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. 2009 May 19;48(19):4074-85.
doi: 10.1021/bi802291y.

SH3 domains of Grb2 adaptor bind to PXpsiPXR motifs within the Sos1 nucleotide exchange factor in a discriminate manner

Caleb B McDonald  1 , Kenneth L Seldeen, Brian J Deegan, Amjad Farooq
Affiliations

SH3 domains of Grb2 adaptor bind to PXpsiPXR motifs within the Sos1 nucleotide exchange factor in a discriminate manner

Caleb B McDonald et al. Biochemistry. .

Abstract

Ubiquitously encountered in a wide variety of cellular processes, the Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites containing PXpsiPXR motifs within Sos1. Here, using isothermal titration calorimetry, we demonstrate that while the nSH3 domain binds with affinities in the physiological range to all four sites containing PXpsiPXR motifs, designated S1, S2, S3, and S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3, and S4 sites, the PXpsiPXR motif at the S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In striking contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site, but their role is not critical for the recognition of S2, S3, and S4 sites. Site-directed mutagenesis suggests that the two additional arginine residues flanking the PXpsiPXR motif at the S1 site contribute to free energy of binding via the formation of salt bridges with specific acidic residues in SH3 domains. Molecular modeling is employed to project these novel findings into the 3D structures of SH3 domains in complex with a peptide containing the PXpsiPXR motif and flanking arginine residues at the S1 site. Taken together, this study furthers our understanding of the assembly of a key signaling complex central to cellular machinery.

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Figures

Figure 1
Figure 1
Domain organization and sequence analysis of Grb2 and Sos1. (a) Grb2 is comprised of a central SH2 (Src homology 2) domain flanked between SH3 (Src homology 3) domains. The sequence alignment of nSH3 and cSH3 domains highlights various structurally equivalent residues within the β-barrel fold. The residues constituting the β1–β6 strands are boxed. Vertical arrows indicate acidic residues whose roles in recognizing Sos1 are being investigated in this study. (b) The proline-rich (PR) domain of Sos1 lies at the extreme C-terminal end. The PR domain contains four distinct sites (designated S1, S2, S3 and S4) characterized by the PXψPXR consensus motif. The complete sequences of these sites are shown. The position of various residues relative to the first proline within the PXψPX motif, which is designated zero, is also indicated. Other domains within Sos1 shown are HF (histone fold), DH (Dbl homology), PH (pleckstrin homology), REM (Ras exchange motif) and Cdc25.
Figure 2
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 Origin software.
Figure 3
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 Origin software.
Figure 4
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 (ΔGTotal) of binding of nSH3 and cSH3 domains to S1 peptide expressed in absolute terms (kcal/mol). The energetic contribution of R+5 (ΔGR+5) was calculated from the relationship ΔGR+5=ΔGR+5A−ΔGTotal, where ΔGR+5A is the free energy of binding of S1_R+5A peptide to the nSH3 or the cSH3 domain. The energetic contribution of R+6 (ΔGR+6) was calculated from the relationship ΔGR+6=ΔGR+6A−ΔGTotal, where ΔGR+6A is the free energy of binding of S1_R+6A peptide to the nSH3 or the cSH3 domain. The energetic contribution of R+7 (ΔGR+7) was calculated from the relationship ΔGR+7=ΔGR+7A−ΔGTotal, where ΔGR+7A is the free energy of binding of S1_R+7A peptide to the nSH3 or the cSH3 domain. (b) Energetic contributions relative to the total free energy (ΔGTotal) of binding of nSH3 and cSH3 domains to S1 peptide expressed as a percentage (%). The energetic contribution of R+5 (%R+5) was calculated from the relationship %R+5=[(ΔGR+5A/ΔGTotal)×100], where ΔGR+5A is the free energy of binding of S1_R+5A peptide to the nSH3 or the cSH3 domain. The energetic contribution of R+6 (%R+6) was calculated from the relationship %R+6=[(ΔGR+6A/ΔGTotal)×100], where ΔGR+6A is the free energy of binding of S1_R+6A peptide to the nSH3 or the cSH3 domain. The energetic contribution of R+7 (%R+7) was calculated from the relationship %R+7=[(ΔGR+7A/ΔGTotal)×100], where ΔGR+7A is the free energy of binding of S1_R+7A peptide to the nSH3 or the cSH3 domain. All energetic contributions were calculated using data provided in Table 1.
Figure 5
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 contribution to binding (TΔS) with the + and − signs indicating entropic gain and entropic penalty, respectively; and (d) Overall free energy of binding (ΔG). All parameters were directly determined from ITC analysis. Error bars were calculated from 2–3 independent measurements. All error bars are given to one standard deviation.
Figure 6
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 with the peptide in red. The backbone of Sos1 peptide is colored green with the sidechains of arginine residues involved in salt bridging with SH3 domains in blue. Two alternative orientations, related by an 180°-rotation about the vertical axis, are shown for clarity and close scrutiny.
Figure 7
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 black. The proteins containing this motif are listed in the far left column, while the far right column provides the corresponding Expasy codes. The numerals hyphenated to amino acid sequence at each end denote the residue number within the protein sequence.
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