(Received for publication, February 3, 1997, and in revised form, April 4, 1997)
From the Laboratory of Molecular Oncology, The
Rockefeller University, New York, New York 10021, the
§ Department of Biochemistry, Mount Sinai School of
Medicine, New York, New York 10029, and the ¶ Department of
Chemistry, Texas A&M University, College Station, Texas 77843
We had previously identified the WW domain as a novel globular domain that is composed of 38-40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of Yes kinase-associated protein which we named WW domain-binding proteins 1 and 2. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of WW domain-binding proteins 1 and 2. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively. In addition, using site-directed mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.
The Src homology (SH)1 2 and SH3 domains have assumed essential roles in furthering the understanding of how an extracellular signal is transmitted from the cellular membrane, through the cytoplasm, and finally into the nucleus where the signal is interpreted through the process of gene-specific transcription. The SH2 domain has been shown to interact specifically with sequences containing a phosphotyrosine residue, whereas the SH3 domain mediates binding to proline-rich sequences with the minimal consensus of PXXP (P represents proline, and X designates any amino acid) (1, 2). The SH2 and SH3 domains thus consist of a common binding core that recognizes phosphotyrosine- or proline-rich motifs, respectively, and which achieve binding specificity through unique flanking sequences (3-5). As a result, these domains determine which proteins can interact, and equally important, in what order the interaction occurs in the closely regulated pathways of signal transduction. Recently, two other important signaling modules were characterized: the pleckstrin homology domain and the protein interaction domain/phosphotyrosine binding domain (6-10). These modular repeats represent true protein domains in that they constitute structurally distinct three-dimensional units that can properly fold and function in the context of other proteins or in isolation (11, 12).
We have previously identified a Yes
kinase-associated protein (YAP) that interacts
in vitro with the SH3 domain of the Yes proto-oncogene
product via a proline-rich region (13). In addition, the YAP sequence
contains a semiconserved region of 38 amino acids which has since been
found in a variety of unrelated proteins including dystrophin
(implicated in Duchenne's and Becker's muscular dystrophy), Pub1 (a
ubiquitin-ligase involved in cell cycle regulation in fission yeast),
Rsp5/Nedd4 (transcriptional regulators with ubiquitin-ligase activity),
and FE65 (an adaptor protein that binds Alzheimer's -amyloid
precursor protein) (14-16). This protein repeat has been named the WW
domain after the two highly conserved tryptophan residues spaced 20-22
residues apart within the consensus sequence. The list of WW
domain-containing proteins continually grows, and these proteins
seem to share the characteristic of being involved in regulatory or
signaling processes (11).
More recently, we have shown that the WW domain of YAP is indeed a novel domain capable of mediating protein-protein interactions (17-19). By screening an expression library constructed from 16-day-old mouse embryo, we identified two putative ligands binding with high specificity and relatively high affinity (Kd = 1-50 mM; Refs. 17 and 20) to the WW domain of YAP, namely WBP-1 and WBP-2 (WW domain-binding protein). Using the technique of Western ligand blotting in performing in vitro binding assays, we further narrowed the binding region of WBP-1 to a proline-rich motif with the sequence Pro-Pro-Pro-Pro-Tyr (PPPPY), which we chose to call the PY motif, interestingly, the only region of perfect homology between WBP-1 and WBP-2. Binding assays using mutant forms of the PY motif established the preliminary minimal binding consensus as XPPXY (where X signifies any amino acid) required for interaction with the WW domain of YAP. Based on these results and the observation that the PY motif failed to bind arbitrarily to selected SH3 domains, we proposed that the PY motif differed from the binding consensus determined for SH3 ligands.
Although the WW and SH3 domains are functionally similar, their
three-dimensional structures are different (for review, see Ref. 11).
The WW domain has its NH2 and COOH termini in close juxtaposition, opposite from the ligand binding surface, allowing the
modular unit to exist close to the protein surface exposed to the
solvent (20). The NMR solution structure solved for the WW domain of
YAP consists of a bent three-stranded antiparallel sheet and a
hydrophobic ligand binding pocket composed of leucine 190, tyrosine
188, and tryptophan 199 (the second conserved tryptophan). The two
central proline residues of the consensus ligand
(XPPXY) contact tryptophan 199 through van der
Waals interactions, whereas the tyrosine residue of the PY ligand fits
into a hydrophobic pocket of the WW domain formed by leucine 190 and
histidine 192.
