From the Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029-6574
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ABSTRACT |
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Phosphotyrosine binding (PTB) domains of the
adaptor protein Shc and insulin receptor substrate (IRS-1) interact
with a distinct set of activated and tyrosine-phosphorylated cytokine
and growth factor receptors and play important roles in mediating
mitogenic signal transduction. By using the technique of isothermal
titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated
NPXY-containing peptides derived from known receptor
binding sites. The results showed that relative contributions of
enthalpy and entropy to the free energy of binding are dependent on
specific phosphopeptides. Binding of the Shc PTB domain to
tyrosine-phosphorylated peptides from TrkA, epidermal growth factor,
ErbB3, and insulin receptors is achieved via an overall entropy-driven
reaction. On the other hand, recognition of the phosphopeptides of
insulin and interleukin-4 receptors by the IRS-1 PTB domain is
predominantly an enthalpy-driven process. Mutagenesis and amino acid
substitution experiments showed that in addition to the
tyrosine-phosphorylated NPXY motif, the PTB domains of Shc
and IRS-1 prefer a large hydrophobic residue at pY-5 and a small
hydrophobic residue at pY-1, respectively (where pY is
phosphotyrosine). These results agree with the calculated solvent
accessibility of these two key peptide residues in the PTB
domain/peptide structures and support the notion that the PTB domains
of Shc and IRS-1 employ functionally distinct mechanisms to recognize
tyrosine-phosphorylated receptors.
Protein tyrosine phosphorylation provides a central control
mechanism in regulating protein-protein interactions and activation of
enzymes in mitogenic signal transduction following activation of
cytokine and growth factor receptors (1, 2). Key events in receptor
signaling are the interactions of signaling molecules such as adaptor
protein Shc and insulin receptor substrate
(IRS-1)1 with activated and
tyrosine-phosphorylated receptors. Binding to the activated receptor
results in tyrosine phosphorylation of these signaling molecules, which
in turn experience specific interactions with downstream signaling
proteins and/or enzymes. For example, in insulin receptor (IR)
signaling, upon binding to the activated receptor, IRS-1 is
phosphorylated on many tyrosine residues, which enables IRS-1 to
interact with various Src homology 2 (SH2) domain-containing proteins,
including phosphatidylinositol 3-kinase, protein tyrosine phosphatase
SH-PTP2, and Grb2 (3). On the other hand, tyrosine-phosphorylated Shc
interacts with the SH2 domain of the adaptor protein Grb2, which in
turn binds via its Src homology 3 (SH3) domains to the guanine
nucleotide exchange factor, SOS, leading to Ras activation (4, 5).
Both IRS-1 and Shc can bind to the activated and
tyrosine-phosphorylated insulin receptor through their phosphotyrosine
binding (PTB) domain (also called PID or SAIN domain) (6). The PTB domain is a recently recognized protein module that can serve as an
alternative to the SH2 domain for binding to tyrosine-phosphorylated proteins (6-9). PTB domains that are structurally and functionally distinct from SH2 domains recognize amino acid residues N-terminal (rather than C-terminal) to the phosphotyrosine (pY) (2, 10, 11). In
particular, PTB domains preferentially bind to phosphorylated proteins
at sites containing a NPXpY motif and hydrophobic amino acids N-terminal to this sequence (12-15). Unlike SH2 domains, PTB
domains show very low protein sequence homology. Different PTB domains
exhibit distinct selectivity for residues N-terminal to the
NPXpY-motif. For example, the IRS-1 PTB domain favors
hydrophobic residues at the pY-6 and pY-8 positions and an Ala at pY-1
for high affinity binding (15, 16), whereas the Shc PTB domain requires
a bulky hydrophobic residue at pY-5 (12-14). Recent structural analysis revealed that the two PTB domains are structurally related but
employ two very different mechanisms for recognizing the
phosphotyrosine and the hydrophobic residues N-terminal to the NPXpY
sequence (17-19). Indeed, except for the insulin receptor (6), the PTB domains of Shc and IRS-1 have been shown to interact with a distinct set of growth factor and cytokine receptors. For example, the Shc PTB
domain binds to activated and tyrosine-phosphorylated TrkA, ErbB2,
ErbB3, and epidermal growth factor (EGF) receptors (20-22), whereas
IRS-1 interacts with the tyrosine-phosphorylated interleukin-4 receptor
(IL-4R) via its PTB domain (23, 24).
