(Received for publication, August 7, 1995; and in revised form, September 13, 1995)
From the
Src homology 2 (SH2) domains recognize
phosphotyrosine-containing sequences, and thereby mediate the
association of specific signaling proteins in response to tyrosine
phosphorylation (Pawson, T., and Schlessinger, J.(1993) Curr. Biol. 3, 434-442). We have shown that specific binding of SH2
domains to tyrosine-phosphorylated sites is determined by sequences
adjacent to the phosphotyrosine. Based on the phosphopeptide
specificity and crystal structures, SH2 domains were classified into
four different groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M.,
Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky,
S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M.
M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C.(1993) Cell 72, 767-778). The
[Medline]
D5 residues of SH2 domains were
predicted to be critical in distinguishing these groups (Songyang, Z.,
Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G.,
King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G.,
R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B.,
and Cantley, L. C.(1993) Cell 72, 767-778; Eck, M. J.,
Shoelson, S. E., and Harrison, S. C.(1993) Nature 362,
87-91). We report here that replacing the aliphatic residues at
the
D5 positions of two Group III SH2 domains (phosphoinositide
3-kinase N-terminal SH2 domain and phospholipase C-
C-terminal SH2
domain) with Tyr (as found in Group I SH2 domains) results in a switch
in phosphopeptide selectivity, consistent with the specificities of
Group I SH2 domains. These results establish the importance of the
D5 residue in determining specificities of SH2 domains.
Stimulation of cellular responses by growth factors and
cytokines is often accomplished by the activation of protein-tyrosine
kinases(1, 6) . One of the major consequences of
tyrosine phosphorylation is to induce a specific set of protein-protein
interactions, and thereby initiate a series of intracellular signaling
cascades. The SH2 domains of cytosolic signaling proteins
mediate the assembly of such complexes by binding to phosphotyrosine
moieties within specific sequence
contexts(1, 6, 7) . Therefore, in order to
understand signal transduction by protein-tyrosine kinases, it is
important to decode the mechanisms by which SH2 domains achieve
specificity in their recognition of phosphotyrosine sites.
The
specificity of SH2 domains was first systematically studied using a
degenerate phosphopeptide library(2, 3) .
Subsequently, crystal structures of SH2 domains complexed with their
high affinity ligands were obtained, providing a structural basis for
phosphopeptide recognition by SH2
domains(4, 5, 8, 9) . From these
studies and related experiments analyzing the in vivo binding
sites of various SH2 domains, it has become evident that 3-6
residues C-terminal of phosphotyrosine dictate the specificity of SH2
domain binding. Importantly, SH2 domains can be divided into four
subgroups on the basis of their specificity and primary
sequences(2) . For example, Group I SH2 domains (SH2 domains of
Src family, Abl, Crk, GRB2, Nck SH2 domains, etc.) select the general
motif Tyr(P)-hydrophilic-hydrophilic-hydrophobic and have an aromatic
amino acid at the D5 position. However, Group III SH2 domains (e.g. SH2 domains of phosphoinositide 3-kinase p85,
phospholipase C-
, and Syp/SHPTP2) select the general motif
Tyr(P)-hydrophobic-X-hydrophobic and have Ile or Cys at the
D5 position (Fig. 1A). In the three-dimensional
structures of Src and Lck SH2 domains, the
D5 Tyr is at the
surface and makes contacts with the side chains of both the pY+1
and pY+3 residues of the bound
phosphopeptide(4, 5) . However, in the
three-dimensional structures of Syp and PLC-
SH2 domains (Group
III), the aliphatic residue at the
D5 position is buried deeper in
the protein, opening a hydrophobic cavity for the pY+1 through
pY+6 residues (8, 9) (Fig. 1B).
To address the importance of the
D5 position, we have investigated
the effect of substituting this residue on the specificity of two Group
III SH2 domains.
Figure 1:
Sequence
alignment and NMR structure of SH2 domains. A, sequence
alignment of Src, p85 N-terminal, and PLC- C-terminal SH2 domains.
Amino acids involved in phosphopeptide recognition are shaded. Residues
whose side chains are shown in B, are indicated on the top. B, position of Cys
D5 within the phosphopeptide binding
site of the PLC-
C-terminal SH2 domain. The nuclear magnetic
resonance-determined structure of the PLC-
C-terminal SH2 domain,
displayed as a blue ribbon, bound to the phosphopeptide
Asp-Tyr(P)-Ile-Ile-Pro (in yellow) is illustrated(8) .
The Cys
D5 is in red, and other residues forming the
binding pocket for the pY+1, +2, and +3 positions of the
peptide are in green.
Surface plasmon resonance
analysis was carried out using a Biacore apparatus (Pharmacia
Biosensor) as described previously(11) . The
phosphotyrosine-containing peptides were immobilized to a biosensor
chip through injection of a 0.5 mM solution of the
phosphopeptide, in 50 mM HEPES, pH 7.5, and 2 M NaCl,
across the chip surface previously activated following procedures
outlined by the manufacturer. Injection of anti-phosphotyrosine
antibody was used to confirm that successful immobilization of the
peptide was achieved. To obtain K values for
GST-SH2 domain binding to various phosphopeptides, solutions (100
µl) containing varying concentrations of GST-SH2 domain fusion
protein in 50 mM sodium phosphate, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, and 2 mM dithiothreitol, were
injected across a surface containing the immobilized phosphopeptide.
