nSH3 and cSH3 domains pose distinct requirements for binding to PXψPXR motifs within Sos1
To further characterize the Grb2-Sos1 interaction in biophysical terms, we measured the binding of nSH3 and cSH3 domains to peptides derived from S1–S4 sites containing the PXψPXR motifs using ITC. and show representative data obtained upon conducting such measurements, while corresponding thermodynamic parameters are reported in . It is clearly evident from our data that the binding of both SH3 domains at S1–S4 sites within Sos1 is exclusively under enthalpic control and, with the exception of binding of cSH3 domain to S1 site as noted previously (22
), binding at all other sites is also accompanied by entropic penalty as is often observed in enthalpically-driven biological processes. This observation is consistent with the notion that enthalpy drives SH3-ligand interactions in general, while entropic changes provide unfavorable contributions to the free energy (19
). That enthalpy drives the SH3-peptide interaction implies that both hydrophobic forces and electrostatic interactions play a key role in the assembly of this signaling complex. However, the dominance of entropic penalty accompanying the SH3-peptide interaction is most likely attributable to the greater loss of degrees of motions available to these molecules when free in solution but becoming more compact upon binding. Close scrutiny of data presented in indicates that both SH3 domains have a preference for binding to S1 site. However, while the nSH3 domain binds to S1–S4 sites with very similar affinities that extend over nearly three folds, the cSH3 domain not only binds to S1 site in an exclusive manner but also with an affinity that is weaker that that observed for the binding of nSH3 domain to any of these sites. The binding of cSH3 domain to S2, S3 and S4 sites occurs with affinities in the millimolar range, implying that the interaction of cSH3 domain with these sites is not likely to be physiologically relevant. It should be noted here that the affinities observed for the binding of nSH3 domain to S1–S4 sites and cSH3 domain to S1 site within Sos1 are consistent with the binding of SH3 domains to their cognate ligands with affinities typically in the 10–100µM range (17
). However, in some cases, SH3 domains have also been shown to bind to their ligands with affinities in the sub-µM range (9
). Given the ubiquitous nature of SH3 domains within the mammalian proteome, it is believed that the rather weak SH3-ligand interactions are central to the temporal and spatial regulation of signaling pathways that constitute the bedrock of cellular communication.
Figure 2 ITC analysis for the binding of nSH3 domain of Grb2 to Sos1-derived peptides S1 (a), S2 (b), S3 (c) and S4 (d). The solid lines show the fit of data to a one-site model based on the binding of a ligand to a macromolecule as incorporated in the Microcal (more ...)
Figure 3 ITC analysis for the binding of cSH3 domain of Grb2 to Sos1-derived peptides S1 (a), S2 (b), S3 (c) and S4 (d). The solid lines show the fit of data to a one-site model based on the binding of a ligand to a macromolecule as incorporated in the Microcal (more ...)
Experimentally determined thermodynamic parameters for the binding of wildtype nSH3 and cSH3 domains of Grb2 to various Sos1 peptides obtained from ITC measurements at 25°C and pH 8.0
The differential behavior of SH3 domains toward their putative sites within Sos1 must correlate with amino acids residing within or flanking the PXψPXR motifs. In view of the fact that one or more basic residues flanking consensus proline-rich motifs provide an additional level of modulating their binding affinity and specificity toward SH3 domains, we reasoned that the differential behavior of nSH3 and cSH3 domains toward S1–S4 sites might be attributable to the presence of arginine residues at positions +6 (R+6) and +7 (R+7) within the S1 site but absent in other sites (). Supporting this credence is the observation that the affinity of S1 peptide lacking all three arginine residues at positions +5, +6 and +7 (S1_AAA) is significantly compromised toward both SH3 domains (). To directly test that the high affinity and specific binding of cSH3 domain to S1 site is largely afforded by the presence of R+6 and R+7, we measured the binding of S1 peptide containing alanine substitutions at these two arginine positions (S1_AA) to SH3 domains. Our results reveal that while the binding of S1_AA peptide to nSH3 domain is only slightly weaker than the S1 peptide, it binds to the cSH3 domain with an affinity that is over an order of magnitude weaker relative to S1 peptide (). This salient observation unequivocally demonstrates that R+6 and R+7 flanking the C-terminal of the PXψPXR motif at S1 site are absolutely required for the binding of cSH3 domain within the physiological context. In a leap of further curiosity, we also analyzed the extent to which R+5, R+6 and R+7 within the S1 site were individually critical for binding to SH3 domains (). The substitution of R+5 to alanine within S1 peptide (S1_R+5A) reduced the binding affinities to both the nSH3 and cSH3 domains by an order of magnitude, implying that R+5 is critical for the binding of both SH3 domains to S1 site. In the case of substitution of R+6 to alanine within S1 peptide (S1_R+6A), the binding to nSH3 domain was virtually indistinguishable from that observed for the S1 peptide but underwent a close to three-fold reduction in affinity upon binding to the cSH3 domain relative to S1 peptide.