In this report, we describe the cloning and characterization of the complete cDNA clones for the murine and human orthologs of WBP-1 and WBP-2. Furthermore, the human genes encoding these two proteins were localized to chromosomes 2p12 and 17q25, respectively. To elucidate further the binding requirements of the PY motif, we replaced various positions of the motif with all possible amino acid residues and then assayed for in vitro binding to the WW domain of YAP using the "SPOTs" technique of multiple peptide synthesis on derivatized cellulose (28, 29). Finally, through site-directed mutagenesis of the conserved residues of the WW domain, we showed that the conservative substitution of the residues tyrosine 188, tryptophan 199, histidine 192, and proline 202 with related amino acids abolished binding, suggesting that these residues are important for binding.
Mouse WBP-1 and WBP-2
partial cDNAs (17) were used as probes to screen a pCEV9
cDNA library derived from M426 human lung fibroblast cells (21) (a
gift from Dr. Stuart Aaronson). The low stringency conditions of
hybridization were as follows: 4 × SSPE, 10 × Denhardt's,
2% SDS, 0.2 mg/ml salmon sperm DNA, and 106 cpm/ml
32P-labeled cDNA at 65 °C overnight. The filters
were washed twice at 60 °C for 20 min with 0.1 × SSC, 0.1%
SDS. The
pCEV9 cDNA library contained phages with a plasmid
portion that carried the insert. The plasmids with inserts were easily
rescued from the
genome following the published protocol (21). The
apparently complete sequence of human WBP-1 cDNA was contained in
one recombinant plasmid pCEV9-hWBP1 (clone 43) with a
BamHI-HindIII insert of 1.2 kilobases. Human
WBP-2 cDNA was contained in one recombinant plasmid pCEV9-hWBP2
(clone 29) with a BamHI-HindIII insert of 1.8 kilobases. Both strands of the cDNA clones were subsequently sequenced directly using the Sanger method (22).
To isolate the remainder of the cDNA for murine WBP-1, a 16-day mouse embryo cDNA library was screened with the partial cDNA clone coding for murine WBP-1 that was isolated previously according to published protocols (17, 23). Plaques representing the library were lifted onto nitrocellulose filters, which were subsequently incubated in hybridization solution (6 × SSC, 0.01 M EDTA, 5 × Denhardt's solution, 0.5% SDS, 100 µg/ml denatured salmon sperm DNA) with random labeled cDNA probe overnight at 68 °C. The filters were then washed in 2 × SSC, 0.5% SDS, and 2 × SSC, 0.1% SDS at room temperature, and then 0.1 × SSC, 0.5% SDS at 65 °C before exposing to film. The longest clone obtained by this method was selected for direct DNA sequencing by the Sanger method (22).
The remainder of the murine WBP-2 cDNA clone was successfully
isolated using the technique of 5 rapid amplification of cDNA ends
(Life Technologies, Inc.). First, the cDNA coding for WBP-2 was
derived from poly(A)+-purified mouse liver RNA with reverse
transcriptase using the following primer to the coding region: 5
dTCCCTTAATGAAGTTCGC. A poly(C) tail was added to the 5
end of the
cDNA following the protocol recommended by the manufacturer. The 5
regions of WBP-2 cDNA were amplified using polymerase chain
reaction with primers annealing to the 5
poly(C) tail
(5
-dCUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG, I denoting
deoxyinosine) and to a region in the coding region of WBP-2 (5
-
dAUGAUCAUAGGACAUUAG). The cDNA fragments were then isolated
according to the manufacturer's recommendations.
Lymphocytes isolated from human blood were cultured in a minimal essential medium supplemented with 10% fetal calf serum and phytohemagglutinin at 37 °C for 68-72 h. The lymphocytes cultures were treated with bromodeoxyuridin (0.18 mg/ml, Sigma) to synchronize the cell population. The synchronized cells were washed three times with serum-free medium to release the block and recultured at 37 °C for 6 h in minimal essential medium with thymidine (2.5 µg/ml, Sigma). Cells were harvested, and slides were made using standard procedures including hypotonic treatment, fixation, and air drying.
