(Received for publication, October 20, 1995; and in revised form, December 19, 1995)
From the
The N-terminal 200 amino acids of SHC constitute a unique
phosphotyrosine (Tyr(P)) interaction (PI) domain that shows no
significant sequence similarity to the other Tyr(P)-recognizing module,
the SH2 domain. We describe the thermodynamic parameters characterizing
PI domain binding to various tyrosyl phosphopeptides, using isothermal
titration calorimetry. The PI domain forms 1:1 complexes of similar
affinity with a 12-mer peptide (ISLDNPDpYQQDF) derived from Tyr-1148 of
the epidermal growth factor receptor (EGFR) (K = 28 nM) and an 18-mer (LQGHIIENPQpYFSDACVH)
derived from Tyr-490 of Trk (K
=
42 nM). Binding of the EGFR-derived peptide was largely
enthalpy-driven at 25 °C, while Trk490 peptide binding was
entropy-driven. Based on the change in heat capacity upon binding,
approximately 700 Å
of nonpolar surface was estimated
to be buried upon interaction. Alteration of the Asn or Pro to Ala in
the NPXpY motif of the EGFR Tyr-1148 peptide increased the K
of PI domain interactions to 238 and
370 nM, respectively. Alteration of a Leu at position -5
(with respect to Tyr(P)) in the EGFR peptide to Gly also reduced the
binding affinity (K
= 580
nM). It is proposed that the PI domain recognizes the
1
turn that is found in NPXpY-containing peptides and also
interacts with a larger segment of the peptide than seen for SH2
domains.
SHC, an adapter protein involved in a number of signaling
pathways, consists of an N-terminal region and a C-terminal SH2 ()domain separated by a region rich in glycine and proline
residues. The glycine and proline rich region exhibits sequence
similarity to
1 collagen and contains a tyrosine phosphorylation
site. SHC exists in three different forms of 46, 52, and 66 kDa, which
differ from each other at the N-terminal region. The 46- and 56-kDa
forms are generated either by alternative splicing or translational
initiation, while the origin of the 66-kDa form is not fully
understood(1) . Each form of SHC binds to growth factor
receptors upon stimulation of cells with various growth factors and
rapidly becomes phosphorylated on
tyrosine(2, 3, 4) . SHC is also
phosphorylated on tyrosine in cells transformed by oncogenic kinases
such as v-Src, v-Fps, and Bcr-Abl(5, 6) . One
phosphorylation site, that at tyrosine 317 of SHC, forms a Grb2 binding
site (pYVNV)(5, 7) . Grb2 is an adapter protein
composed entirely of SH2 and SH3 domains(8) . Its SH2 domain
interacts with SHC, thus recruiting the guanine nucleotide exchange
factor, Sos, which is bound to the Grb2 SH3
domains(9, 10, 11, 12, 13) .
Binding of Grb2 to activated growth factor receptors via SHC recruits
Sos to the membrane surface, where it exchanges GDP for GTP on Ras,
leading to its activation. Activated Ras initiates a kinase cascade
that relays the signal from the cell surface to the
nucleus(14) . Phosphorylation of SHC is also observed upon
stimulation of cell-surface receptors for interleukin 2(15) ,
interleukin 3, granulocyte-macrophage colony-stimulating factor, and
Steel factor(16, 17) , as well as the T cell
receptor(18) .
The SH2 domain of SHC binds to proteins that contain phosphotyrosine in the sequence context pYIXL(19, 20) . However, SHC also binds to several signaling targets that lack this recognition sequence. This binding has been shown to be mediated by the N-terminal region of SHC (21, 22, 23, 24) . The N-terminal 200 amino acids of SHC constitute a unique phosphotyrosine interaction domain (PI domain), also termed the phosphotyrosine binding domain (PTB domain; (25) ). The PI domain shows no significant sequence similarity with the SH2 domain. It is postulated to recognize phosphotyrosine in the sequence context NPXpY. Data base searches have identified a number of proteins with regions that show significant sequence similarities with the SHC PI domain(26) . The function of these conserved regions is not yet understood.
In an effort to understand the interaction of phosphopeptides with the PI domain, we have overexpressed and purified the SHC PI domain and studied its binding to various peptides using isothermal titration calorimetry (ITC). The interaction of the PI domain with NPXpY-containing phosphopeptides is one of the highest affinity interactions thus far observed in the growth factor induced intracellular signaling process and shows an exquisite dependence on the sequence of the peptides studied.
