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INTRODUCTION |
Trp-Trp (WW1) domains
are compact modules of 38-40 amino acids long, folded into a
three-stranded
sheet, and found in single or tandem repeats in over
25 unrelated cell-signaling proteins (1, 2). Although their binding to
proline-based ligands is now well described (3, 4), little is known
about their precise biological function. WW domains form a new family
of protein-protein interaction modules targeted to proline residues,
analogous to the Src homology (SH) 3 domains (5).
Based on their proline-rich sequence binding specificities, WW domains
are classified into five distinct groups (6). The N-terminal WW domain
of the peptidyl-prolyl isomerase Pin1, an essential regulator at
mitotic entry, is grouped among class IV domains, which bind peptides
containing a proline residue preceded by a phosphoserine or a
phosphothreonine (pSer/pThr-Pro motif) (7). This latter motif is found
in several mitotic Pin1-binding phosphoproteins (8), including the
mitotic phosphatase Cdc25 (9, 10) and the microtubule-associated tau
(
) protein (11).
Site-directed mutagenesis experiments indicate that Pin1 binds
phosphoproteins through its N-terminal WW domain and that the binding
site mainly implicates the conserved residues Tyr18 and
Trp29 (7, 11), which constitute a nearly flat hydrophobic
area on the molecular surface of the WW domain. WW domains interacting with the core sequence PPXY, like Yes-associated protein,
use the same hydrophobic surface for molecular recognition (3, 4, 12).
However, this hydrophobic binding site alone is not likely to explain
the phospho-dependent character of the ligand binding to
the Pin1 WW domain.
Recently the x-ray crystal structure of the full Pin1 protein bound to
a doubly phosphorylated peptide (YpSPTpSPS) from the C-terminal domain
(CTD) of RNA polymerase II was reported (13). The protein-peptide
interactions are essentially limited to two regions on the WW domain
surface. First, a phosphate binding pocket, encompassing the side
chains of Ser11, Arg12, and the backbone amide
of Arg12, anchors the interacting phosphate moiety via
several hydrogen bonds. Second, the aromatic pair
Tyr18-Trp29 forms a molecular clamp that
constrains the proline at position +1 of the interacting phosphoserine.
The peptide ligand binds to the Pin1 WW domain in a N- to C-terminal
orientation, in contrast to the C- to N-terminal orientation that is
found for two other proline-rich peptides in complex with a group I WW
domain (3, 14). Other proline recognition domains, such as the SH3
domain of the Caenorhabditis elegans signaling adaptor
protein Sem5, can bind peptidic ligands in both orientations (15). It
therefore remains an open question whether the group IV WW domain of
Pin1 is capable of binding different ligands in different orientations or whether a unique orientation is preferred.
Furthermore it is of major interest to consider the conformation of the
recognized motif and, more specifically, the isomerization state of the
peptide bond in the pSer/pThr-Pro motif. In the crystal structure of
Pin1 complex (13), the peptide ligand binds with both pSer-Pro peptide
bonds in trans conformation. The cis conformation of the peptide would require major rearrangements of the binding site
and is therefore not very probable. However, for a related adaptor
module, the 14-3-3 domain, a recent crystallography study showed that
peptide substrates can be bound with both isomerization states
(cis and trans) (16).
Here we have investigated by solution NMR the binding interactions
between a synthetic Pin1 WW domain and two biologically relevant
monophosphorylated substrates, a Cdc25 peptide model (EQPLpTPVTDL) and
a peptide of the
protein (KVSVVRpTPPKSPS). Based on the structures
of both complexes in solution, we found a similar binding mode as the
one observed in the crystal structure of Pin1 bound to a peptide
isolated from the CTD of RNA polymerase II (13). This observation gives
experimental evidence to a unique binding scheme for Pin1 WW domain to
its multiple substrates. Finally, we found the bound peptides to be in
a trans conformation and discuss the implication of this
aspect in regard of recent data concerning Pin1 molecular function
(17).