Chromosomal Localization by Fluorescence in Situ Hybridization (FISH)The cDNA inserts for WBP-1 and WBP-2 were separately biotinylated with dATP using the Life Technologies, Inc. BioNick labeling kit at 15 °C for 1 h as described elsewhere (24). The procedure for FISH detection was performed as described previously (24, 25). Slides were baked at 55 °C for 1 h. After RNase treatment, the slides were denatured in 70% formamide in 2 × SSC for 2 min at 70 °C followed by dehydration with ethanol. The biotinylated probes were denatured at 75 °C for 5 min in a hybridization solution consisting of 50% formamide and 10% dextran sulfate. The probes were loaded on the denatured chromosomal slides. After an overnight hybridization, the slides were washed and detected as well as amplified. FISH signals and the DAPI banding pattern were recorded separately by photography, and assignment of the FISH mapping data with chromosomal bands was accomplished by superimposing FISH signals with DAPI-banded chromosomes (26).
In Vitro Site-directed MutagenesisThe cDNA encoding
the WW domain of human YAP was subcloned previously in-frame into the
vector pGEX-2TK, allowing it to be expressed as a fusion protein with
glutathione S-transferase (GST) (Pharmacia Biotech Inc.)
(17). This vector in addition allowed the purified fusion protein to be
directly labeled by 32Pi at a protein kinase A
phosphorylation site in the region between the GST and WW portions, as
described elsewhere (17, 18, 27). Substitution of selected amino acids
in the WW domain was achieved using a double-stranded site-directed
mutagenesis kit (Pharmacia). The selection primer changed the unique
ScaI site in the vector to an MluI site, the
introduced mutation in boldface
(5-CTGTGACTGGTGACGCGTCAACCAAGTC-3
). The target primer was
used simultaneously to effect the desired substitutions within the WW
domain, the introduced mutation in boldface (W177F:
5
-GTCTTTGCCATCTCGAAACCTGCTG-3
; Y188F: 5
-GTGATTTAAGAAAAATCTCTGACCA-3
; F189Y:
5
-GATGTGATTTAAATAGTATCTCTGA-3
; H192F:
5
-TGTCTGATCGATGAAATTTAAGAAG-3
; W199F:
CCTGGGGTCCTGGAATGTTGTTGTC-3
; P202A:
5
-ATGGCCTTCCTGAGCTCCTGCCATG-3
). The mutagenesis reactions were performed according to the manufacturer's instructions. Plasmid DNA isolated from individual colonies was purified and sequenced by the Sanger method to confirm each mutation (22).
HeLa cells were grown to confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 10-cm plates and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1% Trasylol, 1 µM leupeptin, 1 µM antipain, 1 mM sodium vanadate). GST or GST-WW-YAP (100 µg) bound to glutathione-agarose was incubated with 200 mg of the cell lysate which was diluted 10-fold in Tris/Tween buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 1% ovalbumin, 1 mM dithiothreitol) at 4 °C overnight. The complexes were then analyzed by Western ligand blotting using 32P-GST-WW-YAP protein as a probe as described elsewhere (17). Binding assays with the mutant WW-YAP protein was performed by western ligand blotting using 32P-GST-GTPPPPYTVG protein as a probe (17).
SPOTs Method of Peptide Synthesis on Derivatized CelluloseThe peptide synthesis was accomplished following the method described by Frank and co-workers (28, 29). Fmoc (N-(9-fluorenyl)methoxycarbonyl) L-amino acids, derivatized cellulose membranes, instruction manual and computer software SPOTs, Release 1.0, for generating various schedules of amino acid applications were all purchased from Genosys Biotechnologies, Inc. Phosphorylated serine, threonine, and tyrosine were obtained from Chem Impex International. The membranes were incubated with the 32P-labeled GST-WW domain protein of human YAP under the conditions described previously (17), except the 5% milk in the blocking solution was replaced with 1 × blocking buffer supplied (as 10 × solution) by the manufacturer. After probing the membranes with 32P-labeled WW proteins, the membranes were regenerated with urea, mercaptoethanol, and acetic acid-containing buffers (as described by the manufacturer), and stained with Coomassie Blue for 10 min and destained using standard protocol for visualizing proteins on SDS-polyacrylamide gels. Uniform light blue staining was observed for the peptides with the exception of those containing Lys, Arg, or His, which were always stained more intensely with Coomassie Blue.