Studies of thermodynamics of protein-ligand interactions can provide
important insights into the structural and functional relationships of
molecular recognition of the system. In an effort to determine further
the structural and dynamic basis of functional differences in the
molecular mechanisms by which the Shc and IRS-1 PTB domains recognize
tyrosine-phosphorylated peptides, we have studied thermodynamics of
peptide binding of the PTB domains using the isothermal titration
calorimetry (ITC) technique. The phosphopeptides used in this study
were derived from known Shc- and IRS-1-binding sites on growth factor
and cytokine receptors. Results from these studies revealed that the
PTB domains of Shc and IRS-1 appear to bind in a thermodynamically
distinct manner to the NPXpY-containing peptides. The
components of the free energy of the interactions show that the high
affinity binding of the Shc PTB domain to the phosphopeptides is an
overall entropy-driven process. In contrast, recognition of the IRS-1
PTB domain to the IR and IL-4R phosphopeptides is achieved
predominantly by a large enthalpy contribution. By using site-directed
mutagenesis and amino acid substitution, we have further quantified the
relative contribution of the pY-5 and pY-1 residues in phosphopeptide
binding to the PTB domains.
Protein Preparation--
The PTB domain of Shc (residues
17-207) was cloned, expressed, and purified using procedures as
described previously (17, 25). Briefly, the protein was subcloned into
the bacterial expression vector pET15b (Novagen), which introduces a
His tag followed by a thrombin cleavage site at the N terminus of the
recombinant protein. The protein was expressed in Escherichia
coli BL21(DE3) cells, which were induced with 1 mM
isopropyl-1-thio-
The PTB domain of IRS-1 used in this study consists of residues
157-267 of the full-length protein. A slightly larger protein (residues 157-278) was subcloned into pET30b plasmid (Novagen) and
expressed in E. coli BL21(DE3pLysS) cells with an additional Leu-Glu-(His)6 sequence at the C terminus as described
previously (18). The cells were grown overnight in LB media, and the
expression of the protein was induced using 1 mM
isopropyl-1-thio- Peptide Synthesis--
The tyrosine-phosphorylated peptides used
in the experiments reported here were synthesized by the Protein Core
Facility at the Mount Sinai School of Medicine, using an Fmoc-based
strategy. Phosphotyrosine was incorporated using the reagent
Fmoc-Tyr(PO3H2) with HBTU/HOAt activation.
Analysis of the purified peptides by analytical high pressure liquid
chromatography demonstrated homogeneity.
Isothermal Titration Calorimetry (ITC)
Analysis--
Calorimetric measurements were performed with an Omega
instrument (Microcal, Northampton, MA) (26). All experiments were carried out at 25 °C in a 50 mM Tris-HCl buffer of pH
8.0 containing 200 mM NaCl, 5 mM
Each titration experiment consisted of 25 10-µl injections of a
peptide into the calorimetric cell containing 1.34 ml of a protein
solution. A 250-s period was allowed between each injection, and there
was an initial 60-s delay at the start of the experiment. Reaction
enthalpies were also measured for injection of buffer into the protein
and the phosphopeptide into the buffer. In each case, the measured
enthalpies were found to be negligible compared with the enthalpy of
the binding of the phosphopeptide to the PTB domains. The mean of the
enthalpy of injection of buffer into the protein was subtracted from
raw titration data prior to curve fitting. The peptide concentration
was determined gravimetrically, whereas the protein concentration was
measured using the Lowry method. Titration curves were fit to an
in-built function by a non-linear least squares method using the ORIGIN
software (Microcal, Northampton, MA). This function is based upon the
binding of a ligand to a macromolecule (26) and contains n