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis--
The Pin1 WW domain was obtained by
peptide synthesis using the t-butoxycarbonyl-benzyl strategy
and the (2,1H-benzotriazolyl-1-yl)-1,1,3,3-tetramethyl uranium
hexofluorophosphate in situ activation protocol on an Applied Biosystems 430A peptide synthesizer (Foster City, CA). Side-chain protections were Arg(Tos), Trp(For), Asn(Trt), Gln(Trt), Ser(Bzl), His(Tos), Tyr(Br-Z), Lys(2-CIZ). The formyl group of tryptophan was removed with a solution of 20% piperidine in dimethyl formamide prior to hydrofluoric acid cleavage. After lyophilization, the crude peptide was purified by reverse phase-high pressure liquid
chromatography on a Nucleosil C18 (Macherey-Nagel, Duren, Germany) column using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Homogene fractions, as checked by reverse phase
on an X-terra RP18 (Waters, Milford, MA) and mass spectrometry on a
Quattro II electrospray mass spectrometer (Micromass, Manchester, UK),
were pooled and lyophilized. The same conditions and procedures were
used for WWR12A and WWS11A mutants.
The phosphopeptide sequences, derived from Cdc25 (AcEQPLpTPVTDL) and
(AcKVSVVRpTPPKSPS) protein sequences, were previously identified by
peptide-scanning experiments as major binding sites for the Pin1 WW
domain (7, 11) and were also supplied by peptide synthesis. Peptide
chemical synthesis was identical as described (18).
NMR Spectroscopy--
Experiments were performed on a Bruker DMX
600-MHz spectrometer and processed with the Bruker software. Spectra
were recorded on a 1 mM sample of the WW domain in a buffer
of 50 mM deuterated Tris-HCl, pH 6.4, 100 mM
NaCl and at a temperature of 12 °C. Two-dimensional TOCSY (19) and
NOESY (20) spectra were acquired with mixing times of 62 and 300 ms,
respectively. Spectral widths were 8400 Hz, with 1024 complex points
for t2, 256 complex points for t1, 32 transients per increment for
TOCSY experiments, and 64 transients for NOESY experiments. The water
signal was suppressed using the Watergate (21) or jump-return (22)
pulse sequences. The data sets were processed with square sine
functions, and zero filling was applied to obtain spectra with a
digital resolution of 16.4 Hz/point in the F1 dimension and 8.2 Hz/point in the F2 dimension. The classical homonuclear NMR procedure
for resonance assignment in polypeptides was used (23). The
stereospecific assignment was assisted by chemical shift calculations
(24) computed from the x-ray structure of Pin1 WW domain (Rutgers
University Protein Data Bank code 1PIN (25)). Proton chemical
shifts were measured in parts per million (ppm) and referenced relative
to the methyl proton resonances (put at 0.0 ppm) of the internal
standard trimethyl silyl propionate.
NMR Titration--
Increasing amounts of phosphopeptide ligands,
with Cdc25 or
sequences, were added to a 1 mM sample of
WW domain to give molar ratios ranging from 1:0.0 to 1:4.5 for the WW
domain-Cdc25 peptide sample or from 1:0.0 to 1:11.0 for the WW
domain-
peptide sample. The pH of each sample was systematically
controlled and, if necessary, adjusted to a value of 6.4 by addition of
an aliquot of dilute NaOD or DCl solutions. For each titration
point, a one-dimensional NMR spectrum was recorded. In addition,
two-dimensional NMR TOCSY and NOESY experiments were acquired at low
ligand concentration (typically at a ratio of 1:0.5), at equimolar
concentration and finally at substrate saturation. For the latter
ratio, NOESY spectra with a high number of scans per increment
(i.e. 416 and 320, respectively, for the WW domain-Cdc25 and
WW domain-
samples) were acquired, to detect possible weak
intermolecular NOEs. The binding constant was calculated by fitting to
the following equation,
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(Eq. 1)
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where 
is the observed chemical shift,

max is the maximum shift at ligand saturation, [WW]
is the protein concentration, Kd is the dissociation
constant, and X is the molar ratio of peptide substrate to
protein concentration [WW].