We described previously the
isolation of partial clones for murine WBP-1 and WBP-2 which were each
missing part of the 5-coding regions (17). In this paper, the
remainder of the murine cDNA encoding these proteins was
determined. Additionally, the human orthologs of WBP-1 and WBP-2 were
isolated by screening a human cDNA library (M426 lung fibroblasts)
under low stringency conditions with the corresponding murine cDNA
probes. Human and murine WBP-1 share an 84% sequence identity (Fig.
1A), whereas human and murine WBP-2 are 94%
identical (Fig. 1B), not considering those residues that are
biochemically similar. The WBP-1 and WBP-2 sequences are dissimilar
except for the conserved PY motif. Data-base searches using the FASTA
program again did not reveal any known proteins with significant
homology to WBP-1. However, human WBP-2 shared 26 and 29% homology
with two proteins in Caenorhabditis elegans (unnamed, with
GenBank accession numbers [GenBank] and [GenBank]) respectively, as predicted
from the open reading frames. These two C. elegans proteins
have yet to be characterized. Interestingly, the most extensive region
of homology between WBP-2 and the C. elegans proteins
contains the PY motif (marked by an asterisk). The PY motif
in the C. elegans proteins lacks a proline in the first
position but still conforms to the XPPXY
consensus that has been established as the minimal sequence that can
bind to the WW domain of YAP. Additionally, another region (marked by two asterisks) contains a sequence that has the
XPPXY consensus. The above sequence comparison
implies that these regions have been relatively well conserved through
evolution because of their functional significance, presumably in
mediating protein-protein interactions. A third region containing PPPPY
sequence (indicated with arrows, Fig. 1B) is
conserved between human and mouse WBP-2 sequences, but it is not
present in the C. elegans protein.
Chromosomal Localization of WBP-1 and WBP-2
Under the
conditions described, the hybridization efficiency for the WBP-1
cDNA probe was approximately 81% (among 100 mitotic figures
examined, 81 showed signals on one pair of chromosomes). Similarly, the
efficiency for the WBP-2 cDNA probe was approximately 76%. The
detailed positions of the loci were determined further based on the
summary from 10 FISH signal photographs/probe; representative hybridization pictures are shown in Fig. 2, A
and D. By superimposing the signals obtained from FISH onto
a representation of DAPI-banded chromosomes, we localized the WBP-1
locus to chromosome 2p12 and the WBP-2 locus to chromosome 17q25 (Fig.
2, C and F, respectively). Some hybridization of
the WBP-1 cDNA occurred at chromosome 2p11.2 as well, but this
signal was considerably weaker compared with hybridization at region
p12.
Filter Binding Assays with Mutant WW Domains
To determine the
residues of the WW domain which are important for binding, we performed
in vitro site-directed mutagenesis of selected residues in
the WW domain fused to GST (Fig. 3). The most conserved
residues in the domain were chosen, including Trp177,
Tyr188, Phe189, His92,
Trp199, and Pro202. Since we were interested in
elucidating the residues directly involved in forming the binding
surface and contacting the ligand, we replaced the above residues with
amino acids similar in structure and size. Replacing with amino acids
too dissimilar in size would have risked disturbing the natural
tertiary structure of the WW fold. We conducted filter binding assays
in which these GST-WW mutant proteins were transferred to a
nitrocellulose membrane that was subsequently probed with
32P-labeled GST-PY protein. The WW mutants Y188F, H192F,
P202A, and W199F failed to bind to the ligand probe (Fig.
