Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins

Abstract

Edited by Norma AllewellFull activation of T cells and differentiation into effector T cells are essential for many immune responses and require costimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptorbound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N-and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

Assembly of novel, nuclear dimers of the PI3-Kinase regulatory subunits underpins the pro-proliferative activity of the Cdc42-activated tyrosine kinase, ACK

“…To confirm that the cSH2 domains were functional, binding was tested to a known target peptide from CD28 ( Figure 8C and D). Binding affinities similar to those previously reported were observed, indicating that both cSH2 domains were capable of binding phosphopeptides (31). Thus pTyr607 supports an interaction with p85α and p85β nSH2 domains, with higher affinity for the p85β pY607 peptide.…”

“…We propose a mechanism for increased p85 stability mediated by an interaction involving the p85 nSH2 domain and the ACK phosphorylation site (Tyr607 or equivalent). This interaction likely occurs via a binding mode similar to those already described for the p85 nSH2 domain (31), as shown by abrogated binding in the R358M SH2 null mutant. It is likely that the pTyr607-nSH2 interaction masks the iSH2 region, the target site on p85 for the degradative machinery (30).…”

“…The availability of only the cSH2 might be expected to have variable effects on the ability of a pTyr607-nSH2 p85 dimer to bind to RTKs. However the cSH2 domain is often seen to have higher affinity for receptor sequences than the nSH2 (31,42,43). Thus competition could be the mechanism underpinning the decrease in PIP 3 we observe.…”

“…On the other hand, the cSH2 would still be available to interact with RTKs ( Figure 12). p85 interactions with RTKs are complex, with the nSH2 and cSH2 having different affinities for different pTyr motifs (31,42,43). The availability of only the cSH2 might be expected to have variable effects on the ability of a pTyr607-nSH2 p85 dimer to bind to RTKs.…”

“…An arginine residue is evolutionarily conserved amongst SH2 domains and forms a hydrogen bond with the phosphate group of the phosphotyrosine residue on binding targets (32). Mutation of this arginine to methionine in p85α completely abolishes phosphopeptide binding without disrupting the characteristic SH2 fold (31). Site-directed mutagenesis was used to generate SH2 mutants p85α R358M and p85β R349M and the purified domains were analysed in binding assays.…”