(reaction stoichiometry), KD (dissociation
constant), and NMR Spectroscopy--
All NMR spectra were acquired at 30 °C
on a Bruker DRX-500 NMR spectrometer. Uniformly 15N-labeled
proteins of the IRS-1 PTB domain were prepared for the NMR experiments
by growing bacteria that overexpress the PTB domain in an M9 minimal
medium containing 15NH4Cl as the sole nitrogen
source. The NMR samples of wild-type and the Met-257 Phosphopeptide Binding by the Shc PTB Domain--
We used the same
TrkA receptor peptide (HIIENPQpYFSDA) in the isothermal titration
calorimetry studies as the one used in our recent structural analysis
of the Shc PTB domain-TrkA phosphopeptide complex by NMR (17). From a
peptide titration experiment using ITC, one can obtain thermodynamic
information of the binding process (29, 30). The parameters include
binding affinity, binding stoichiometry, enthalpy of binding
(
To determine how the Shc PTB domain interacts thermodynamically with
other NPXpY-containing phosphopeptides, we measured
thermodynamic parameters of the Shc PTB domain binding to
tyrosine-phosphorylated peptides derived from EGF, ErbB3, and insulin
receptors (Table I). The results indicated that while the enthalpy of
binding (either exothermic or endothermic reaction) is
phosphopeptide-specific, change of entropy
(T
To study further the thermodynamics of the PTB domain of Shc binding to
tyrosine-phosphorylated peptides, we conducted ITC measurements using a
phosphopeptide derived from IL-4 receptor (pY497). This IL-4R
phosphopeptide is not a biological ligand for the Shc PTB domain as Shc
has not been linked to IL-4R signaling. On the other hand, the IL-4R
peptide represents a biologically relevant binding site for the IRS-1
PTB domain (23, 24). The ITC results showed that the PTB domain of Shc
binds to the IL-4R peptide much weaker than to those phosphopeptides
from the known Shc binding sites (Table I). Interestingly, the Shc
binding of the IL-4R peptide is also dictated by a large favorable
entropic contribution. Taken together, our ITC results suggest that
under the conditions of our study, binding of the Shc PTB domain to the
NPXpY-containing phosphopeptides of the TrkA, EGF, ErbB3, and insulin receptors appears to be an overall entropy-driven process.
Phosphopeptide Binding of the IRS-1 PTB Domain--
We performed
an ITC titration using a phosphopeptide from the IL-4R (pY497)
(LVIAGNPApYRS) which is a known binding site for IRS-1 (23, 24). As
shown in Fig. 1B, binding of the IRS-1 PTB domain to the
IL-4R peptide (see Table II) is
exothermic (
Binding of the IRS-1 PTB domain to the IR-pY960 peptide (LYASSNPEpYLS)
also appears to be governed mainly by an enthalpy contribution ( Binding Specificity of the IRS-1 and Shc PTB Domains--
The
major difference in amino acid sequence between the IL-4R and IR
phosphopeptides is the residue at pY-1. To determine the contribution
of the pY-1 residue to binding of the IRS-1 PTB domain, we substituted
the Glu pY-1 in the IR-pY960 peptide by an Ala. The latter amino acid
corresponds to the Ala pY-1 in the IL-4R phosphopeptide. The ITC
measurements showed that this single amino acid substitution led to a
38-fold increase of the binding affinity to 2.32 µM (Fig.
1D and Table II), which is nearly the same as that of the
IL-4R peptide binding to the IRS-1 PTB domain (KD = 1.82 µM). It is interesting to note that this significant increase of the peptide binding affinity results largely from a
reduction of the entropy penalty (T
To investigate the size preference of the favored hydrophobic residue
at pY-1, we substituted the Glu pY-1 by amino acid Ile or Phe in the
IR-pY960 phosphopeptide. The measured thermodynamic parameters showed
that the phosphopeptide containing either Ile or Phe at pY-1 binds to
the IRS-1 PTB domain about 3-4-fold weaker than the IR(A-1)-pY960 but
~10-13-fold stronger than the wild-type phosphopeptide which
contains a Glu at pY-1.