4A). However, the constructs W177F and F189Y
were still able to bind to the ligand, and the former construct may
bind at a slightly higher affinity than even the wild type GST-WW
protein (Fig. 4A). Thus, according to these results, the
residues Tyr188, His192, Trp199,
and Pro202 seem to be involved in establishing the binding
surface of the WW domain. Alternatively, some of these residues may be
critical for maintaining the three-dimensional structure of the binding site on the nitrocellulose membrane, should a renatured form of the
GST-WW protein be required to retain binding function.
Binding of the WW Mutants in Solution
The binding of the WW domain while in its natural tertiary shape in solution may more closely resemble conditions encountered in vivo. As a result, coprecipitation assays were conducted in which GST-WW proteins coupled to glutathione-agarose were incubated with cell lysates to isolate the endogenous ligand to the WW domain of YAP. Using this method, the residues His192, Pro202, and Trp199 were found to be important for the WW domain to bind its ligand in solution (Fig. 4B). Remarkably, residue Tyr188, previously found to be important in filter binding assays, does not seem to be implicated in the binding activity of the WW domain in its natural form since the construct Y188F still was able to coprecipitate the endogenous ligand (Fig. 4B).
Mutational Analysis of the PY MotifEach position of the
target ligand,
Gly1-Thr2-Pro3-Pro4-Pro5-Pro6-Tyr7-Thr8-Val9-Gly10,
corresponding to residues 170-179 of the mouse WBP-1 sequence, was
substituted with all possible amino acids using SPOTs peptide synthesis
to determine the binding requirements of the YAP WW domain ligand.
Certain points can be inferred from the results of the SPOTs analysis:
The replacement of the Pro4 residue with leucine, serine,
valine, or alanine resulted in weak binders to the WW domain of YAP.
However, substitution with other amino acids in this position abolished
binding completely, in agreement with the consensus established earlier
(Fig. 5). When the Pro5 position was
replaced with a tyrosine (Fig. 6, spot 20), the peptide
bound with moderate affinity. All other substitutions at
Pro5 were negative. Tyr7 could not be replaced
with phenylalanine, tryptophan, histidine, or with any other amino acid
without disrupting binding activity. Also, a target peptide containing
a phosphorylated tyrosine failed to interact with the WW domain,
suggesting a method of regulating binding activity by tyrosine
phosphorylation. The Tyr7 residue seems to be the least
tolerant of any substitution. Moreover, the target peptide with the
reverse sequence (GVTYPPPPTG) does not bind to the WW domain of YAP
(Fig. 5).
Although the lack of perfect normalization of the SPOTs membrane
because of difficulties in precise measurements of the amount of
peptide in each individual spot precludes a rigorous quantitation of
the binding affinities, certain conclusions can be drawn. Based on the
Coomassie Blue staining of the membranes, we assume that there is no
significant difference in the amount of peptide between different
spots. From the data shown in Figs. 5, 6, 7 we can observe that the replacement of Pro3, Pro6,
Val9, and Gly10 with lysine, and
Thr8 with proline increase the interaction with the
GST-WW-YAP domain. Cysteine at position Gly1 and a cysteine
or proline at position Thr2 seems to increase the binding
slightly. In contrast, an isoleucine at position Pro3 and
acidic residues at position Pro6, Thr8,
Val9, or Gly10 reduce the interaction with the
WW domain. To prove these conclusions, however, selected peptides would
have to be synthesized, and the Kd value of their
interaction with the WW domain, in solution and on solid supports,
would have to be determined. Preliminary results obtained with an
isothermal titration microcalorimeter indicate that peptide analogs
containing lysine and proline substitutions at the carboxyl-terminal
end of the parent peptide show at least 5-fold lower binding constants
compared with the Kd of the parent peptide binding
to the WW domain of YAP.2
We have cloned the human homologs of the ligands to the WW domain of YAP, WBP-1 and WBP-2. These proteins allowed us to categorize the WW motif as a novel polyproline-binding module (17). The amino acid sequences of WBP-1 and WBP-2 have been well conserved through evolution, considering the high degree of homology between mouse and human orthologs. The degree of homology shared by the orthologs, especially in the region of the PY motif, suggests that the function of these proteins has been conserved between species. The PY motif, a region mediating protein interaction, is perfectly conserved between the mouse and human forms of WBP-1 and WBP-2 and represents the most conserved region shared between WBP-2 and two C. elegans proteins. This further supports the hypothesis that the biological function of these proteins depends on their ability to participate in protein interactions.