Solution Structure Calculations--
Three-dimensional models of
WW domain and of complexes between phosphothreonine peptides and WW
domain were determined with interproton restraints derived from NOE
spectra. NOEs within the protein and between the protein and ligand
were grouped into four distance ranges: 1.8-3.0, 1.8-4.0, 2.0-5.0,
and 2.5-6.0 Å, corresponding to strong, medium, weak, and very weak
NOEs, respectively. To account for the higher apparent intensity of
methyl resonances, 0.5 Å was added to the upper distance limits for
NOEs involving methyl protons. Protein backbone hydrogen-bonding
restraints (two per hydrogen bond; rHN-O = 1.5-2.5 Å; rN-O = 2.5-3.5 Å) within areas of
regular secondary structure were introduced during the final stages of
refinement. No torsion angle restraints were used.
The structures were calculated via a hybrid distance
geometry/simulated annealing protocol (26) as implemented in the
program XPLOR (27). The conformers with no distance violations greater than 0.5 Å and r.m.s. differences for bond and angle deviations from
ideality less than 0.01 Å and 2°, respectively, were further submitted to a restrained energy refinement. CVFF was used as a
force field in the Discover modules (MSI software) (28), with the
experimental intermolecular NOEs used as distance constraints. The
minimization procedure consisted of 50 steps of steepest descent followed by steps of Quasi-Newton-Raphson optimization (29) until the
maximum energy derivative was lower then 0.05 kcal mol
1
Å
1. Finally, the 20 structures with the lowest overall
energies were selected as the representative structures. To compare our results with x-ray structures we used the structure, named the reference structure, whose average r.m.s. deviation with respect to the
19 other structures was minimum. The stereochemical quality of
the structures was evaluated by the PROCHECK-NMR program (30).
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RESULTS |
NMR Assignment and Structure of the Isolated WW
Domain--
Sequence-specific 1H NMR assignments of the WW
domain, based on homonuclear TOCSY and NOESY spectra recorded in
H2O (23), were straightforward due to the large
1H NMR chemical shifts dispersion observed, ranging from
10.57 ppm for the Hn
1 of Trp6 to the most upfield
resonance for the H
1 of Asn21 at
0.64 ppm. Chemical
shift calculations starting from the WW domain x-ray structure
confirmed the atypical resonance frequencies of several protons and
indicated the ring current shifts associated with the high number of
aromatic residues in the WW domain as the primary source. In
particular, the chemical shifts of protons Asn21 H
1
(
0.64 ppm), Asn21 H
1 (4.23 ppm), Pro32
H
(0.03 and 0.62 ppm), and Pro32 H
(0.79 and 0.87)
showed large upfield shifts due to the ring current effect of the
Trp6 indole ring. In the same way the proximity of the
aromatic rings of Tyr18 and Tyr19 contributed
to the upfield shifts of protons Arg9 H
1 (0.10 ppm) and
Arg31 H
(2.68 ppm), respectively.
On the basis of 643 experimental NOE constraints, we derived the
solution structure of the WW domain. The structural and energetic statistics on the 20 favorable WW conformers are showed in Table I. The overall fold of the synthetic WW
peptide, an antiparallel
-sheet composed of three
-strands, was
very similar to the x-ray crystallography model (Protein Data Bank code
1PIN (25)), with an r.m.s. backbone deviation of 1.38 Å.
NMR Titration with Ligands--
The binding interaction between
phosphothreonine peptides and the WW domain was examined by means of
one-dimensional 1H NMR spectra (Fig.
