Structural and functional properties of Grb2 SH2 dimer in CD28 binding

Abstract

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a critical role in cellular signal transduction. It contains a central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Binding of Grb2 SH2 to the cytoplasmic region of CD28, phosphorylated Tyr (pY) containing the peptide motif pY-X-N-X, is required for costimulatory signaling in T cells. In this study, we purified the dimer and monomer forms of Grb2 SH2, respectively, and analyzed their structural and functional properties. Size exclusion chromatography analysis showed that both dimer and monomer exist as stable states. Thermal stability analysis using circular dichroism showed that the dimer mostly dissociates into the monomer around 50°C. CD28 binding experiments showed that the affinity of the dimer to the phosphopeptide was about three fold higher than that of the monomer, possibly due to the avidity effect. The present crystal structure analysis of Grb2 SH2 showed two forms; one is monomer at 1.15 Å resolution, which is currently the highest resolution analysis, and another is dimer at 2.00 Å resolution. In the dimer structure, the C-terminal region, comprising residues 123–152, was extended towards the adjacent molecule, in which Trp121 was the hinge residue. The stable dimer purified using size exclusion chromatography would be due to the C-terminal helix “swapping”. In cases where a mutation caused Trp121 to be replaced by Ser in Grb2 SH2, this protein still formed dimers, but lost the ability to bind CD28.

The dynamics of free and phosphopeptide-bound Grb2-SH2 reveals two dynamically independent subdomains and an encounter complex with fuzzy interactions

“…There are two main regions in conformational exchange, subdomain I which involves α2 and β5, and subdomain II, at α3. These data are complemented by R 1 , R 2 and 1 H- 15 N heteronuclear NOE ( Figure S2) and by 15 N CPMG-RD experiments at 5, 10, and 17 °C ( Figures S3, S4, S5, and S6). Fig.…”

“…1B and 2B, which represents an expected average value of R 2eff for residue without exchange contribution 21 . The typical plot of 15 N CPMG data observed for fast exchange regimes is where all the points in a flat profile are above the dotted line, meaning that much larger values of ν CPMG would be necessary to refocus the exchange contribution to R 2eff ∞ (Fig. 2B).…”

“…We chose the EGFR derived phosphopeptide EpYINSQV (pY-pep) because it leads to the dissociation of the Grb2, as described by Yuzawa et col. (2001) 18 . We measured the 15 N nuclear spin relaxation parameters R 1, R 2 and 1 H- 15 N-heteronuclear NOE ( Figure S1), which showed that Grb2-SH2 domain is mostly rigid, displaying only a few residues involved in thermal motion (pico-to nanosecond timescale). They are mainly in the loop between α2 and β5.…”

“…3C) is enthalpically favorable and entropically unfavorable at 298 K. This is typical behavior of a conformational fluctuation involving the exposure of hydrophobic residues to the solvent 24 . Knowing that the conformational exchange involving loop β8/α3 and α3 may be associated with the observation, in several crystal structures, of a domain swap involving α3, which opens to interact with the adjacent subunit to form the swapped dimer [13][14][15][16] . The question that remains is whether the minor state is dimeric or monomeric.…”

Recent Advances in Allogeneic CAR-T Cells

“…Another co-stimulatory domain for CAR-T activity is from CD28 and is known as a receptor for CD80 and to stimulate production of various interleukins upon T cell stimulation [19]. Phosphorylation at tyrosine 170 in the intracellular domain of CD28 is responsible for its binding to SH2-domain containing proteins such as PI3K, Grb2, and Gads, resulting in increased production of IL2 and activation of T cells [20][21][22]. Comparative analysis with various CAR constructs to see the effect of TNFRSF9 or CD28 co-stimulation domains identified a higher expansion of CAR-T cells with either co-stimulation domain than with the CD247 domain alone when cocultured with cancer cells expressing the target antigen.…”