On the basis of these observations, the most straightforward conclusion is that while R+6 is important for the binding of cSH3 domain to S1 site, it is redundant in the case of nSH3 domain. Finally, in the case of the S1 peptide containing an alanine substitution at R+7 (S1_R+7A), the binding to both SH3 domains suffers between two-to-three fold reduction, implying that R+7 plays a non-redundant role in the binding of both the nSH3 and cSH3 domains to S1 site. Taken together, our data demonstrate that while all three R+5, R+6 and R+7 arginine residues are critical for the binding of cSH3 domain to S1 site, the nSH3 domain only strictly requires R+5 and R+7.
D33-R+7 salt bridge enhances the binding of nSH3 domain to S1 site
Our data presented above suggest strongly that both R+5 and R+7 arginine residues within S1 site are required for optimal binding of the nSH3 domain. The simplest mechanism by which these basic residues in the S1 peptide are likely to contribute to the free energy of binding is by virtue of their ability to engage in the formation of ion pairs or salt bridges with specific acidic residues in the nSH3 domain. It should be noted that such charged residues do not exist in solitude but in a symbiotic relationship with counterions when free in solution. Upon the formation of ion pairs with oppositely charged residues, usually through intermolecular association, the release of counterions into solution contributes to the free energy of binding through entropic gain. We have previously shown that R+5 ion pairs with D15 in the nSH3 domain (22
). But which acidic residue in the nSH3 domain does R+7 ion pair with?
Analysis of available 3D atomic coordinates of nSH3 domain in complex with a Sos1-derived peptide flanking the S1 site reveals that the two most likely suspects for this role could be either E31 or D33 () (18
). Both of these acidic residues are located close to the exit of the hydrophobic groove in the nSH3 domain that accommodates the peptide and lie within stretching distance of R+7. To determine which of these two potential acidic residues is actually responsible for neutralizing the positive charge on R+7, we measured the binding of mutant nSH3 domains containing either an alanine substitution at E31 (nSH3_E31A) or at D33 (nSH3_D33A) to S1 peptide using ITC (). Our data reveal that while nSH3_E31A binds to S1 peptide with an affinity that is very similar to that observed for the binding of nSH3 domain, the binding of nSH3_D33A to S1 peptide occurs with an affinity that is over four-fold weaker relative to nSH3 domain. This finding suggests that it is D33 and not E31 that engages in the formation of a salt bridge with R+7. To further demonstrate that this is so, we also measured the binding of S1_R+6A and S1_R+7A peptides to nSH3_E31A and nSH3_D33A mutant domains (). The fact that the S1_R+6A peptide binds to nSH3_E31A and nSH3_D33A mutant domains with affinities that are similar to those observed for the binding of S1 peptide implies that R+6 is not involved in the formation of an ion pair as noted above. In contrast, the S1_R+7A peptide binds to nSH3_E31A with an affinity that is nearly two-fold weaker than that observed for the binding of S1 peptide, implying that this reduction in affinity is most likely due to the disruption of an ion pair involving R+7 with an acidic residue in the nSH3_E31A domain other than E31. This mysterious acidic residue involved in the formation of an ion pair with R+7 is thus likely to be D33 due to the fact that the nSH3_D33A mutant domain binds with very similar affinities to both the S1 and S1_R+7A peptides.
Experimentally determined thermodynamic parameters for the binding of mutant cSH3 domains of Grb2 to various Sos1 peptides obtained from ITC measurements at 25°C and pH 8.0
In light of these considerations and data reported previously (22
), the binding of nSH3 domain to S1 site in Sos1 is driven by the formation of D15-R+5 and D33-R+7 salt bridges. However, the D33-R+7 salt bridge is not critical allowing the nSH3 domain to also bind to S2, S3 and S4 sites in Sos1 that are devoid of R+7.