The preference for a small hydrophobic residue at pY-1 by the IRS-1 PTB
domain agrees with our recent NMR structural analysis of the PTB
domain-IL-4R peptide complex (18). The NMR structure revealed that the
Ala pY-1 in the NPApY motif interacts with a hydrophobic binding site
formed by three methionines, i.e. Met-257, Met-260, and
Met-209. To determine the relative contribution of this hydrophobic
site to phosphopeptide binding, we mutated Met-257 of the IRS-1 PTB
domain to Ala for calorimetric studies. A comparison of
1H/15N heteronuclear single quantum coherence
NMR spectra of the mutant Met-257
The requirement of an Ala at pY-1 for high affinity phosphopeptide
binding appears only unique to the IRS-1 PTB domain, because amino acid
residues at this corresponding site are highly variable in the known
Shc PTB domain binding sites in various mitogenic receptors. Instead,
the PTB domain of Shc prefers a large hydrophobic residue at pY-5 as
suggested by our NMR structural analysis of the Shc PTB domain-TrkA
peptide complex (17). The NMR structure revealed that in addition to
the NPXpY motif, the Ile pY-5 of the TrkA peptide interacts
extensively with residues in the hydrophobic core of the PTB domain of
Shc. To examine the contribution of the Ile pY-5 to the high affinity
peptide binding, we substituted Ala for Ile pY-5 in the TrkA peptide.
The ITC measurements showed that this substitution results in a 4-fold
reduction in the peptide binding affinity (Table I). Furthermore, as
compared with the TrkA phosphopeptide, binding of the TrkA(A-5) peptide
showed decreased enthalpy and entropy of binding to 2.78 kcal/mol
(
To test whether the preference of a large hydrophobic residue at pY-5
is specific for the Shc PTB domain, we performed the ITC studies of the
IRS-1 PTB domain binding to the tyrosine-phosphorylated peptides
derived from known Shc PTB domain binding sites on TrkA, EGF, and ErbB3
receptors. The ITC data revealed that these Shc-specific NPXpY-containing peptides showed very weak or no binding to
the IRS-1 PTB domain (Table II). This finding can be explained by the
IRS-1 PTB domain structure (18), which shows there is insufficient space in the PTB domain to accommodate large, hydrophobic side chains
at pY-5 in the phosphopeptides. These results, which agree with
structural and biochemical studies (12, 17), suggest that in addition
to the NPXpY motif, the PTB domains of Shc and IRS-1
recognize differentially specific amino acid residues N-terminal to the
pY in order to achieve their distinct binding specificity.
Solvent Accessibility of the Peptide Residues in the PTB
Domain/Peptide Structures--
To understand further the nature of
hydrophobic interactions between the PTB domains and phosphopeptides,
we calculated solvent-accessible surface area (SASA) for the
phosphopeptide residues in the PTB domain/phosphopeptide structures.
The results indicated that the extent of the solvent accessibility of
the phosphopeptide residues agrees with the degree of their
interactions with the PTB domains (Fig.
3, A and B). For
example, the Ala pY-1 of the IL-4R peptide is nearly completely buried
in the IRS-1 PTB domain-IL-4R peptide complex, as its calculated SASA
in the PTB domain-bound form is about 1% that in a hypothetical
tri-peptide state. This result is in agreement with the importance of
the Ala pY-1 in determining the peptide binding specificity and binding
affinity of the IRS-1 PTB domain.
The SASA analysis of the IRS-1 PTB domain has revealed that Met-257,
Met-260, and Met-209 that are involved in intimate interactions with
the Ala pY-1 are largely buried with SASA less than 20%. Other peptide
residues important for binding to the PTB domain also show low SASA,
such as 9.2% for Asn pY-3 and 16.4% for Ala pY-5. The SASA of the pY
and the hydrophobic residues at pY-6 and pY-7 is 25 and 42-47%,
respectively. These results are consistent with the PTB domain
structure which shows that the binding sites for these peptide residues
are located on the surface of the protein. Furthermore, the SASA
analysis of the Shc PTB domain (Fig. 3B) suggests that the
peptide residues of the pY, Asn pY-3, Ile pY-5, and Phe pY+1 are
involved in extensive interactions with the protein.
