the C.I.C. of the University of Granada for mass spectrometry measurements is
Role of WW Domains in Human YAP Isoforms
PLOS ONE | DOI:10.1371/journal.pone.0113828 January 21, 2015 18 / 22

acknowledged. We also would like to specially thank our colleagues Amjad Farooq and Virgin-
ia Mazak for very valuable comments on the first version of the manuscript.
Author Contributions
Conceived and designed the experiments: MS IL. Performed the experiments: MIB FC ESC
TO. Analyzed the data: MIB FC ESC TO MS IL. Contributed reagents/materials/analysis tools:
MS IL. Wrote the paper: MIB FC ESC MS IL.
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