D187-R+6 and D190-R+7 salt bridges cooperate to drive the binding of cSH3 domain to S1 site
While the nSH3 domain only requires the ion pairing of R+5 and R+7 in binding to S1 site, R+6 also appears to be necessary for the binding of cSH3 domain (). In a previous study, we demonstrated that R+5 forms a salt bridge with E171 in the cSH3 domain (22
). Here we set out to identify the potential partners of R+6 and R+7 in the cSH3 domain. The most likely candidates for this role are D187 and D190 within the cSH3 domain in agreement with structure-based sequence alignment with nSH3 domain (). To test this hypothesis, we introduced alanine substitutions within the cSH3 domain at D187 (cSH3_D187A) and D190 (cSH3_D190A) and measured the binding of these mutant domains to S1 peptide (). The fact that both cSH3_D187A and cSH3_D190A mutant domains bind to S1 peptide with about two-fold reduction in binding affinity relative to the wildtype cSH3 domain suggests strongly that both D187 and D190 are involved in forming ion pairs with R+6 and R+7.
Experimentally determined thermodynamic parameters for the binding of mutant cSH3 domains of Grb2 to various Sos1 peptides obtained from ITC measurements at 25°C and pH 8.0
To identify the specific residues involved in the formation of these ion pairs, we also measured the binding of cSH3_D187A and cSH3_D190A mutant domains to S1_R+6A and S1_R+7A peptides (). The fact that the cSH3_D187A binds to the S1_R+7A peptide with an affinity that is over four-fold weaker than that observed for its binding to the S1_R+6A peptide implies that it is R+6 and not R+7 that must ion pair with D187. This observation is further corroborated upon the binding of cSH3_D190A to S1_R+6A peptide with an affinity that is nearly two-fold weaker than that observed for its binding to S1_R+7A, implying that it is R+7 and not R+6 that forms an ion pair with D190. Taking these observations together in light of the previous data (22
), the binding of cSH3 domain to S1 site in Sos1 is driven by the formation of E171-R+5, D187-R+6 and D190-R+7 salt bridges. Given that all of these three salt bridges are critical for the binding of nSH3 domain to S1 site implies that it cannot bind to S2, S3 and S4 sites in Sos1 devoid of R+6 and R+7.
Arginine residues within S1 site contribute differentially to the free energy of binding of nSH3 and cSH3 domains
The foregoing argument suggests strongly that the arginine residues R+5, R+6 and R+7 within the S1 site play a key role in driving the Grb2-Sos1 interaction. As illustrated in , it can be further seen that these arginine residues contribute between 30–40% of the total free energy available to drive the binding of nSH3 and cSH3 domains to S1 site in a distinct manner. Thus, while R+5 is the major contributor to the overall free energy of binding of nSH3 domain to S1 site, R+7 and R+6 play lesser roles, with the contribution of the latter falling within the experimental error of the measurements being reported here. In contrast, while R+5 also heavily contributes to the overall free energy of binding of cSH3 domain to S1 site, R+6 and R+7 clearly play significant roles and, together, the energetic contributions of R+6 and R+7 almost match those of R+5. The specificity of nSH3 and cSH3 domains toward Sos1 thus seems to be largely due to the distinct contributions of arginine residues R+5, R+6 and R+7 to the free energy of binding. In other words, electrostatic interactions in lieu of hydrophobic contacts appear to define the distinguishing features of the binding of nSH3 and cSH3 domains to Sos1. This is further corroborated by the fact that the S1_AAA peptide, in which all three arginine residues are substituted by alanine, binds to both nSH3 and cSH3 domains with very similar affinities, albeit in a non-physiologically-relevant manner ().
Figure 4 Comparison of energetic contributions of arginine residues R+5, R+6 and R+7 within the S1 site to the free energy of binding of nSH3 (shaded columns) and cSH3 (unshaded columns) domains. (a) Energetic contributions relative to the total free energy (ΔG (more ...)
D15 underscores the high-affinity binding of nSH3 domain to PXψPXR motifs within Sos1
Our data presented above provide the rationale underlying the binding of nSH3 domain to all four S1–S4 sites with Sos1, while the cSH3 domain can only bind to S1 site. But what features within the nSH3 domain enable it to only strictly require R+5, while the cSH3 domain has an obligate requirement of all three R+5, R+6 and R+7 arginine residues within the putative binding sites in Sos1? Given that the binding affinity of S1_R+5A peptide to nSH3 domain is reduced by more than an order of magnitude relative to S1 peptide (), we reasoned that the energetic contribution resulting from the formation of D15-R+5 salt bridge may be largely responsible for driving the binding of nSH3 domain to all four S1–S4 sites in contrast to cSH3 domain that can only bind to S1 site. This seems logical in light of the fact that the cSH3 domain contains a glycine (G173) instead of an acidic residue, let alone an aspartate, at the structurally equivalent position occupied by D15 in the nSH3 domain (). The cSH3 domain rather relies on a neighboring glutamate (E171) to engage in the formation of a salt bridge with R+5. Because D15 and E171 are structurally and chemically non-equivalent, it is very likely that such differences also translate into D15-R+5 and E171-R+5 salt bridges being energetically non-equivalent and thus may underlie the distinct behavior of nSH3 and cSH3 domains toward S1–S4 sites. As discussed above and illustrated in , this is indeed the case.