In this study we have characterized the thermodynamics of binding
of the PTB domains of Shc and IRS-1 to the NPXpY
motif-containing phosphopeptides. Our ITC results demonstrated that the
binding of IRS-1 PTB domain to tyrosine-phosphorylated IL-4R and IR
peptides is an enthalpy-driven process, whereas the binding of Shc PTB domain to tyrosine-phosphorylated peptides of TrkA, EGF, ErbB3, and
insulin receptors appears to be largely governed by entropic factors.
Since the phosphopeptides used in this study are all derived from known
Shc- and IRS-1-binding sites in cytokine and growth factor receptors,
our thermodynamic results should provide new insights into the dynamic
nature of interactions of the PTB domains with the
tyrosine-phosphorylated receptors.
It should be noted that Mandiyan et al. (31) have recently
reported ITC studies of the Shc PTB domain binding to phosphopeptides from TrkA and EGF receptors. Mandiyan and colleagues (31) reported a
KD of 42 nM, The enthalpy-driven nature of the IRS-1 PTB domain binding to the
tyrosine-phosphorylated peptides is consistent with the structure and
protein dynamics studies of the IRS-1 PTB domain-IL-4R peptide complex
(18). Formation of hydrophobic and electrostatic contacts and hydrogen
bonds between the protein and peptide residues could account for the
largely exothermic reaction ( Phosphopeptide binding of the Shc PTB domain appears thermodynamically
different from that of the IRS-1 PTB domain. The thermodynamic parameters of the Shc PTB domain, however, cannot be readily explained using its three-dimensional structure. The NMR structure of the Shc PTB
domain-TrkA peptide complex showed that the PTB domain interacts with
fewer peptide residues than does the IRS-1 PTB domain, yet Shc exhibits
higher phosphopeptide binding affinity in general. Whereas the
contributions of enthalpy and entropy to the free energy of binding
depend on PTB domain interactions with specific phosphopeptides, the
high affinity peptide binding to the Shc PTB domain is achieved mainly
by a large favorable entropy change. This entropy-driven nature of the
phosphopeptide binding suggests that the binding of the Shc PTB domain
to the NPXpY-containing peptides may involve the burial of
more hydrophobic surface and/or an increase in the conformational
freedom of the protein upon complex formation.
The change of hydrophobic surface area ( The PTB domains of IRS-1 and Shc show very little sequence homology but
share a common structure fold of pleckstrin homology (PH) domains
(17-19, 37). The PH domains have been shown to bind to In summary, we have demonstrated in this study that the two
structurally homologous but functionally distinct PTB domains of Shc
and IRS-1 recognize the NPXpY motif-containing
phosphopeptides in a thermodynamically distinct manner. Results from
the ITC analysis provide new insights into the dynamic nature of the
interactions of the phosphopeptides with the Shc and IRS-1 PTB domains.
Our results support the credence that residues in and around the
NPXpY motif are directly involved in determining the
specificity of phosphopeptide interaction with the Shc and IRS-1 PTB
domains. Such specificity may be the basis of the differences in the
biological functions of these signaling proteins. Furthermore, our
thermodynamic data suggest that, in addition to enthalpic contribution,
entropic factors may also play a pivotal role in the formation of
PTB-peptide complexes involved in cellular signal transduction.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-D-galactopyanoside for 4 h at
37 °C. The His-tagged protein was purified by affinity chromatography on a nickel-NTA column (Qiagen) and was treated with
thrombin to remove the His tag.
-D-galactopyanoside at 25 °C for
6 h. The cells were then disrupted using a French press. The
His-tagged protein was purified by a nickel-NTA column. Subsequent
cleavage of this protein with thrombin at a natural cleavage site
(267-268) removed the C-terminal His-tag and the extra amino acids to
give the PTB domain of IRS-1 (residues 157-267). The IRS-1 PTB mutant
Met- 257
Ala was prepared as described previously, using the
Chameleon Double-Stranded, Site-directed Mutagenesis Kit (Stratagene
Cloning Systems, La Jolla, CA), and the template plasmid used in the
mutagenesis was pET30b-IRS1 (18). Expression and purification of the
mutant IRS-1 PTB domain was accomplished as described for the wild-type protein.