Using the cDNA of these clones, we localized the genes encoding human WBP-1 and WBP-2 to chromosomes 2p12 and 17q25, respectively. The chromosome region 2p12 has been shown to be potentially involved in several disorders. Chromosomal abnormalities are found in the majority of cases of non-Hodgkin's lymphoma, a subset of which shows a translocation involving the regions 3q27 and 2p12 (30). In addition, the gene for familial juvenile nephronophthisis, a leading genetic cause of juvenile end-stage renal failure, has been narrowed to the region 2p12 based on human linkage analysis (31). However, more work must be done to determine if WBP-1 can be implicated in these human conditions.
The chromosome region 17q25 has been shown to be involved in certain forms of human carcinogenesis. Some cases of alveolar soft part sarcoma show a consistent abnormality of 17q25 (32). However, the region of 17q25, also occasionally translocated in chronic myelogenous leukemia, has not yet been associated with any known oncogenes (33). Additional genetic and biochemical studies would need to be performed to demonstrate the importance of WBP-2 as a potential oncogene in the etiology of these human cancers.
Using site-directed mutagenesis we have implicated specific residues in
the WW domain of YAP which are important to maintain its ability to
bind to the PY motif. Our results complement the recent data on the NMR
solution structure of the WW domain of YAP in complex with the
proline-rich ligand (20). The three-dimensional structure consists of a
three-stranded, antiparallel sheet with residues Tyr188
and Trp199 (the second conserved tryptophan) on the concave
side and residues Phe189 and Trp177 on the
convex side. The concave aspect contains an almost flat hydrophobic
binding surface represented by the residues Tyr188,
Trp199, His192, and Leu190.
Moreover, the NH2 and COOH termini of the domain appose
each other on the convex side in the manner of a hydrophobic buckle which presumably helps maintain the stable folded structure of the
domain. This buckle forms when Pro174 and
Pro202 pack against Trp177, the interaction of
which is further stabilized by the presence of Ile167.
These results from Macias and co-workers (20) support the binding
consensus of the PY ligand established earlier as
XPPXY (17). Moreover, the mutagenesis results
presented here are for the most part in agreement with the NMR
structure. Conservative substitution of the residues
Tyr188, His192, Trp199, and
Pro202 abrogated the binding function of the WW domain in
our filter binding assays, implying in light of the NMR data that the
hydrophobic binding surface is disrupted by the first three mutations
or that the fold of the WW domain is severely compromised by the
Pro202 substitution. Nuclear Overhauser effects between
His192 and ligand residues Tyr7 and
Val9, Trp199, and Pro5, and between
Tyr188 and the carbonyl group of Pro6 provide
corroborating evidence for the importance of these residues. The
replacement of the histidine residue at position 192 with a
phenylalanine suggests the participation of a hydrogen bond located
between the imidazole nitrogen acceptor of histidine and the hydroxyl
hydrogen donor of Tyr7 (Fig. 8). Elimination
of this hydrogen bond likely destabilizes this structure, as does the
introduction of the bulkier phenylalanine residue into such a confined
space. Lack of ligand binding by the W199F and P202A mutants are not
manifested through variations in charges or hydrogen bonds but likely
reflect alteration in the architecture of the surface topology and
changes in side chain packing. The removal of tryptophan at position
199 and replacement with a phenylalanine may eliminate enough of the
surface recognition site to inhibit ligand binding. Alternatively, the
newly introduced phenylalanine residue may not pack efficiently into
the cavity designated by Pro4 and Pro5 (Fig.
8). Likewise, molecular van der Waals packing interactions are central
to the P202A mutation. The incorporation of an alanine diminishes this
tight hydrophobic packing which may ultimately destroy the molecular
"fastener" thought to be responsible for maintaining the integrity
of the core domain
sheet (Fig. 8). One should point out that the
stability of the WW domain could be considered marginal (20) because
the domain is only one
sheet layer thick and thus may not have a
well defined hydrophobic core.