To further test the extent to which the ability of nSH3 domain to bind to all four S1–S4 sites is attributable to D15, we introduced alanine substitution at G173 in the cSH3 domain (cSH3_G173D) and measured the binding of cSH3_G173D mutant domain to S1–S4 peptides containing PXψPXR motifs. Comparison of affinities and various associated thermodynamic parameters for the binding of nSH3 and cSH3_G173D domains to S1–S4 peptides is provided in . It is clearly apparent from our data that the G173D substitution renders the cSH3 domain to behave very much like the nSH3 domain in its ability to recognize all four S1–S4 sites with binding affinities in the physiologically relevant range. This suggests strongly that D15 is largely responsible for the ability of nSH3 domain to recognize all four S1–S4 sites, while the placement of a glycine residue at this structurally equivalent position within the cSH3 domain deprives it of recognizing S2, S3 and S4 sites.
Figure 5 Comparison of energetics of binding of Sos1-derived peptides S1–S4 to wildtype nSH3 domain (shaded columns) and cSH3_G173D mutant domain (unshaded columns). (a) Binding affinity (Kd); (b) Enthalpic contribution to binding (ΔH); (c) Entropic (more ...)
It is of worthy note that although the nSH3-mimetic cSH3_G173D may appear to exhibit similar binding energetics (), the underlying thermodynamic forces are quite distinct (). Thus, while the binding of nSH3 domain to all four S1–S4 sites is under enthalpic control accompanied by entropic penalty, this only holds true in the case of cSH3_G173D binding to S1 site, whereas binding of cSH3_G173D to S2, S3 and S4 sites is accompanied by favorable entropic contributions. This is indicative of the fact that although D15 may be largely responsible for the ability of nSH3 domain to bind to all four S1–S4 sites, other residues may also play a role in defining its binding specificity.
3D atomic models offer structural insights into the distinct mechanisms employed by SH3 domains in binding to Sos1
In an attempt to rationalize the distinct mechanisms employed by SH3 domains of Grb2 in recognizing Sos1, we modeled 3D structures of nSH3, cSH3 and cSH3_G173D domains in complex with S1 peptide (). In these models, the SH3 domains adapt the characteristic β-barrel fold and the peptide is accommodated into a hydrophobic groove with a relatively open left-handed polyproline type II (PPII) helical conformation. Although the nature of intermolecular hydrophobic forces stabilizing nSH3-peptide and cSH3-peptide complexes is virtually identical, as discussed earlier (22
), the major differences surface in the nature of intermolecular electrostatic forces. Thus, while the nSH3 domain employs respectively D15 and D33 in the formation of ion pairs with R+5 and R+7 in the peptide (), the cSH3 domain relies on E171 and D190 in accomplishing what may seem to be the same feat but it is far from that (). The reason being that while D190 is structurally and chemically equivalent to D33 with the effect that the D33-R+7 and D190-R+7 salt bridges are more or less energetically equivalent, D15 and E171 are structurally non-equivalent and chemically distinct, with G173 in the cSH3 domain being structurally equivalent to D15. Such differences within the two SH3 domains result in the D15-R+5 and E171-R+5 salt bridges being energetically non-equivalent and therefore being the major source of the differential behavior of these two domains toward various potential binding sites within Sos1. As if these residues were insufficient to distinguish their biological roles, the cSH3 domain entertains one additional trick to further differentiate its behavior from the nSH3 domain. Such coup de grace is delivered in the form of D187 involved in the formation of a third salt bridge with R+6 in the peptide ().
Figure 6 3D structural models of S1 peptide in complex with nSH3 (a), cSH3 (b), and cSH3_G173D (c) domains of Grb2. The β-strands in SH3 domains are shown in yellow with loops depicted in gray and the sidechains of acidic residues involved in salt bridging (more ...)