1A). Addition of increasing
amounts of peptide ligands caused chemical shift changes for
several protons in the WW domain. During the NMR titration some proton
resonances, such as the Hn of Arg12 and the Hn
1 of
Trp29, rapidly became broader, until they disappeared from
the spectra to reappear at large excess of ligand. This broadening can
be ascribed to slow exchange, with the difference in chemical shift for
the proton concerned in its free and bound state being of the same
order as the exchange rate (31). Other resonances, such as the amide
proton of Ser11, moved gradually, whereas many others
exhibited practically no chemical shift perturbations. Resonance
frequencies for protons of the WW domain were similarly affected upon
binding of the Cdc25 and
peptide ligands, indicating an equivalent
binding mode for both phosphothreonine peptides. The isolated
Ser11 amide proton resonance, in rapid exchange on the NMR
time scale and hence apparent throughout the titration, was used to
estimate a binding constant of 117 and 230 µM for the
Cdc25 and
phosphopeptides, respectively (Fig. 1B).
Finally, the non-phosphorylated variants of peptide ligands did not
induce modifications of the WW domain resonances (data not shown),
confirming the phospho-dependence of the interaction.

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Fig. 1.
A, chemical shift changes observed in
the amide regions of the 1H NMR spectrum of Pin1 WW domain
in presence of increasing amounts (from bottom to
top) of Cdc25 phosphopeptides. The extrapolated molar ratio
WW domain:ligand and the resonance assignments are indicated.
B, titration curve corresponding to chemical shift changes
of backbone amide proton of Ser11 (shown by a dotted
line in a) was used to calculate the binding constant
(see "Experimental Procedures").
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NMR Binding Maps--
To obtain a precise description of the
complex, two-dimensional NMR spectra of the ligand-saturated WW protein
were recorded and completely reassigned. Comparison with the
assignments of the free WW domain allowed a precise mapping of the
binding surface (Fig. 2). The chemical
shift perturbations were essentially grouped in two regions of the WW
domain: the loop region between the strands
1 and
2
(Ser11-Gly15) and the C terminus of the strand
3 (Gln28-Glu30). The largest chemical shift
changes were observed for the Hn
1 of Trp29 and the
backbone amide proton of Arg12, indicating thereby their
presence in the immediate vicinity of the binding site. Noteworthy, an
exceptional value of 11.4 ppm was found in the bound form for the
resonance of Arg12 backbone amide proton. When displayed on
the WW domain surface (Fig. 2), those two most perturbed regions
delineated in an unambiguous fashion the phospho-ligand binding
site.

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Fig. 2.
Plot of backbone amide chemical shift changes
observed for each residue of WW domain in the presence of a large
excess of Cdc25 or peptide ligand. The
three -strands constituting the WW domain fold are shown by
arrows. The very large shift (2.10 ppm) of
Hn-Arg12 resonance is indicated. On the left
side, a ribbon drawing of the WW domain, with residues
displaying the largest chemical shifts rendered in detail, is shown.
Note that due to some overlapped resonances we were not able to
unambiguously assign chemical shifts of Ser13 and
Ser14 in the bound form with ligand.
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The fact that the WW domain folds into a small compact structure and
that only few residues were strongly affected by addition of
phosphopeptides suggests that chemical shift changes were caused by
direct interaction between the WW domain and peptide ligands and not by
some ligand-induced conformational changes. The
-sheet twist
modification upon phosphopeptide complexation, reported from x-ray
crystal data (13), appears thus as a minor conformational change during
the binding leading to no substantial changes in the pattern of
intramolecular NOEs.
