Photon Factory Activity Report 2016 #34 (2017)

BL-5A, 17A/2014G585

Crystal structure analysis of CD28 family molecules complexed with signaling
proteins
Nobutaka Numoto*
Medical Research Institute, Tokyo Medical and Dental University (TMDU),
Bunkyo-ku, Tokyo 113-8510, Japan
1 Introduction
T cells play a central role in immune system. Two setsof signals are needed to activate T cells. One is the T cell
receptor (TCR) signal that is mediated by the major
histocompatibility complex (MHC), and the other is theco-signal provided by CD28 family receptors. The
intracellular domain of CD28 consists of about 40 aminoacids and has no enzymatic activity. A tyrosine kinase isrecruited to the intracellular domain of CD28, and thenphosphorylated YMNM motif (pYMNM) is recognizedby the SH2 domains of signaling molecules such as Grb2,
Grb2-related adaptor downstream of Shc (Gads), and p85
subunit of phosphoinositide 3-kinase (PI3K). The
consensus SH2-binding sequence of Grb2 and Gads ispYXNX, whereas that of p85 is pYXXM. Thus, althoughthese three adaptor molecules activate different signaling
pathways downstream of CD28, the SH2 domains of all
the molecules can bind to CD28 at the same site pYMNM.
To elucidate the structural bases of the molecular
recognition mechanisms between CD28 and thesesignaling molecules, we have performed crystal structure
analyses of three SH2 domains of Gads, N- and Cterminal SH2s of p85, which are complexed with the pYcontaining peptide derived from CD28.

forms hydrogen bond with the SH2 protein. In contrast,
CD28 peptide reveal a more straight conformation in thestructures of p85 nSH2 and cSH2 complexes (Fig. 1a).
Met at pY+3 of CD28 peptide in the p85 nSH2 and cSH2
complexes forms hydrophobic contacts with the SH2
protein and Asn at pY+2 loses contact with the protein.
These facts are consistent with the difference of theconsensus sequence between Gads (pYXNX) and p85(pYXXM). Moreover, comparison among the threestructures reveals the variation of the environments
around the bound phosphorylated tyrosine of CD28
peptide (Fig. 1b).
Our detailed models may useful for development ofcompounds that specifically inhibit the association ofCD28 with these SH2 domains of the adaptor proteins tosuppress excessive T cell responses, such as in allergies
and autoimmune diseases.

2 ExperimentThree SH2 domains (Gads SH2, p85 nSH2, and cSH2)
were co-crystalized with the synthesized pY-containing
peptide (SDpYMNMTP) derived from CD28.
Crystallization conditions were screened using
commercially available screening kits (HamptonResearch) with the hanging-drop vapor diffusion method.
X-ray diffraction experiments were performed atbeamelines 5A and 17A at PF, KEK. The data wereprocessed and scaled using program XDS [1] and weretruncated using the CCP4 program suite. [2] The structure
were solved by the molecular replacement method using
PHASER. [3] Several cycles of manual model rebuilding
and refinement were performed by using the program
COOT [4] and PHENIX, [5] respectively.

b3 Results and Discussion
The crystal structures of Gads SH2-CD28 peptide, p85
nSH2-CD28 peptide and p85 cSH2-CD28 peptide weredetermined at 1.2, 0.9, and 1.1 Å resolution, respectively.
[6] A bent conformation of CD28 peptide in the complex
with Gads SH2 is observed as previously reported
structure of the complex of Grb2 SH2-CD28 peptide. [7]Asn at pY+2 of CD28 peptide in the Gads SH2 complex

aFig. 1: (a) Overall structures of Gads SH2 (green), p85
nSH2 (cyan), and cSH2 (magenta) complexed with CD28
peptide (yellow, stick model). (b) Close up view of theinteractions of the phosphorylated tyrosines. Hydrogenbonds are indicated as yellow dash lines.
References[1] W. Kabsch, Acta Cryst. D66, 125 (2010).
[2] M. D. Winn et al., Acta Cryst. D67, 235 (2011).
[3] A. J. McCoy et al., J. Appl. Cryst. 40, 658 (2007).
[4] P. Emsley et al., Acta Cryst. D66, 486 (2010).
[5] P. D. Adams et al., Acta Cryst. D66, 213 (2010).
[6] S. Inaba et al., J. Biol. Chem., 292, 1052 (2017).
[7] K. Higo et al., PLoS One, 8, e74482 (2013).
* numoto.str@mri.tmd.ac.jp