Isothermal titration calorimetry involves the
direct measurement of the heat of a reaction, and permits an accurate
determination of the enthalpy (H) associated with the
binding of a ligand to a macromolecule(27, 28) . By
observing the saturation of the potential binding site from sequential
injections, the stoichiometry of the interaction can also be determined
with confidence. By fitting the obtained binding data, the binding
constant K
can be determined, from which the free
energy (
G) and entropy change (
S) upon
ligand binding can be calculated using the relationship shown by .
R is the gas constant, and T is the absolute temperature. These parameters permit more detailed characterization of nature of the binding reaction than is possible from the measurement of binding constants alone(27, 28) .
The PI domain of SHC was expressed as a glutathione S-transferase fusion protein that was subsequently cleaved with thrombin and purified using a series of chromatographic steps. The protein was more than 95% pure as determined by SDS-polyacrylamide gel electrophoresis (data not shown). The SHC PI domain employed for these studies contains amino acids 1-209 of SHC, with 5 additional amino acids at both the N and C termini.
Figure 1: Typical examples of isothermal titration experiments for the binding of the PI domain to the EGFR1148 peptide (A) and the Trk490 peptide (B). The titration involved 16 15-µl injections of phosphopeptide (concentration) into a solution of purified SHC PI domain (30 µM in A, 20 µM in B) in the calorimeter cell. The points correspond to the change in enthalpy per injection plotted against the molar ratio. The data were fit using a nonlinear least square algorithm within the program ORIGIN (27) .
The SHC PI domain also binds to
the nerve growth factor receptor (TrkA), and the site for this binding
has been identified as phosphorylated tyrosine 490(31) . The
Trk490 peptide that we have synthesized contains 18 amino acids
(LQGHIIENPQpYFSDACVH) flanking this tyrosine. ITC studies of PI domain
binding by this peptide show that it is endothermic at 25 °C (Fig. 1B), by contrast with the exothermic reaction of
the EGFR1148 peptide. Binding of the Trk490 peptide is therefore
entropy-driven (S° = 41.7 cal mol
K
), suggesting that it involves the
burial of more hydrophobic surface and/or more significant
conformational rearrangements. Curve fitting of the titration curves
obtained gave a binding constant of 2.36
10
M
or a K
of 42
nM (Table 1), similar to the value observed for binding
of the EGFR1148 peptide.
Figure 2:
Effect of temperature on H for binding of the SHC PI domain ito the EGFR1148 peptide (A) and Trk490 peptide (B). Titrations were performed
as described in Fig. 1at various temperatures. The
concentration of the PI domain was 30 µM (Fig. 2A) and 40 µM (Fig. 2B). The line was fit for values below 25
°C (see text). The resulting
C
°
values for the EGFR1148 peptide and Trk490 peptide were -185 and
-207 cal mol
K
,
respectively.
C
° values for a binding event have
been correlated with the nonpolar surface area buried upon binding
(
A
)(32, 33) .
A
for binding of the peptides to PI domain
can be estimated following this
analysis(33) .
The A
estimated for binding of the
EGFR1148 peptide to the PI domain is 560-780 Å
,
while that estimated for Trk490 peptide binding is 620-860
Å
. It should be noted that the correlation between
nonpolar surface area buried and the measured
C
° has been called into question in other
studies(34) . However, for comparing the binding of different
peptides to the same protein, this analysis appears to be worthwhile.
Binding of both peptides involves the burial of an approximately equal
nonpolar surface area of 700 Å
. The ratio of buried
polar surface area (
A
) to nonpolar surface
area can be approximated as 0.59(32) , indicating that the
total surface area buried (
A
+
A
) upon binding of the EGFR1148 and Trk490
peptides to the SHC PI domain is 1100-1200 Å
.