In contrast, mutations of Trp177 or Phe189 had no observable effect in the binding assays, suggesting that structural changes possibly imparted by these substitutions were insignificant to the overall function of the domain. The replacement of Tyr188 with phenylalanine failed to attenuate the binding ability of the domain in solution, as illustrated by the coprecipitation assays (Fig. 4B). Based upon NMR data, the hydroxyl proton of this tyrosine residue is within hydrogen bonding distance of the carbonyl oxygen of ligand residue Pro6 (Fig. 8, A and B). The ligand binding studies presented within suggest that such an interaction is not required to manifest ligand binding in solution. Comparison of the mutant Y188F structure with that of the wild type does not reveal any significant structural distinction. We also cannot rule out the possibility that another portion of the domain may compensate for or "mask" this mutation, depending on the conformation of the domain while bound to nitrocellulose membrane compared with its state in solution.
Mutational analysis of the target peptide (GTPPPPYTVG) for the WW
domain of human YAP led us to the following conclusions. The data
confirm our preliminary consensus
Xaa-Pro4-Pro5-Xaa-Tyr7 generated by
the "alanine scan" mutagenesis (17). In addition, these results are
in agreement with the structure of the WW domain of human YAP in
complex with the target peptide (20). The PY motif ligand (peptide
GTPPPPYTVG) fits into the binding surface in the form of a polyproline
type II helix. The Pro4 and Pro5 of the ligand
are involved in the binding interface by forming van der Waals contacts
with the second conserved Trp199. Moreover, the
Tyr7 of the ligand peptide is accommodated by a hydrophobic
surface containing conserved residues including Leu190 and
His192 in the WW domain. Interestingly, mutations P5L and
Y7H in the PY motif are analogous to those found in the
amiloride-sensitive sodium channel subunit of two Liddle's
syndrome patients (34, 35). The proline-rich motif of
amiloride-sensitive sodium channel is a target of deletions and point
mutations resulting in Liddle's syndrome of hypertension. It is likely
that the two mutations P616L and Y618H (Refs. 34 and 35), which are
analogous to P5L and Y7H, result in the complete lack of binding to the
WW domain of Nedd4. In addition, the fact that the WW domain does not
bind to a target peptide with the reverse sequence provides suggestive
evidence that the YPPPP motif in the WBP-2 protein (amino acids
179-183 of human WBP-2) may be nonfunctional in terms of binding to
the WW domain of YAP.
The WW domain appears to be involved in various physiological and
pathophysiological processes. The disruption of the WW-PY interaction
may in fact be the molecular deficiency present in a form of inherited
hypertension known as Liddle's syndrome (36-39). Furthermore,
dystrophin, a protein implicated in Duchenne's and Becker's muscular
dystrophy, forms a stable complex via its WW domain to a proline-rich
motif in -dystroglycan in vitro (40). In addition, the
Gag protein of retroviruses contains a PY motif that when mutated
severely curtails the ability of the virus to bud from the host
cell membrane (40-43).
Because of the relatively small size of the PY motif, low molecular weight mimotopes may be designed to fit into the binding pocket of the WW domain, to interfere with the viral life cycle, for example. Additional work on the WW domain will provide answers to fundamental biological questions and will ultimately lead to the development of therapeutic applications.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U40825[GenBank] (mouse WBP-1), U40826[GenBank] (mouse WBP-2), U79457[GenBank] (human WBP-1), and U79458[GenBank] (human WBP-2).
We thank Dr. Hidesaburo Hanafusa for valuable comments and advice and the members of the Laboratory of Molecular Oncology at the Rockefeller University as well as the Department of Biochemistry at the Mount Sinai School of Medicine for stimulating discussions. We owe special thanks to Dr. Mark Lemmon for performing microcalorimetric measurements of WW domain-ligand interactions, to Drs. Matti Saraste and Hartmut Oschkinat for constructive discussions regarding the structure of the WW domain of human YAP, to Dr. Peer Bork for help in sequence analysis, and to Jason Paragas for critical comments on the manuscript.