-mercaptoethanol, and 1 mM EDTA. This condition was
optimal for protein stability of the PTB domains of Shc and IRS-1, as
there was no sign of significant protein aggregation for up to 0.5-1
mM protein concentration as determined by NMR spectroscopy.
Both the PTB domains and the phosphopeptides were dissolved in the same
buffer. The concentrations of protein and phosphopeptide were typically
of 30-300 µM and 1-2 mM, respectively. To
optimize the ITC measurements, the c value
(c = [PTB domain]/KD) was
controlled in the range of 10-200 for all the ITC experiments, except
for the weak binding of the IRS-1 PTB domain to the phosphopeptides of
IR-pY960 (KD = 87.07 ± 3.84 µM)
and TrkA-pY490 (KD = 678 ± 96.53 µM) (Table I).
H (reaction enthalpy) as the variable
parameters. These parameters can thus be directly determined from curve
fitting. From the values of KD and
H,
the free energy (
G) and entropy change (
S)
upon peptide binding can be calculated using the relationship:
RT ln(1/KD) =
G =
H
T
S, where R
is the universal molar gas constant and T is the absolute temperature.
Ala mutant of
the IRS-1 PTB domain were prepared at a concentration of 0.5 mM in 50 mM Tris-d11/HCl
buffer of pH 6.5, containing 50 mM NaCl and 5 mM dithiothreitol-d10 in 90% H2O, 10% 2H2O. Two-dimensional
1H/15N heteronuclear single quantum coherence
spectra were acquired with 96 and 1024 complex points in
1 and
2, respectively. The NMR spectra
were processed and analyzed using the NMRPipe (27) and NMRView (28) programs.
RESULTS
H), and free energy change (
G) by a
nonlinear fit of the binding isotherm, as well as entropy of binding
(
S) from a difference between the free-energy change and
the enthalpy of binding. A representative calorimetric isotherm and the
corresponding titration curve of the Shc PTB domain binding to the TrkA
peptide (Fig. 1A) show that a
heat absorbance is associated with the peptide
binding, indicating that the interaction is endothermic at 25 °C
(
H = 3.63 kcal/mol). The heat absorbance upon
addition of the phosphopeptide to the protein solution underwent a
sharp change at 1:1 molar ratio of the protein to peptide, suggesting that the Shc PTB domain binding to the TrkA peptide is very tight, and
the stoichiometry of this interaction is 1:1. By using these ITC data,
we calculated a dissociation constant (KD) to be 190 nM (Table I). Furthermore,
the thermodynamic titration data revealed that the high affinity
binding of the Shc PTB domain to the TrkA peptide is achieved by an
overall entropy-driven process as the free energy of binding
(
G =
9.12 kcal/mol) results predominantly from a
large favorable entropic contribution (T
S = 12.75 kcal/mol).
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Fig. 1.
Isothermal titration calorimetric data for
binding of the Shc PTB domain to TrkA-pY490 (A) and
the IRS-1 PTB domain to tyrosine-phosphorylated peptides of IL4R-pY497
(B), IR-pY960 (C), and IR(A-1)-pY960
(D). The solid lines show the fit of
the data to a function based on the binding of a ligand to a
macromolecule using the software ORIGIN (26).
Thermodynamic parameters obtained for binding of the Shc PTB domain to
NPXpY-containing phosphopeptides at pH 8.0 and 25 °C
H
were calculated directly from the curve fitting of the titration data
to a function based on the binding of a ligand to a macromolecule (22),
using ORIGIN. In this fitting procedure, the values for
KB,
H, and n (reaction
stoichiometry) were all allowed to float. The mean value for
n was found to be 1 ± 0.1. Errors quoted for
KD and
H are standard deviations from
the three ITC experiments, whereas errors on
T
S and
G are propagated errors.