Alteration
of the NPXpY motif to NAXpY decreased
H of binding from -5.46 ± 0.15 kcal
mol
to -8.39 ± 0.18 kcal
mol
(Table 1). This reflects an increase in
the number of bonds formed and/or a reduction in those broken upon
association of this mutated peptide with the PI domain. The reduction
in the value of
S° (1.3 cal mol
)
suggests that the entropy of the unbound state (free peptide) is
increased by this mutation and/or that the entropy of the complex is
decreased. The affinity itself is reduced by the Pro
Ala change (K
is increased from 28 to 370 nM). The
observed data can be rationalized in the light of structural studies
that indicate that the NPXY motif forms a type I
-turn(35) . These structural studies also showed that a
Pro
Ala mutation disrupts the
turn(35) . The
thermodynamic effects of this mutation in the EGFR1148 peptide are
precisely those that would be anticipated if the structure of the free
peptide were disrupted by the mutation, becoming more ordered upon
binding to the PI domain.
Mutation of NPXpY to APXpY also reduced the binding affinity (Table 1)
and H changed from -5.46 ± 0.15 kcal
mol
in the wild type case to -2.03 ±
0.15 kcal mol
in the Asn-1148
Ala peptide.
Since
S° is increased by this mutation (Table 1), the Asn
Ala mutation is unlikely to lead to
significant disordering of the free peptide, unless the bound
conformation is a greatly less structured than that of wild-type. We
therefore suggest that the Asn side chain is directly involved in
stabilizing the peptide-PI domain complex and that the interactions
responsible for this are lost in the Asn-1148
Ala peptide.
Change of the Leu at -5 to Gly also significantly weakened
binding of the peptide to the PI domain (K = 580 nM).
H for binding of the
Leu-1148
Gly peptide to the PI domain is very similar to that
for the wild type peptide. However, the entropic advantage of wild type
peptide binding (
S° = 16.2 cal
mol
K
) is significantly
reduced in the case of Leu-1148
Gly (
S°
= 7.3 cal mol
K
).
This would be true if the number of hydrophobic residues buried upon
binding of the peptide to the PI domain is reduced. We therefore
suggest that this Leu is involved in hydrophobic interactions with the
PI domain and that these are removed by the mutation.
From studies reported here as well as from others(21, 22, 23, 24, 29, 31) , it is now clear that SHC contains two phosphotyrosine recognition regions: the PI domain (also called the PTB domain; (22) ) at the N terminus and the SH2 domain at the C terminus. The intervening segment between these two domains contains the unique tyrosine phosphorylation site that functions as a binding site for the Grb2 SH2 domain. The SH2 domain of SHC protects phosphotyrosine 1173 of the EGF receptor from dephosphorylation, and the EGFR1173 phosphopeptide was found to compete for SH2 binding to the EGF receptor(20) . Failure of this peptide to bind to the SHC PI domain indicates that the binding specificity of the two domains is different.
The isolated PI domain of SHC protects phosphotyrosine 1148 of the EGF receptor from dephosphorylation(29) . Comparison of the sequence of this site (QISLDNPDpYQQDF) with other receptor kinases that interact with SHC in vivo indicates a common motif of NPXpY(36) . Previous studies have shown that the SH2 domain of SHC does not bind to this sequence(20) . The EGF receptor contains two phosphorylation sites with NPXY sequences, at Tyr-1086 and Tyr-1148(37) . We have recently shown that the SHC PI domain protects only Tyr(P)-1148 from dephosphorylation, and not Tyr(P)-1086(29) . Indeed, the NPXpY sequence alone is not sufficient to form a high affinity binding site for the PI domain. Additional residues are clearly required, since only the 12-mer EGFR1148 peptide (ISLDNPDpYQQDF), and not shorter peptides, could block binding of the PI domain to the EGF receptor. Another phosphopeptide (DNPDpYQQDFFPKEAK), which still contains the NPXpY sequence but lacks 3 amino acids at the N terminus, also failed to bind to the PI domain(29) . These experiments suggest that the SHC PI domain recognizes a larger segment of phosphotyrosine-containing peptides than do SH2 domains. Furthermore, amino acids N-terminal to the phosphotyrosine are critical for binding to the PI domain, while SH2 domains recognize amino acids C-terminal to the phosphotyrosine for high affinity binding(19) . Crystal structures of various SH2 domains with bound peptides have shown that the peptides are bound in extended conformations(38, 39, 40) .
