SH3 Domains of Grb2 Adaptor Bind to PXψPXR Motifs Within the Sos1 Nucleotide Exchange Factor in a Discriminate Manner

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 PXψPXR 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 PXψPXR motifs, designated S1, S2, S3 and S4, the cSH3 domain can only do so at S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXψPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXψPXR motifs at S2, S3 and S4 sites, the PXψPXR motif at S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of cSH3 domain. In striking contrast, these two additional arginine residues augment the binding of nSH3 domain to 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 PXψPXR motif at 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 PXψPXR motif and flanking arginine residues at S1 site. Taken together, this study furthers our understanding of the assembly of a key signaling complex central to cellular machinery. KeywordsGrb2 adaptor; Sos1 nucleotide exchange factor; SH3 specificity and promiscuity; SH3-ligand thermodynamics Grb2-Sos1 interaction, mediated by the canonical binding of N-terminal SH3 (nSH3) and Cterminal SH3 (cSH3) domains of Grb2 to proline-rich motifs within Sos1, plays a central role in relaying external signals from receptor tyrosine kinases (RTKs) at the cell surface to downstream effectors and regulators such as Ras within the cytosol (1-4). Comprised of a central SH2 domain flanked between nSH3 and cSH3 domains (Figure 1a), Grb2 recognizes activated RTKs by virtue of its SH2 domain to bind to tyrosine-phosphorylated (pY) sequences in the context of pYXN motif located within the cytoplasmic tails of a diverse array of receptors, including EGF and PDGF receptors (5,6). Upon binding to RTKs, the SH3 domains live up to Grb2's reputation and grab a wide variety of proteins, containing proline-rich sequences, in an attempt to recruit them to the inner membrane surface -the site of initiation of a plethora of signaling cascades (3,(7)(8)(9)(10)(11)(12)(13)(14). Among them, the guanine nucleotide exchange factor Sos1 is by

Multivalent Binding and Facilitated Diffusion Account for the Formation of the Grb2–Sos1 Signaling Complex in a Cooperative Manner

“…Within each nSH3 hydrophobic groove, the PXψPXR motifs are stabilized by an extensive network of intermolecular contacts as reported earlier (26, 32, 33). In particular, the aliphatic sidechains of consensus ψ residue within S1 (V1153) and S4 (V1292) sites are sandwiched between benzyl rings of F9 and Y52 located within each nSH3 hydrophobic groove through van der Waals contacts.…”

“…While the β-barrel is comprised of a pair of nearly-orthogonal β-sheets, with each β-sheet containing three anti-parallel β-strands, the peptide adopts a relatively open left-handed polyproline type II (PPII) helical conformation upon binding. Although our previous studies have shown that the isolated nSH3 domain of Grb2 can potentially bind to peptides derived from all four S1–S4 motifs in a physiologically-relevant manner (26, 32, 33), the precise mechanism of the assembly of Grb2-Sos1 signaling complex remains hitherto poorly understood. In light of the knowledge that Grb2 exists in a dimer-monomer equilibrium in solution (34), it is tempting to postulate that Grb2 could bind to Sos1 in a multivalent manner so as to generate higher-order Grb2-Sos1 multimers rather than a simple binary complex.…”

Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms

“…pET102 bacterial expression plasmids for wildtype and mutant cSH3 domains of human Grb2 were cloned and expressed in Escherichia coli Rosetta2(DE3) strain as described earlier (McDonald et al, 2008a; McDonald et al, 2009). Recombinant proteins were purified to apparent homogeneity using a combination of Ni-NTA and size-exclusion chromatographic procedures and further characterized as reported previously (McDonald et al, 2008a; McDonald et al, 2009).…”

“…Recombinant proteins were purified to apparent homogeneity using a combination of Ni-NTA and size-exclusion chromatographic procedures and further characterized as reported previously (McDonald et al, 2008a; McDonald et al, 2009). 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.…”

“…The experiments were initiated by injecting 25 × 10µl aliquots of 2–8 mM of each peptide from the syringe into the calorimetric cell containing 1.8ml of 50–200 µM of an cSH3 domain solution at 25 °C. All other control experiments were performed as described earlier (McDonald et al, 2008a; McDonald et al, 2009). To extract binding affinity (K d ) and binding enthalpy (ΔH), the ITC isotherms were iteratively fit to the following built-in function by non-linear least squares regression analysis using the integrated ORIGIN software: $$\mathrm{q}(\mathrm{i})=(\mathrm{n}\mathrm{\Delta }\text{HVP}/2){\{[1+(\mathrm{L}/\text{nP})+({\mathrm{normalK}}_{\mathrm{normald}}/\text{nP})]-{[[1+\mathrm{L}/\text{nP})+{\mathrm{normalK}}_{\mathrm{normald}}/\text{nP})]}^2-(4\mathrm{L}/\text{nP})]}^{1/2}\}$$ where q(i) is the heat release (kcal/mol) for the ith injection, n is the binding stoichiometry, V is the effective volume of protein solution in the calorimetric cell (1.46 ml), P is the total protein concentration in the calorimetric cell and L is the total concentration of peptide ligand added for the ith injection.…”