S) always favors the binding. Moreover, this large favorable entropic contribution appears to be the major determinant for the high affinity of the Shc PTB domain binding to the
phosphopeptides. This observation is consistent not only with the
phosphopeptides that contain the consensus sequence of
XNPXpY (where
pY-5 is a hydrophobic
residue) known for the high affinity binding to the Shc PTB domain but
also with the IR phosphopeptide that contains large hydrophobic
residues at pY-6 to pY-8 instead of pY-5.
H =
9.43 kcal/mol) and involves an
unfavorable change of entropy (T
S =
1.63 kcal/mol). Curve fitting of the ITC data gave a dissociation constant KD of 1.82 µM for this PTB
domain-peptide complex. Thus, the IL-4R peptide interaction with the
IRS-1 PTB domain is enthalpy-driven, which is in sharp contrast to the
entropy-driven binding of the Shc PTB domain.
Thermodynamic parameters obtained for binding of the IRS-1 PTB domain
to NPXpY-containing phosphopeptides at pH 8.0 and 25 °C
H are
standard deviations from the three ITC experiments, whereas errors on
T
S and
G are propagated errors.
H =
10.74 kcal/mol and
T
S =
5.22 kcal/mol). However, binding affinity of the IR-pY960 peptide to the IRS-1 PTB domain
(KD = 87.07 µM) is ~50-fold weaker
than that of the IL4R-pY497 peptide (KD = 1.82 µM) (Fig. 1C, Table II). This marked reduction of the peptide binding affinity correlates with an increased entropy penalty.
S) of ~2
kcal/mol. Furthermore, the non-phosphorylated form of the IR(A-1)-pY960
peptide showed no interaction with the protein as probed by isothermal
titration calorimetry (Table II), indicating that binding of the IRS-1
PTB domain to the NPApY motif is tyrosine
phosphorylation-dependent.
Ala and wild-type IRS-1 PTB
domain suggested that the mutation did not cause any significant
structural perturbations (Fig. 2,
A and B). The binding affinity of this mutant to
IL4R-pY497 and IR(A-1)-pY960 peptides was almost an order of magnitude
lower than that of the wild-type protein (Table II).
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Fig. 2.
Comparison of two-dimensional
1H/15N heteronuclear single quantum coherence
NMR spectra of wild-type (A) and the mutant Met-257
Ala of the IRS-1 PTB domain (B).
H) and 11.16 kcal/mol (T
S),
respectively. Nevertheless, the TrkA(A-5) peptide binding to the Shc
PTB domain is still largely an entropy-driven process.
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Fig. 3.
Analysis of SASA of the phosphopeptide
residues complexed to the PTB domains of IRS-1 (A) and
Shc (B). Calculations of the residue-based SASA
were performed for the averaged energy-minimized NMR structures of the
IRS-1 and Shc PTB domain-phosphopeptide complexes using X-PLOR program
(43). The percentage of the SASA was determined based on the
solvent-exposed surface area of a particular amino acid residue in a
hypothetical tri-peptide form.
DISCUSSION
H of 2.36 kcal/mol, and T
S of 12.44 kcal/mol for the
TrkA (pY490) peptide and a KD of 28 nM,
H of
5.46 kcal/mol, and T
S of
4.84 kcal/mol for the EGF receptor (pY1148) peptide. Our ITC data agree
with their results of the TrkA peptide in general but differ from those
of the EGF receptor peptide (Table I). The discrepancies between the
two studies could be due to differences in the c values
(c = [protein]/KD) used in the ITC
measurements. The c values used in our study for these two
phosphopeptides were between 20 and 150, which are considered to be
optimal for the ITC measurements (26), whereas their c values were in
the range of 320-1070.