Because the key role of Trp29 in the binding feature was
previously established for other WW domains (3, 12), we focused on the
influence of the loop Ser11-Gly15. Two WW
domain mutants, Ser11
Ala and Arg12
Ala, were synthesized. After confirmation of their structural integrity
by circular dichroism and NMR spectra (data not shown), we performed an
NMR titration with the Cdc25 phospho-peptide. For both mutants, we
observed chemical shift changes upon addition of phosphorylated peptide
(Fig. 3), confirming their capacity to
bind the Cdc25 peptide. However, chemical shift pertubations were less
important, and a 3-fold excess of peptide was not sufficient to drive
the Arg12 amide proton resonance of the Ser11
Ala WW mutant into the fast exchange regime with concomitant line
narrowing (Fig. 3). Therefore, mutations in the loop appeared to weaken
the interaction without destroying it completely. The binding affinity
between phospho-substrates and Pin1 WW domain seems thus generated by a
sum of at least two energetically favorable interactions, the
charge-charge interaction between the phosphate group and the
positively charged WW loop and the proline-aromatic interaction, with
none of the individual interactions appearing as absolutely essential
(13).

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Fig. 3.
1H NMR spectrum of amide regions
for wild type WW mutants (bottom), Arg12
Ala mutant (middle), and Ser11 Ala
mutant (top), either in the solution free state
(left side) or bound to Cdc25 phosphothreonine peptide
(right side) at a protein:ligand molar ratio of
1:3. Chemical shift perturbations for the Hn 1 of
Trp29 and the backbone amide proton of residue 11 are
larger in the wild type WW domain then in both mutants.
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Conformation of the Bound Peptide--
The conformation of the
recognized motif on the peptide ligands and, more specifically, the
isomerization state of the peptide bond separating the phospho-residue
and proline, is of great interest. We set out to distinguish both
isoforms first in the free peptides and then in the complexes. For the
peptide, the cis form of the Pro8 following
the pThr7 residue could not be detected, in agreement with
a recent study that gave an upper limit of 3% for the cis
form of the same Pro residue in a somewhat longer peptide that was
phosphorylated on Thr7 and Ser11 (32). For the
Cdc25 peptide, however, the cis form of Pro6 was
clearly detectable, and the complete spin system starting from the
protons could be easily assigned for both trans and cis forms (Fig. 4). Upon
addition of the WW domain, the spin system of the cis form
of this Pro6 did not change, whereas the spin system
corresponding to the trans form, which was nine times more
intense for the free peptide, completely disappeared due to exchange
broadening (Fig. 4). This result clearly shows that the WW domain
selects for the pThr-Pro motif with the peptidic bond in the
trans conformation.

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Fig. 4.
Spin system of Pro6 of Cdc25
peptide connecting its protons with its
and protons in the free
state (top) and in a 1:4.5 complex with WW domain
(bottom). The cis (cP6) and
trans (tP6) conformations are depicted by dotted
lines when detectable.
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Intermolecular NOE Data and Complex Structures--
On the basis
of NOESY analysis on a ligand-saturated WW sample, 14 and 22 unambiguous protein-ligand NOEs were identified for the Cdc25 and for
the
peptide, respectively. Contacts with pThr7,
Pro8, and Pro9 of the
peptide represent
intermolecular NOEs that helped to determine the orientation of the
peptide ligand in the recognition site of the WW domain. Other
intermolecular NOEs were detected between protons of the WW
Arg9, Arg12, Phe20, and
Trp29 residues and protons of Val5,
pThr7, Pro8, and Pro9 of the
peptide and the WW Arg12, Tyr18, and
Trp29 residues and protons of pThr5,
Pro6, and Val7 of the Cdc25 peptide.
These NOEs sets were translated into intermolecular proton-proton
distances and formed the basis for construction of the structure of the
complexes. Table I gives the structural statistics of the complex
models. The WW domain conformation was not significantly modified upon
ligand binding, except for a different relative orientation of the loop
Ser11-Gly15. This change is brought about by a
few distinct intermolecular NOEs observed for the two peptides, which
equally induced a slightly different positioning of the peptide ligands
on WW domain surface (Figs. 5 and
6). However, besides these small
differences, we found a similar N to C orientation of the bound
peptides and the same main ligand-protein interactions that could be
decomposed into two major contributions (Fig. 6). The WW domain side
chain of aromatics Trp29 makes a van der Waals stacking
with the conserved proline at position +1 of the interacting
phosphothreonine and with hydrophobic residue at +2 (Pro or Val, if one
considers
or Cdc25 peptides, respectively). The substrate phosphate
moiety is bound to the side chain and backbone of Arg12 of
the WW domain. The chemical shift perturbations that were observed upon
ligand binding by the WW domain were in accordance with this model.