The amount of bound GST-SH2 domain was estimated from the
steady-state surface plasmon resonance signal (RU). The data
were analyzed using the equation RU = C - K
(RU/[GST-SH2]), where RU is the
steady-state signal, [GST-SH2] is the concentration of SH2
domain, and C is a constant. Prior to each run the
phosphopeptide surface of the Biosensor chip was regenerated using 2 M guanidinium HCl. Peptide inhibition experiments were
performed using solutions (100 µl) containing 1 µM GST-SH2 domain fusion protein and the indicated concentrations of
soluble phosphopeptide that were injected across a Biosensor surface to
which the phosphopeptide DNDpYEEFLPDPK was immobilized. The amount of
bound GST-SH2 domain was estimated from the surface plasmon resonance
signal at a fixed time following the end of the injection and the
percentage bound, relative to injection of GST-SH2 domain alone,
calculated.
Figure 2:
A comparison of phosphopeptide
specificity of the wild-type and D5 mutant (C715Y) PLC-
CSH2
domains. Results are from the fifth, sixth, and seventh cycle of the
sequence (i.e. the pY+1, +2, and +3 positions).
The value represents the ratio of the amount of each amino acid eluted
from GST-SH2 bead columns divided by that of the control GST bead
columns at the same cycle. Amino acids are presented in single-letter code.
Conversion of the D5 Ile of
the p85 NSH2 domain to Tyr resulted in a domain with similar
selectivity to the mutant PLC-
CSH2 domain (C715Y). While the
wild-type p85 NSH2 domain selected for Tyr(P)-Met-X-Met, the
I383Y mutant selected for Tyr(P)-Glu-Gln-Phe (Table 1).
To
further test the specificity of the mutant PLC- CSH2 domain and
the prediction of the peptide library, two additional peptides were
constructed. The pYIIF (DNDpYIIFLPDPK) peptide has a Phe rather than a
Pro at the pY+3 position, which is predicted to increase the
affinity for the mutant PLC-
(compared to pYIIP) but lower the
affinity for the wild-type protein (Fig. 2). The results in Table 2(part B) are in agreement with this prediction. Similarly,
substituting a Glu at the pY+1 position (pYEIP: DNDpYEIPLPDPK)
increases the affinity for the mutant protein (compared to pYIIP) and
lowers the affinity for the wild-type protein, as predicted from the
results in Fig. 2.
One result in Table 2(part B) is
not predicted by the peptide library result. The pYEEF peptide had a
significantly higher affinity than the pYEIP peptide for the wild-type
PLC- CSH2 domain. One possible explanation for this result is that
the Glu at pY+1 forces the peptide out of the normal binding
groove such that favorable interactions for the Ile and Pro at the
pY+2 and pY+3 positions are precluded. If the peptide is on
the surface of the SH2 domain then the more hydrophilic Glu at
pY+2 would be selected (as is the case for Group I SH2 domains
such as Src and Lck).
In summary, we have demonstrated here that the
D5 residue is crucial in determining the binding specificity of
SH2 domains. We were able to switch the specificity of two group III
SH2 domains (p85 NSH2 and PLC-
CSH2 domains) to that of Group I
SH2 domains by substituting their wild-type
D5 residues (Ile or
Cys) with tyrosine found at the
D5 positions of Group I SH2
domains. A comparison of three-dimensional structures of Group I (Src
and Lck) and Group III SH2 domains (Syp and PLC-
) with bound
ligands indicates a major difference in ligand
binding(4, 5, 8, 9) . In the
structures of Src and Lck SH2 domains complexed with pYEEI peptide,
only the phosphotyrosine and three residues (Glu-Glu-Ile) immediately
C-terminal to it make specific contacts with the SH2 domain backbone.
Among these three residues, pY+3 Ile plugs into a hydrophobic
binding pocket while pY+1 and +2 Glu residues form salt
bridges and hydrogen-bonds with amino acids on the surfaces of the SH2
domain. The Syp and PLC-
SH2 domain structures, however, show a
rather ``open'' groove configuration. The aliphatic residues
at the
D5 position are buried in the protein such that a cavity is
opened between the phosphotyrosine pocket and the pY+3 binding
site. This allows the accommodation of hydrophobic amino acids at the
pY+1 position (Fig. 1B). In addition, the distance
between the BG and EF loops is wider than that of Src and Lck SH2
domains providing additional binding pockets for pY+4 and
pY+5 residues. Therefore, up to five residues of the bound
peptides are embedded in a hydrophobic channel.
The prediction of
our model is that substituting a more bulky aromatic residue (Tyr) at
the D5 position, as found in Group I SH2 domains, will disrupt the
hydrophobic cavity and force the pY+1 and pY+2 residues to
lie on the surface, as found for Group I SH2 domains. The selection of
the
D5 Tyr mutants of PLC-
CSH2 and p85 NSH2 for Glu residues
(rather than hydrophobic residues as in the wild-type proteins) at
pY+1 and pY+2 are consistent with this model. The results in Table 2argue that substituting Tyr at
D5 did not create new
contacts for the phosphopeptide but rather eliminated the selection for
hydrophobic amino acids at the pY+1 and pY+2 positions. This
conclusion is supported by the observation that the optimal
phosphopeptide for the PLC-
CSH2 mutant (pYEEF) had a slightly
higher affinity for wild-type PLC-
CSH2 domain than for the
mutant. As discussed above, we suspect that this is due to the ability
of the pYEEF peptide to bind in a conformation in which the glutamate
residues at pY+1 and pY+2 are on the surface of the SH2
domains. However, this peptide does not have as high affinity as the
pYIIP peptide for wild-type PLC-
CSH2.
The data presented here, together with our earlier work involving mutations in the Src SH2 domain (10) and p85 NSH2 domain(12) , indicate the potential to modulate the specificity of SH2 domains by making substitutions at sites predicted to bind side chains of associated phosphopeptides. This approach deepens our understanding of SH2-binding specificity. Furthermore, it should be feasible in the nearest future to design novel SH2 domains with pharmacological applications.