H <0) observed in the ITC
measurements. On the other hand, the entropic penalty (T
S <0) in the peptide binding may result
from the following: 1) a decrease of conformational entropy of amino
acid residues that are directly involved in the peptide binding and
become more rigid upon the complex formation; and 2) a reduction of
translational entropy of the protein and peptide molecules upon complex
formation. By using NMR relaxation measurements, we have recently
characterized the dynamics of the backbone amides of the IRS-1 PTB
domain in both the free protein and the protein when complexed to the
IL-4R phosphopeptide (32). The results showed that the motion of
several residues becomes restricted after ligand binding, including a few residues that do not make direct contacts with the peptide. Such
changes in the motional properties of these residues upon ligand
binding could contribute to change of the conformational entropy of the
system. It is interesting to note that the observed change of entropy
(
S = approximately
5.5 cal/mol/K) in the IRS-1 PTB
domain binding to the IL-4R peptide appears to agree with the recently
reported translational entropy cost for protein-protein association
(
S =
5 ± 4 cal/mol/K) that was measured
using the dimeric subtilisin inhibitor from Streptomyces
(33). Therefore, these results suggest that the observed entropic
penalty of the IRS-1 PTB domain binding to the IL-4R peptide may
largely result from the change of the translational entropy of
the system.
A) of a protein
upon binding to its ligand can be estimated by measurement of the change of heat capacity (
C), which can be determined from
enthalpy of binding (
H) measured at different
temperatures (34, 35). By using the ITC technique, we measured the
C of the IRS-1 PTB domain binding to the IR(A-1)-pY960
peptide to be about
240 cal/mol/K (data not shown). Interestingly,
this value is very similar to the values reported by Mandiyan et
al. (31) for the Shc PTB domain binding to the TrkA-pY490 (
207
cal/mol/K) and EGF receptor -pY1148 (
185 cal/mol/K). It should be
pointed out that an accurate analysis of the change of hydrophobic
surface area (
A) using the
C values may be
difficult (36). Nevertheless, these similar
C values may
indicate that the change of hydrophobic surface area (
A)
upon phosphopeptide binding could be similar for the PTB domains of Shc
and IRS-1. Therefore, the favorable entropic contributions observed in
the Shc PTB domain binding to the phosphopeptides likely result from an
increase in the conformational freedom of the protein upon complex
formation. Indeed, more recently our NMR structural studies of the Shc
PTB domain of the free form suggest that the protein undergoes
conformational change upon binding to the tyrosine-phosphorylated
peptides.2 Further work is
required to determine how the conformational rearrangement of the Shc
PTB domain contributes to the overall entropy-driven nature of its high
affinity binding to phosphopeptides.
subunits
of G proteins and acidic phospholipids and are important for localizing
proteins that contain the PH domain to the membrane surface (37, 38).
Indeed, we have recently shown that the PTB domain of Shc can bind to
the tyrosine-phosphorylated proteins and receptors and to
phospholipids, and both events are essential for tyrosine
phosphorylation of Shc following receptor activation (39). More
recently, it has been reported that "PTB domain-like" proteins
contained in other signaling molecules can also mediate protein-protein
interactions in a phosphotyrosine-independent manner. These include
signaling proteins of X11, FE65, Numb, and SNT (40-42). Taken
together, these results strongly suggest that this conserved PH
domain-structure fold can be utilized for diverse functions for
protein-protein or protein-lipid interactions via distinct molecular mechanisms.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen W. Fesik for the cDNA plasmids of the PTB domains of Shc and IRS-1. We are grateful to Drs. Christophe Dhalluin and Carlos Escalante for technical advice on the isothermal titration calorimetry experiments, Dr. Imre Wolf for phosphopeptide synthesis, and Dr. Diomedes E. Logothetis for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by discretionary funds from the Mount Sinai School of Medicine (to M.-M. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Wellcome International Prize Travelling Research Fellowship.
§ To whom correspondence should be addressed: Structural Biology Program, Dept. of Physiology and Biophysics, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1677, New York, NY 10029-6574. Tel.: 212-824-8224; Fax: 212-849-2456; E-mail: zhoum{at}inka.mssm.edu.
2 A. Farooq and M.-M. Zhou, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IRS-1, insulin receptor substrate 1; IR, insulin receptor; IL-4R, interleukin-4 receptor; ITC, isothermal titration calorimetry; NTA, nitrilotriacetic acid; pY, phosphotyrosine; PTB, phosphotyrosine binding; SASA, solvent-accessible surface area; SH2 Src homology-2, Fmoc, N-(9-fluorenyl)methoxycarbonyl; EGF, epidermal growth factor; PH, pleckstrin homology.
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REFERENCES |
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