What are the
characteristics of the NPXpY motif that are recognized by the
SHC PI domain? Structural studies of a nine amino acid peptide that
constitutes an internalization sequence in the LDL receptor and include
an NPXY motif (CNPVYQKTT) was shown to form a
reverse turn in solution(35) . The side chains of the Asn and
Tyr were found to be close, with a hydrogen bond between the NH of the
Asn and the backbone CO of the Tyr. Indeed, Asn is a strong turn
promoter, and being followed by Pro is anticipated to form a 1
turn(41) . It can therefore be postulated that the
NPXpY-containing peptides employed in this study assume
similar turn structures. Phosphorylation of the Tyr could alter the
structure or stability of this turn, and the altered form might be that
recognized by the PI domain. Alternatively, the phosphate and the turn
structure may be recognized simultaneously. The inability of the the
8-mer and 10-mer peptides to bind to the PI domain could result in
their inability to adopt the turn structure recognized by the PI
domain.
Analysis based on changes in heat capacity upon peptide
binding to PI domain suggests that around 1100-1200 Å of total surface is buried in the interaction. These values are
larger than the approximately 600 Å
of surface area
shown to be occluded in the crystal structure of the Src SH2 domain
bound to a cognate phosphopeptide(38) . However, our value for
the buried surface area is only very approximate. Correlations of
surface area and
C
° are not always
reliable(34) . Indeed, measurement of
C
° for binding of the Fyn SH2 domain to
the same peptide bound to the Src SH2 domain in the crystallographic
studies suggested a surface area of around 1600
Å.(
)
Nonetheless, all of these values are
in the range observed for buried surface areas in tight
complexes(42) .
Alterations in the peptide sequence and
analysis of their effect on binding to the PI domain shed some light on
the specificity of the interaction. Changing Pro in NPXpY to
Ala reduced the binding affinity (K = 370
nM). Structural studies of mutated NPXY peptides from
the LDL receptor indicate that the Pro
Ala mutation destroys the
1 turn (35) . The effects of this mutation on the
thermodynamics of EGFR1148 peptide binding to the PI domain are
consistent with disruption of such a turn structure and the requirement
for its reformation upon binding.
Mutation of Asn in NPXpY
to Ala also reduced the binding affinity, and we suggest that this is
mainly the result of a loss of interactions involving the Asn side
chain. Structural studies in the LDL receptor peptide indicate that the
turn is destroyed on changing Asn to Ala(35) , which
would not be consistent with our interpretations. This inconsistency
may suggest that the structure adopted by the EGFR1148 peptide is
different from that seen in the LDL receptor and is not greatly
disrupted by this mutation. Indeed, there are several examples where
structures of a similar nature are adopted by sequences that do not
have Asn at this position(43, 44) . Mutation to Gly of
the Leu at the -5 position with respect to Tyr(P) reduces the
affinity of binding. We suggest that this results either from
structural alterations or from the loss of hydrophobic interaction (the
Leu side chain) with the PI domain. This, together with the analysis of
binding by peptides of different lengths, indicates that sequences
N-terminal to Tyr(P) are critical for phosphopeptide binding to the PI
domain.
We conclude by suggesting that the PI domain binds to peptides that contain turn structures, while SH2 domains have been shown to recognize peptides in extended peptide conformations. The precise nature of the interactions awaits the analysis of the three dimensional structure of the PI domain and the PI domain-phosphopeptide complex. It is interesting that none of the mutations analyzed here reduced the affinity by more than 20-fold. Since several of the mutations would be expected to have a profound effect upon the formation of any defined structure in the peptide, these results suggest that the PI domain simultaneously recognizes several characteristics of the peptide. SHC binding to the EGF receptor in vivo may be mediated through the cooperative interaction of both the PI and SH2 domains with their respective binding sites. In receptors like Trk, which lacks the SH2 binding site, binding of SHC through the PI domain may facilitate the recruitment of other phosphotyrosine-containing proteins via its SH2 domain(31) . Thirteen different potential PI domains have been identified so far in GenBank(TM)(26) . However, it is not clear whether these molecules are involved in growth factor signaling processes. It is possible that PI domain recognizes turn structures in general. Phosphorylation of residues in these turns could enhance binding affinity to the PI domain, and this may have been exploited as a mode of controlled interaction in intracellular signaling.
Addendum-The structure of the SHC PI domain complexed
with a Trk490 peptide was recently determined(45) . The
predictions that the PI domain interacting peptides assume turn
conformations and that the hydrophobic amino acid at the -5
position is involved in hydrophobic interactions were confirmed in this
study(45) .