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Fig. 5.
Stereo view of the overlaid backbone traces
of the 20 final conformers of the complex between the Pin1 WW domain
and a Cdc25 peptide ligand. Superposition was done on residues
(4-32) of the WW domain.
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Fig. 6.
Ribbon drawing of the NMR reference structure
of the complex between Pin1 WW domain (in light blue)
and phosphopeptide ligand (in
red), in comparison with the orientation of the CTD
peptide (in violet) from the crystallographic model of
the complex (13). The image was obtained by backbone
superimposition of WW domains from our NMR complex and from the CTD
peptide/Pin1 complex (13). Only the WW domain from this study and both
phosphopeptide ligands are represented. Side chains implicated into the
binding interface are labeled (in white for the WW domain
residues and yellow for the ligand residues) and depicted in
detail, as well as the amino acid pair
Trp6-Pro32 of the WW domain. N atoms are
blue and P atoms are violet. C atoms are
green in the WW domain and orange in the tau
ligand.
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DISCUSSION |
The reversible protein phosphorylation on Ser/Thr-Pro motifs acts
as an important molecular switch in the cell cycle and is operated by a
number of Pro-directed protein kinases (such as Cdc2, Cdk2, Wee1, and
Myt1; for a review, see Ref. 33) and phosphatases, such as Cdc25 and
PP2A (34, 35). Although atomic level structural data of all substrates
in their phosphorylated and non-phosphorylated state are still lacking,
one example of the structural influence of phosphorylation is given by
Cdk2 itself, where addition of a phosphate group on the
Thr160 side chain completes the reorganization of the
substrate binding site that was started by cyclin binding (36). A
second mechanism of imposing structural changes is brought about by the
isomerization of the prolyl peptide bond, and a prolyl
cis/trans isomerase, Pin1, has been shown to be
essential to achieve a cell cycle (37, 38). A remarkable fact of both
Cdk2 and Pin1 is that they associate with small binding modules that
are believed to help targeting the catalytic domain more efficiently to
their substrates. Cdk2 recruits the CKS, that indeed binds through its
anion binding site to the pThr-Pro motif of a Cdc25 peptide (39),
whereas human Pin1 contains in its sequence itself already such a
binding protein, the WW domain (7, 37). Both functional (40) and structural (39) data indicated a molecular competition of both of
binding modules for the same Cdc25 substrate, and this competition indeed might be part of the tight regulation of the cell cycle.
In a related cellular process, both Cdk2 and Pin1 interact with the
protein. Cdk2 phosphorylates
proteins, in an Alzheimer-like way,
leading to microtubule depolymerization (41). Conversely, Pin1 can
restore the tubulin polymerization function of these hyperphosphorylated
before they aggregate into filaments in Alzheimer neuronal tangles (11).
contains about 17 Thr/Ser-Pro motifs, which can all be phosphorylated in the cellular context (42).
Amazingly, however, only the
pThr131-Pro132
motif was found to be recognized by Pin1, with a high affinity of 40 nM (11). This unusual high affinity for a binding module was not confirmed by our NMR titration data, because we obtained similar dissociation equilibrium constants in the order of 100 µM for both Cdc25 and
ligands. Except for differences
in methodology, the reason for these differences in
Kd values could be due to the fact that we used the
isolated WW in our binding experiments, instead of the full-length
PIN1. An NMR titration on WW domain-
peptide samples without 100 mM NaCl yielded an increase of binding affinity
(Kd = 29 µM), stressing the importance
of ionic strength on the binding affinity of Pin1 related WW domains.