Taking these considerations into account, it would suffice to add that the nSH3 and cSH3 domains employ two-prong and three-prong mechanisms to engage in electrostatic interactions with S1 site in Sos1, respectively. Clearly, the lack of R+6 and R+7 at S2, S3 and S4 sites in Sos1 would imply that not only nSH3 would bind to these sites in an exclusive manner but that it would do so through the engagement of a single D15-R+5 salt bridge, albeit with no effect on the nature of intermolecular hydrophobic forces. It is also of worthy note that while the nSH3 domain and its mimetic cSH3_G173D mutant domain bind to S1–S4 peptides with very similar affinities, the latter displays distinct contributions from the underlying enthalpic and entropic components (). Although structural thermodynamics is still in its infancy, it is interesting to note that such differences in the underlying thermodynamic components may be due to the fact that while nSH3 domain engages in only two intermolecular salt bridges (), the cSH3_G173D mutant domain employs three (). Additionally, the R+5 residue in S1 peptide likely bifurcates the E171 and G173D ion pairs within the cSH3_G173D mutant domain, while R+5 merely engages in the formation of an ion pair with D15 — the residue that is structurally equivalent to G173D — due to the substitution of an alanine (A13) at the structurally equivalent position occupied by E171 in the cSH3_G173D mutant domain. In short, our 3D atomic models offer a closer glimpse into the structural basis of the exclusivity of S1 site for binding to cSH3 domain, while the nSH3 domain can bind to all S1–S4 sites indiscriminately.
SH3 domains appear to combine fidelity and promiscuity under the flagship of Grb2 in cellular signaling
The data presented herein suggest strongly that the cSH3 domain strictly requires the PXψPXRRR motif for binding to Sos1, while the nSH3 domain will do so only upon the presentation of PXψPXR motif under physiological context. In light of these observations, it is tempting to add that the SH3 domains may have evolved to bring the best of both worlds to Grb2. Thus, while nSH3 domain may be ideally suited for signaling promiscuity due to requirement of only one arginine residue within its proline-rich ligands, the cSH3 domain most certainly imparts signaling fidelity upon Grb2. To further confirm this notion, we searched the human proteome for the occurrence of PXψPXR and PXψPXRRR motifs. Our findings reveal that while there are 753 PXψPXR motifs within the human proteome across nearly as many proteins, only six of them contain the PXψPXRRR motif (). In addition to Sos1, these include GPC2, MDM1, OBSL1, SETD5 and SPTA1 (50
). With the exception of Sos1, none of these proteins contain the PXψPXR motif, implying that the cSH3 domain of Grb2 may bind to them in an exclusive manner and thereby leading to build-up of weak interactions. Such a design may be an important aspect of transient signaling allowing Grb2 to fine-tune various overlapping cascades via its interaction with the aforementioned partners.
Figure 7 Amino acid sequence alignment showing the cccurrence of PXψPXRRR motif in human proteome. Absolutely conserved residues within the PXψPXRRR motif are shown in red, the ψ residue is depicted in blue and all other residues are colored (more ...)
Our analysis presented above clearly suggests that while the interaction of Grb2 with other cellular partners through its nSH3 domain may be highly promiscuous, the cSH3 domain provides a striking contrast by virtue of its ability to bind to only a handful of partners adding much needed fidelity to signaling via Grb2. It should be borne in mind that while the proteins containing the PXψPXRRR motif should be expected to bind to the cSH3 domain, the lack of such motif in cellular proteins may not necessarily rule out their interaction with Grb2. This is due to the fact that SH3 domains have also adapted an alternative mechanism independent of the consensus proline-rich sequence PXXP for recognizing some of their partners. In particular, the cSH3 domain of Grb2 has been shown to bind to key signaling modulators Gab1 and SLP76 via the recognition of a non-consensus PXXXRXXKP motif (9
). Structural analysis reveals that the PXXXRXXKP motif binds to the cSH3 domain in a manner akin to the binding of PXψPXRRR motif, with the both motifs sharing the same binding groove and orientation (48
). However, there are some discernable differences. While the PXψPXRRR motif adapts a relatively open left-handed polyproline type II (PPII) helical conformation, the PXXXRXXKP motif adapts a 310
-helical conformation upon binding to the cSH3 domain. The respective conformations are required to orient the critical residues within each motif for optimal interactions within the binding groove of the cSH3 domain. Overall, the PXXXRXXKP motif engages in additional contacts within the binding groove of cSH3 domain relative to the PXψPXRRR motif and, by virtue of these distinguishing interactions, the cSH3 domain binds to Gab1 and SLP76 with higher affinity than Sos1. Such differential binding of cSH3 domain to Gab1 and SLP76 versus Sos1 may be an important determinant of monitoring the ratios of Grb2-Sos1 pool versus Grb2-Gab1 and Grb2-SLP76 pools with consequences for activation of Ras versus other signaling pathways.