Still, considering the ionic concentration in the cell, our calculated Kd appears more relevant, and confirms that the
interaction of Pin1 with Cdc25 and
are related molecular processes.
Despite the reported selectivity of Pin1, we found that both Cdc25 and
phosphopeptides bind to Pin1 WW domains in a similar manner, close
to the described binding pattern in the CTD peptide-Pin1 x-ray model
(13). However, the crystal complex of the CTD peptide-Pin1 protein
shows a larger contact surface between protein and ligand than what was
observed in our NMR models. The anchoring zone of the Cdc25 and
peptide ligands to the WW domain was only composed of the
phospho-residue and residues +1 and +2 (Figs. 5 and 6). We did not
observe any significant interaction with the Tyr18 of the
WW domain, neither in the chemical shift perturbation assay, nor by the
observation of intermolecular NOEs.
The three-dimensional NMR complex model shows that the phosphopeptide
ligands are principally fixed by a charge-charge interaction and a
proline-aromatic stacking. The participation of the backbone amide
group in complexation of the phosphate moiety could explain partly why
the mutant Arg12
Ala persists to fix ligand. The same
argument could help to interpret the phosphopeptide binding properties
of other Pin1-related WW domains, such as Nedd4 or Rsp5 proteins, that
lack a basic residue in the
1-
2 loop. In addition, it must be
stressed that the intermolecular stacking structure between
Trp29 and the proline at position +1 of pThr ligand is
analogous to the intramolecular stacking between conserved residues
Trp6 and Pro32 of domain WW (Fig. 6). Because
several mutagenesis studies (10, 43-45) have established the
importance of the Trp6-Pro32 interaction for
the folding and the stability of WW domains, we think that a similar
strong intermolecular Trp-Pro interaction is used to promote
protein-protein interactions between the WW domain and its substrates
(46).
The molecular mechanisms of the Pin1 function very recently became
clearer through an elegant study by Zhou et al. (17), who
showed that Pin1 enhances in vitro the dephosphorylation of Cdc25C by the major transPro-directed phosphatase PP2A.
Moreover, they showed that overexpression of the catalytic domain alone can be sufficient for cell survival, whereas at lower levels of expression this is not the case (7). At the molecular level, Pin1 would
catalyze the conversion of the cis bond in the pThr-Pro motif of Cdc25C, turning the peptide into a suitable substrate of PP2A.
Although direct evidence of such a cis peptide bond in the
complete Cdc25 or
lacks, their biochemical evidence that Pin1
up-regulates the dephosphorylation of both Cdc25 and
convincingly sustains this hypothesis. However, the results of our NMR study unambiguously define the pThr-transPro motif in both Cdc25
and
peptide ligands as the binding surface recognized by the Pin1 WW domain, in agreement with the earlier crystal structure of full Pin1
with a doubly phosphorylated CTD peptide (13). Moreover, because our
NMR results confirm the absence of interaction between the Pro residue
in the cis conformation and the WW domain, it is unlikely
that the latter would assist the catalytic domain by targetting it to
its pThr/Ser-cisPro substrate, be it on Cdc25,
or other
protein targets.
The available data can be combined into several scenarios. The WW
domain could specifically target Pin1 to the pThr231 of
or pThr47 and pThr68 of Cdc25, followed by
isomerization of neighboring pThr-Pro bonds through the catalytic
domain (47). In another scenario, the catalytic domain could induce the
conformational transition from cis to trans of
the pThr-Pro motif prior to any WW binding. Before a return to the
cis/trans equilibrium situation, the WW domain could then immediately bind to the pThr-trans conformation,
thereby stabilizing this conformation and simultaneously increasing the local concentration of Pin1. Both scenarios solve the dilemma that the
WW domain has an at least 10-fold higher affinity as does the catalytic
domain for the same substrates (7, 13) and, consequently, would bind
first to the epitope before catalysis could happen. Further studies
will be necessary to investigate this possibility of prolyl
cis/trans isomerization anterior to WW binding.